ROR1 CAR T cells and lenvatinib cooperatively target anaplastic thyroid carcinoma

ROR1 CAR T cells and lenvatinib cooperatively target anaplastic thyroid carcinoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article ROR1 CAR T cells and lenvatinib cooperatively target anaplastic thyroid carcinoma Christine Dierks, Oleksandra Skorobohatko, Dmitry Chernyakov, and 16 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8690827/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Anaplastic thyroid carcinoma (ATC) is a rare thyroid malignancy with poor prognosis and very limited treatment options. Therefore, the development of novel therapies is urgently needed. Here, we identified the Receptor Tyrosine Kinase like Orphan Receptor 1 (ROR1) protein as a specific CAR T cell target for ATC. ROR1 is part of the Wnt signalling pathway, mainly expressed during embryogenesis, but mostly absent in differentiated adult tissue. In ATCs, ROR1 is strongly overexpressed (RNA/protein level, surface expression) and high expression levels are associated with reduced survival. ROR1 CAR Ts specifically target ATC cell lines in 2D and 3D spheroid cultures, reduce the quantity of circulating tumour cells and block tumour metastases in different ATC mouse models. While small tumours were completely eliminated by ROR1 CAR T cells alone, larger tumours required the combination of ROR1 CAR T cells with the multikinase inhibitor lenvatinib. Lenvatinib blocked primary tumour growth, reduced the quantity of immunosuppressive cancer associated fibroblasts (CAF-S1) in the microenvironment and enhanced CAR T cell functionality and activation. Overall, we validated ROR1 as a prime target for CAR T cell therapies in ATC and identified lenvatinib as highly valuable combination partner, which is able to improve CAR T cell functionality. Health sciences/Oncology/Cancer/Cancer therapy/Cancer immunotherapy Health sciences/Endocrinology/Endocrine system and metabolic diseases/Endocrine cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Key points ROR1 is specifically overexpressed in ATC and functions as a prime target for CAR T cell therapy. While ROR1 CAR T cells efficiently target circulating tumour cells and tumour metastases, lenvatinib blocks primary tumour growth and enhances CAR T cell functionality and activation. Introduction Anaplastic thyroid carcinoma (ATC) is a devastating disease with a median survival of 4 months despite multimodal therapy including surgery, irradiation and chemotherapy. 1–3 Although ATC accounts for only 1% of thyroid carcinomas (TC), it is responsible for 50% of the deaths caused by thyroid cancer. ATCs arise from the follicular epithelium of the thyroid gland, with 50% developing de novo and 50% with a pre-existing differentiated TC. Tumours are high proliferative and show a very fast and infiltrative growth into local structures like oesophagus, trachea or the carotid artery, with 50% having distant metastasis already at diagnosis. Genetic alterations include mutations in TP53, HRAS, KRAS, PIK3CA, PTEN and only 20–30% carry targetable mutations in BRAF or have NTRK or RET fusions. 4–7 Since ATCs usually have high PD-L1 levels and a strong inflammatory tumour microenvironment (TME), 8 we and others have developed novel treatment strategies combining kinase inhibitors with immune checkpoint inhibitors (lenvatinib/pembrolizumab), which achieve improved responses and an increased median survival compared to chemotherapy. 9 Despite some progress, the majority of patients relapse and urgently need alternative treatment options. CAR T cells are engineered T cells which carry a synthetic receptor, which is directed against a tumour antigen. In 2017, the first CAR T cells against CD19 were approved for the treatment of B cell lymphoma and B cell acute lymphoblastic leukaemia. 10,11 While CAR T cells are highly effective in hematologic malignancies, several obstacles exist for CAR T cells in solid tumours that must be overcome. These include the identification of specific tumour antigens, the hypoxic and acidic TME, immunosuppressive mechanisms and the compactness and desmoplasia of many carcinoma entities, which prevent CAR T cell access to its target cells. 12 Previous efforts in differentiated TCs include CAR T cell targeting of the TSH receptor 13 , but this option cannot be used for ATCs as its expression is lost during the dedifferentiation process. The tyrosine protein kinase transmembrane orphan receptor 1, ROR1, is part of the non-canonical Wnt signalling pathway and can be activated via Wnt5a. 14 It is mainly expressed during embryonic development and reactivated in various neoplasms. 15–17 In this context, ROR1 can influence several intracellular signalling cascades like pro-apoptotic signalling pathways, MAP kinase signalling, phosphoinositide 3-kinase or NF-κB leading to an elevated cell survival, migration and proliferation promoting tumour growth and resistance to apoptosis. 18–20 Furthermore, ROR1 was shown to be overexpressed on cancer stem cells and to be predominantly expressed in undifferentiated tumour types, which positively correlates with occurrence of tumour metastasis and relapse. 21,22 Due to its specificity especially for un- and dedifferentiated malignant neoplasias, while sparing most normal adult tissues, ROR1 represents an interesting target for an adoptive immunotherapy based on chimeric antigen receptor T cells (ROR1 CAR T cells). 23 ROR1 CAR T cells were shown to specifically target some hematological malignancies, such as e.g. CLL and solid tumours. 17,24–26 Furthermore, their safety was evaluated in various preclinical models 27,28 and in a phase I clinical trial in hematologic malignancies and solid tumours. 29 Kinase inhibitors, like lenvatinib, sorafenib and cabozantinib, are approved for the treatment of radioiodine refractory differentiated TCs. 30–33 Their common feature is targeting the VEGFR signalling axis and therefore preventing neoangiogenesis and thus reducing tumour growth. Besides neoangiogenesis, VEGFR inhibitors can also improve T cell function in immune checkpoint inhibitor therapies and can transform the immunosuppressive microenvironment in several tumour entities. 34 Based on these data, we hypothesized that VEGFR inhibitors might also support CAR T cell function. In the study shown here, we identified ROR1 as a prime target for anaplastic thyroid carcinoma and identified lenvatinib as an ideal combination partner, which blocks tumour growth, improves CAR T cell functionality and reduces local immunosuppression. Results ROR1 is overexpressed in ATC compared to normal thyroid tissue and high expression levels are associated with reduced patient survival ROR1 expression was determined via transcriptome analysis, IHC and ROR1 surface expression. By using RNAseq data from fresh-frozen own ATC/PDTC patient samples (n = 20) compared to goiter, we found ROR1 RNA to be overexpressed in anaplastic and poorly differentiated TC compared to goiter (Fig. 1 A). Through an NCBI dataset (GSE76039) 7 which includes transcriptome data (Affymetrix U133 plus 2.0 array) from fresh frozen and histologically confirmed ATC (n = 18) and PDTC (n = 17) samples compared to PTCs we could determine that ROR1 was significantly higher expressed in ATCs (n = 18) compared to PDTCs (n = 17) (Fig. 1 B, Figure S5 A-C). TCs with a BRAF-like signature or BRAF mutations (n = 275) had significantly higher ROR1 levels than RAS-like or RAS mutated thyroid tumours (n = 118) respectively (Figure S5 A, B). High expression levels for ROR1 were associated with a significantly shorter survival (median survival ROR1 high = 5.9 month, ROR1 low = 102.1 month, Fig. 1 C). ROR1 expression was inversely correlated with the expression of the TSH receptor , indicating it as a marker for undifferentiated thyroid cancer (Fig. 1 D, Figure S5 C). Also gene clustering analysis on primary frozen ATC/PDTC and goiter showed that the clusters with the most undifferentiated tumours (GSEA cluster undifferentiated cancer) within ATCs (ATC cluster II) are associated with the highest ROR1 transcript levels, while losing differentiation markers like thyreotropin stimulating hormone receptor (TSHR) , thyreoperoxidase ( TPO) , thyreoglobulin (TG) and others (Fig. 1 E, F, Figure S6 D-F). Histologically, the ATC areas displayed high ROR1 levels, while adjacent normal thyroid tissue from the same patient sample showed low to absent ROR1 expression (Fig. 1 G, H). Conversely, ATC areas were negative for TSHR, which was always expressed on normal thyroid tissue (Fig. 1 I, J). ATC cell lines, which were either BRAF V600E mutated (8505C, SW1736) or HRAS G13R mutated (C643) completely recapitulated the features of primary ATC patient tissues and showed high ROR1 surface expression and absent TSHR expression (Fig. 1 K, Figure S7 A-D). (A) ROR1 transcript levels of fresh-frozen tissue samples from patients with ATC (n = 12) compared to goiter (n = 8); mean ± SD, unpaired t-test (*** p ≤ 0.0001). Patient data is available in table S1. (B) NGS analysis of ROR1 expression in ATC (n = 18 ●) versus PDTC (n = 17 ●) using the GSE76039 dataset 7 ; mean ± SD, unpaired t-test (*** p ≤ 0.0001). (C) Overall survival (OS) of ATC and PDTC patients with high (above median) and low (below median) ROR1 -expression from the GSE76039 dataset 7 ; log-rank (Mantel-Cox) test (**** p ≤ 0.0001). (D) Pearson’s correlation of ROR1 and TSHR RNA expression in the Halle patient cohort (Table S1). (E) Principle component analysis and cluster analysis of the NGS data of goiter patient samples (n = 8) and ATC patient samples (n = 12). (F) Heatmap of the expression of the thyroid-tissue specific markers and ROR1 . TG – Thyroglobulin, IYD – Iodotyrosine Deiodinase, TSHR – Thyroid Stimulating Hormone Receptor, NKX2-1 – NK2 Homeobox or Thyroid Transcription Factor-1. (G, I) Representative examples for ROR1 and TSHR IHC DAB-staining in healthy thyroid tissue (HTT) and ATC within the same patient (n = 20); black scale bar corresponds to 200 µm, red – to 50 µm. (H, J) H Score of the ROR1 and TSHR in ATC tissue samples (ATC) ● and healthy thyroid tissue samples (HTT) ○ from the same patient (n = 20); paired t-test (*** p ≤ 0.001; **** p ≤ 0.0001). (K) ROR1 surface expression as measured by flow cytometry in 3 different ATC cell lines (8505C, C643, SW1736) and one ovarian cancer cell line (A2780). The values in red represent median of ROR1-PE expression of each cell line. Experiments were performed in triplicates. Taken together, ROR1 is specifically overexpressed at the RNA, protein and surface level in both, primary ATC tissues and ATC cell lines and high ROR1 expression levels are associated with a reduced patients’ survival. Since previous studies have shown absence or low levels of ROR1 expression in healthy tissues, ROR1 represents an interesting and specific novel CAR T cell target for ATCs. 3,17,27,35,36 ROR1-directed CAR T cells effectively and specifically lyse ROR1-positive ATC cells in vitro Second-generation ROR1 CAR T cells were developed by the laboratory of Prof. M. Hudecek and the ROR1 antigen targeting CAR is expressed in the lentiviral targeting vector R12 4-1BB CD3 zeta (Fig. 2 A). ROR1 CAR T cells were previously shown to be effective against their target; a high target specificity and can be safely applied to animals and humans. 17,27 Confirming these results in ATC, co-culture of ROR1 CAR T cells with any of the 3 different ROR1 + ATC cell lines induced efficient and fast tumour cell lysis (PI staining for dead cells and SRB-based viability assay; Fig. 2 B, C), while ROR1-negative ovarian cancer cells were not affected and also untransduced control T cells (UTD) did not induce tumour cell lysis. In addition, ROR1 + ATC cell lines induced specific activation of ROR1 CAR T cells including production of inflammatory cytokines, like INF-α and GM-CSF (Fig. 2 D, Figure S8). Target specificity was tested via CRISPR-Cas9-induced knockout (KO) of ROR1 in all 3 ATC cell lines, which was verified via sequence analysis and flow cytometry for ROR1 surface expression (blue graph; Fig. 2 E). The ROR1 KO completely abolished tumour cell lysis (Fig. 2 F, right columns), indicating target specificity of the ROR1 CAR T cells in ATCs. Next, ROR1 CAR T cell efficacy was tested in 3D spheroid cultures, where T cells need to invade into a semisolid structure under hypoxic and acidic conditions. ROR1 CAR T cells were able to efficiently infiltrate the spheroids formed by C643 and SW1736 cells, destroyed the spheroid structure including the spheroid barriers and efficiently lysed ROR1 + tumour cells (Fig. 2 G, H), which was documented via live cell imaging over 24 hrs (supplementary video 1). ROR1 KO ATC cells were also able to form spheroids, but were not affected by ROR1 CAR T cells confirming target specificity also in 3D cultures. (A) Schematic representation of the targeting principle using ROR1 CAR T cells with ROR1 + ATC (anaplastic thyroid carcinoma) cell lines. Right picture shows the CAR construct with the single chain variable fragment (scFv) domain, the CD28 transmembrane domain (TMD), the 4-1BB costimulatory domain and the CD3ζ intracellular domain (ICD). 25,37,38 (B) Representative fluorescence and HD phase image (Incucyte) of the ROR1-expressing ATC-cell line 8505C co-incubated with untransduced (UTD) T cells (upper image) or ROR1-targeting CAR T cells for 24 hours. Propidium iodide (PI) staining in red clusters represents dead cancer cells (scale bar 400 µm). (C) Relative number of living cells using a SRB assay (measurement of relative cellular protein content) for ROR1-expressing ATC cell lines (8505C, SW1736, C643) and the ROR1-negative ovarian cancer cell line A2780 after co-incubation with two- and fivefold numbers of UTD or ROR1 CAR T cells; n = 3, mean ± SD, multiple t-tests ( *** p ≤ 0.001, ** p ≤ 0.01; * p < 0.05; ns ≥ 0.05). (D) Quantification of cytokines like interferon-γ using the MACSPlex Cytokine 12 Kit from Miltenyi Biotec. Cytokine levels were assessed in the supernatant of co-cultures of ROR1 + ATC cell lines (8505C, SW1736, C643) or ROR1- ovarian cancer cells (A2780) with control, UTD (untransduced T cells) or ROR1 CAR T cells. N = 3, mean ± SD, multiple t-tests, (*** p ≤ 0.001, ** p ≤ 0.01, * p < 0.05; ns ≥ 0.05). (E) Flow cytometry analysis of ROR1 expression in wild type ATC cell lines (8505C, C643, SW1736, red graph) and the respective ROR1-KO ATC cell line (blue graph) generated with Crispr-Cas9 against the ROR1 sequence. The values in red represent median fluorescence intensity (MFI) of the ROR1-PE staining of each cell line. (F) SRB-mediated relative viability assay measuring the cellular protein content in ATC cell monolayers and treated with either mock, UTD or ROR1 CAR T cells. n = 3, mean ± SD, One-Way ANOVA. (*** p ≤ 0.001, ns ≥ 0.05) (G) Luminescence-based viability assay of the ROR1-expressing ATC model cell line C643 and SW1736 in 3D culture as spheroids and its ROR1-KO counterpart (3D) co-incubated with mock, untransduced T cells (UTD T) and ROR1-targeting CAR T cells (CAR T). Luminescence was measured with Tecan Spark® Multimode Mikroplate Reader. N = 3, mean ± SD, two-Way ANOVA. (*** p ≤ 0.001, ns ≥ 0.05). (H) Visualisation of the co-incubation of C643 spheroids as well as C643 with ROR1-KO with UTD control T cells or CAR T cells (scale bar 200 µm). The experimental setup is identical to that described in (G). For the visual analysis and video generation (Supplemental video 1) with the Incucyte, the T cells were labelled with CellTracker™ Red CMTPX according to the manufacturer's instructions. ROR1 CAR T cells eliminate small ATC xenograft tumours in vivo Next, ROR1 CAR T cell efficacy was tested in different ATC xenograft models in vivo . While the ROR1-expressing BRAF-mutated ATC cell line 8505C was subcutaneously (s.c.) injected into the left flank of immunocompromised NSG (NOD.Cg-Prkdcscid Il2rgtm1Sug/JicTac) mice (Fig. 3 A-C), the RAS mutated ATC cell line C643 was injected retroorbitally and cells were mainly detected at the injection site and within the lungs (Fig. 3 D-F). After 2 or 7 days 5 *10 6 ROR1 CAR T cells (CD4/CD8 mixed 1:1) or vehicle were injected intravenously (i.v.) and tumour cell expansion was monitored via volume determination (s.c. model) and luminescence imaging (i.v. model). ROR1 CAR T cells efficiently blocked tumour development independent of the cell line, mode of engraftment and detection method (volume and luminescence detection), injection time point and mutation status (BRAF- or RAS mutated) (Fig. 3 A-F). Next, we aimed to establish a more physiological situation, like in patients, where CAR T cells are used when tumours are already established with sizes at least beyond 100 mm³. Therefore, tumour cells were injected and tumours were allowed to develop for 25 days until a medium start volume of 165.9 mm 3 was reached (Fig. 3 G, H). At day 25, mice received either a control injection with PBS (n = 7), a one-time ROR1 CAR T cell injection (CD4:CD8 ratio 1:1) (n = 8) or weekly ROR1 CAR T cell injections (n = 7), respectively. In contrast to the previous experiments, the one-time injection of ROR1 CAR T cells was not able to significantly reduce the tumour mass of these already established, fast growing ATC tumours (Fig. 3 H). In contrast, repeated CAR T cell injections (3 x once weekly) showed improved effects, i.e. tumour volumes were reduced by one third (control mean = 957 mm³, 3 x CAR T cells = 630 mm³, p = 0.0254) and tumour weight to about half (control mean = 1.0 g, 3 x CAR Ts = 0.58 g; p = 0.0002, Fig. 3 I, J). To visualize the infiltration into the tumour, CAR T cell, were labeled with IVISense 680 Fluorescent Cell Labeling for further experiments prior to injection into mice. Extracted tumours (40 days after injection) showed CAR T cell fluorescence signals on the tumour surface and also within the tumour indicating that CAR T cells can indeed penetrate the tumour (Fig. 3 K), which was confirmed by immunohistological stainings (Fig. 3 L, M). (A) Experimental scheme for NSG mice subcutaneously injected with BRAF V600E + mutated 8505C ATC cells (3×10⁶ 8505C cells transduced with a luciferase vector) and treated with intravenously (i.v.) applied ROR1 CAR T cells 2 or 7 days after tumour cell injection. The administration of 100 µL of CAR T cells (5×10⁶ CAR T cells per NSG mouse with a CD4 to CD8 ratio of 1:1) or vehicle (PBS) was conducted via the tail vein. (B, C) Exemplary imaging analysis (luminescence imaging, IVIS Spectrum) before (experimental day 2) and 26 days after CAR T injection (day 28) and tumour volume analysis with a digital caliper gauge are shown in Figures (B) and (C), respectively. (D) Experimental scheme for NSG mice retroorbitally injected with RAS-mutated C643 ATC cells (1×10⁵ in 100 µL PBS) and treated with control (n = 3) or ROR1 CAR T cells (5×10⁶, CD4 to CD8 ratio of 1:1, retroorbital opposite site, n = 3) two days after tumour cell injection. (E, F) Representative imaging analysis (luminescence imaging, IVIS Spectrum) before (experimental day 2) and 12 days after CAR T cell injection (day 14) and tumour volume analysis with a digital caliper gauge are shown in figures (E) and (F), respectively. N = 3, mean ± SD, two-way ANOVA (**** p ≤ 0.0001). (G) Experimental design for CAR T cell treatment in already established 8505C tumours. 3×10⁶ 8505C cells were injected in the left flank of NSG mice and grew over 25 days (mean volume 163.7 ± 53.7 mm 2 ), then mock (n = 7) or 5×10⁶ ROR1 CAR T cells were injected intravenously in the tail vein once (n = 8) or 3-times weekly (n = 7). (H) Mean tumour volume over time; (I) mean tumour volume on day 42 (end of control group) and (J) mean tumour weight at the end of the experiment 42 days after tumor cell injection and 17 days after first CAR T cell injection; n = 7–8, mean ± SD, unpaired t-test (* p ≤ 0.05, *** p ≤ 0.001). (K) Fluorescent images of tumours treated either with control of IVISense 680 Fluorescent Cell labelled ROR1 CAR T cellss. Images show either front view (surface) or cross sections. (L) Percentage of ROR1 CAR T cells within tumour tissue analysed by IHC = 7–8, mean ± SD, unpaired t-test (*** p ≤ 0.001). (M) Images of tumour sections stained with a CD5-antibody marking the infiltrating ROR1 CAR T cells. The IHC slides were analysed using the QuPath 0.4.3 software, red-marked cells represent CD5 + CAR T cells, blue-marked cells represent CD5- tumor and other cell types. (N) Percentage of ROR1 CAR T cells within tumour tissue analysed by flow cytometry of mouse peripheral blood; n = 7–8, mean ± SD, unpaired t-test (*** p ≤ 0.001). As shown in Fig. 3 M, ROR1 CAR T cells (in red) are located mainly around the tumour blood vessels, but are not equally distributed over the complete tumour mass. Quantification shows that CAR T cells account for about 2–3% of total cells within a tumour (Fig. 3 L). In addition, circulating CAR T cells are found in the peripheral blood of mice 19d after CAR T cell treatment (Fig. 3 N). Lenvatinib and other kinase inhibitors can improve CAR T cell functionality in vitro From previous preclinical work and from the clinical ATLEP trial in ATC and PDTC patients, we know that multikinase/VGFR inhibitors, like lenvatinib, enhance the efficacy of endogenous T cells in the context of immune checkpoint inhibitor therapies and can change the microenvironment. 39–43 Based on these results, it was hypothesized that multikinase inhibitors might also improve CAR T cell functionality and tumour invasion. To test this, co-cultures of tumour cells and ROR1 CAR T cells were treated with the multikinase inhibitors lenvatinib, sorafenib and cabozantinib for 24 hrs (Fig. 4 A). Gene expression analysis of the CAR T cells in co-culture demonstrated strong effects of the kinase inhibitors on CD4 + CAR T cells demonstrated by enhanced expression of genes related to T cell activation, T cell proliferation, T cell differentiation, adaptive immune response, TCR signalling, T cell-mediated toxicity and T cell migration, while CD8 + CAR T cells were less affected (Fig. 4 B, Figure S10-11). Lenvatinib showed a distinct cluster profile from sorafenib and cabozantinib (Figure S12) with strongest effects in the most physiological setting with CD4/CD8-mixed CAR T cell cultures (Fig. 4 C). From all 3 inhibitors tested, lenvatinib had the strongest outcome on major T cell functions, like T cell proliferation, TCR pathway activation, T cell mediated cytotoxicity, T cell cytokine production and T cell migration (Fig. 4 C, blue). The VEGFR inhibitors lenvatinib and cabozantinib neither directly influenced the viability of tumour cells nor altered ROR1 receptor expression (Fig. 4 D, E; Figure S13). High sorafenib concentrations reduced the viability of the RAS-mutated cell line C643. None of the inhibitors negatively affected tumour cell killing of ROR1 CAR T cells (Fig. 4 F; Figure S14 A-C) (A) Schematic overview of the experimental setup to analyse the influence of TKIs on activated CAR T cells. 8505C WT cells (0.01×10⁶) were seeded in a 24-well plate and incubated for 24 hours before treatment with 0.02% DMSO (mock) or 1 µM lenvatinib, 1 µM cabozantinib, or 1 µM sorafenib, respectively, CAR T cells (1.2–1.5×10⁶) were added 24 hrs later for another 24 hrs, followed by RNA isolation from CAR T cells and subsequent RNA sequencing. (B) Heatmap of upregulated and downregulated genes in activated CAR T cells (left CD4 + T cells only and right CD8 + T cells only) after treatment with different TKIs in comparison to mock from the experiment described in (A). ( https://software.broadinstitute.org/morpheus ), L = Lenvatinib, C = Cabozantinib, S = Sorafenib. (C) Radar charts illustrating RNA expression in key biochemical pathways for the T cell immune response from experiment (A). Charts were generated using the radar chart function from the fsmb package. T cell-related GO terms were selected from Gene Set Enrichment Analysis (GSEA) 44 , and mean gene expression was calculated for each CAR T cell treatment group, with mock samples as the baseline. Percent expression differences relative to the mock group are shown as coloured lines in the radar charts. (D) Relative numbers of viable cells measured with an SRB-based viability assay in which ROR-1 expressing ATC cell lines were co-incubated with different TKIs (n = 3, mean ± SD, t-test, ** p < 0.01) (E) Flow cytometric analysis for ROR1 expression of ATC cell lines treated with vehicle or lenvatinib at 1 or 2 µM concentration for 72 hrs, respectively. (F) SRB-based viability assay was performed using the ROR1-expressing 8505C ATC cell line co-incubated with UTD or CAR T cells at 1:2 and 1:5 ratios with or without additional lenvatinib treatment for 24 hrs. N = 4, mean ± SD, t-test (ns ≥ 0.05). Lenvatinib reduces the quantity of immunosuppressive CAFs S1 in ATC fibroblast cultures In order to understand whether lenvatinib not only alters CAR T cell differentiation, but also changes the immunosuppressive TME, cancer-associated fibroblasts (CAFs) from 6 different patients with ATC or PDTC were isolated and their subtypes determined. As shown in Fig. 5 A, B, the patient-derived CAFs comprise of all different CAF subtypes, such as normal fibroblast-like (CAF-S2 and CAF-S3), immunosuppressive CAF-S1 and CAF-S4 known to drive tumour metastasis. Treatment of patient-derived ATC fibroblasts with lenvatinib did not alter CAF density or phenotype (Fig. 5 C), and did not reduce total quantity of CAFs (Fig. 5 D), but strongly reduced the immunosuppressive CAF-S1 subtype (Fig. 5 E, F). (A) Scheme presenting the different phenotypes of cancer-associated fibroblasts (CAFs) including their proposed function according to the literature. 45–47 (B) FACS gating strategy for CAF subpopulations of ATC patient material. (C) Images showing patient- derived fibroblasts from thyroid preparations from ATC/PDTC patients. (D, E) Quantification of total CAFs (D) or total immunosuppressive CAF-S1 (E) with and without lenvatinib treatment over seven days (7d, 1 µM), n = 6, unpaired t-test (** p ≤ 0.01, ns p ≥ 0.05). (F) Flow cytometric images showing CAF-S1 cells (EpCAM-CD45-CD31-FAP high CD29 high ) with and without lenvatinib treatment. Phenotypic changes in ATC and PDTC patient-derived fibroblasts after treatment in vitro with 1 µM lenvatinib for one week. Lenvatinib improves ROR1 CAR T cell efficacy in vivo To test, if lenvatinib can indeed improve the efficacy of ROR1 CAR T cells in vivo , we combined ROR1 CAR T cell with lenvatinib in the 8505C xenograft mouse model (Fig. 6 A). Again, ROR1 CAR T cells alone were not able to stop tumour growth in the exponential growth phase (Fig. 6 B, red line) compared to vehicle control (grey line). Lenvatinib alone (blue line) significantly reduced tumour volume and delayed disease development, but did not completely inhibit tumour growth (Fig. 6 B, C). In contrast, the combination of lenvatinib with ROR1 CAR T cells (pink line) not only efficiently blocked tumour development, but was also able to induce regression of already established tumours (Fig. 6 B-D). Results were further improved by combination of lenvatinib treatment with repeated ROR1 CAR T cell injections (Fig. 6 E-H, violet lane) resulting in about one-third tumour volumes (control = 1458.3 mm 3 , lenvatinib alone = 750.3 mm³, lenvatinib + 1 x CAR T cells = 522.4 mm³, lenvatinib + weekly CAR T cells = 390.3 mm³). Next, we investigated the effect of the combination treatment on tumour metastasis. Tumour metastasis was measured by bioluminescence imaging in different organs (Fig. 6 I). Interestingly, lenvatinib treatment alone did not influence tumour metastasis and could not prevent spreading of cancer cells into the different organs like lungs, liver or spleen (Fig. 6 J, K; Figure S15). In contrast, ROR1 CAR T cell treatments were able to nearly completely block tumour metastases, both as single treatments and even better as weekly repeated treatments (Fig. 6 J-L; Figure S15). In concordance, ROR1 CAR T cells significantly reduced the numbers of circulating tumour cells by around 70% determined in the peripheral blood of treated mice (Fig. 6 M). The combination of ROR1 CAR T cells with lenvatinib could further enhance the anti-metastasis effects and reduced the quantitiy of metastasis in lung and liver (Fig. 6 J, K). Our results indicate that lenvatinib is an ideal combination partner for ROR1 CAR T cells supporting their effect on reducing primary tumour growth and tumour metastases. (A) Experimental scheme for the in vivo treatment of NSG mice with the lenvatinib/ROR1 CAR T cell combination. 8505C cancer cells (3×10⁶ cells/mouse) were injected into the right flank of NSG mice and grew until a median volume of 177.8 mm³ was reached. Then lenvatinib (5 mg /kg BW) or vehicle treatment was started for one week, followed by ROR1 CAR T injections (5×10⁶, CD4:CD8 = 1:1) once or weekly. (B) Mean tumour volume as measured by calliper gauche over time from 4–6 mice per group. (C) Mean tumour volume on experimental day 42 (end of control group). (D) Mean tumour volume on day 46 (end of lenvatinib treatment groups); n = 4–6, mean ± SD, two-way ANOVA for (B), one-way ANOVA for (C), t-test for (D), **** p ≤ 0.0001, ** p ≤ 0.01, * p < 0.05; ns ≥ 0.05. (E) Scheme for lenvatinib/ROR1 CAR T cell treatments with multiple CAR T cell injections. 8505C cancer cells (3×10⁶ cells/mouse) were injected into the right flank of NSG mice and grew until a median volume of 118.2 mm³ was reached. (F) Mean tumour volume over time (n = 4–5 per group). (G) Mean tumour volume on day 46 (end of control group), n = 4–5, mean ± SD, (** p ≤ 0.01, * p < 0.05). (H) Mean tumour volume on day 49 (end of lenvatinib group); n = 4–5, mean ± SD, (* p < 0.05). (I) Experimental scheme to monitor tumour metastasis into different organs using the luciferase IVIS imaging system. Lung imaging results are shown in (J) and liver results in (K) from all treatment groups, other organs are depicted in Figure S15. N = 3–4, mean ± SD, one-way ANOVA (* p < 0.05, ns ≥ 0.05). (L) Quantified lung metastasis results are shown for day 46 (end of control groups) and day 49 (end of lenvatinib treatment groups); n = 3–4, mean ± SD, One-Way ANOVA (* p < 0.05). (M) Analysis of circulating tumour cells by flow cytometry in the peripheral blood of NSG mice; n = 3–4, unpaired t-test (** p ≤ 0.01). Lenvatinib enhances CAR T cell quantity and activity in vivo To understand, how lenvatinib affects ROR1 CAR T cell activity in vivo in our ATC mouse models, we monitored human ROR1 CAR T cells in the peripheral blood of NSG mice via flow cytometry (Fig. 7 A). Lenvatinib significantly enhanced the quantity of ROR1 CAR T cells circulating in the peripheral blood of xenografted mice compared to ROR1 CAR T cell treatment alone (Fig. 7 B). Then, we investigated the activation and differentiation profile of the circulating CAR T cells (Fig. 7 C). In concordance with the in vitro data, CAR T cells in the lenvatinib treated group showed enhanced expression of activation markers (CD25, CD137) and reduced expression of exhaustion markers (CD279, CD366) (Fig. 7 D-G). Furthermore, compared to the CAR T cells only group the frequency of circulating CAR T cells in the lenvatinib group shifted towards increased numbers of more differentiated CAR T cells like effector T cells (from 0.8*10 − 3 % to 6*10 − 3 %, 7 fold increase), effector memory T cells (from 2*10 − 2 % to 4.3*10 − 2 %, 2 fold increase) and central memory (from 0.7*10 − 4 % to 5.9*10 − 4 %, 8 fold increase) T cells (Fig. 7 H-K). (A) Gating strategy for circulating ROR1 CAR T cells in the peripheral blood of ATC mice. (B) Quantification of circulating ROR1 CAR T cells in the peripheral blood, n = 7 per group, mean ± SD, unpaired t-test (*** p ≤ 0.001). (C) Surface markers determining activation and exhaustion profiles of circulating CAR T cells. (D-G) Analysis of activation and exhaustion surface markers of circulating ROR1 CAR T cells in mice with or without lenvatinib treatment; n = 7, mean ± SD, unpaired t-test (**** p ≤ 0.0001, ** p ≤ 0.01, ns p ≥ 0.05). (H) Differentiation profile of circulating CAR T cells determined by different surface markers. T N –naïve T cells; T E – effector T cells; T EM – effector memory T cells; T CM – central memory T cells. (I-K) Quantity of circulating CAR T cells in different differentiation stages with and without lenvatinib treatment; n = 7, mean ± SD, unpaired t-test (**** p ≤ 0.0001, *** p ≤ 0.001). Discussion ATC is a devastating disease and despite great progress with immune-kinase inhibitor therapies the majority of patients die due to cervical tumour progression or metastasis into critical organs. CAR T cell therapies at least in hematologic malignancies hold the promise to induce complete tumour regression in part of the patients and can prevent tumour relapse over a long time period due to the formation of memory CAR T cells. 48,49 In contrast to hematologic malignancies, the effect of CAR T cells in solid tumours is quite limited. Best responses are seen in glioblastoma 50 , papillomavirus-associated cervical cancer 51,52 , lung cancer 53 , breast cancer 54 and several other entities, but effects are often short-lived and more of preventive nature. To achieve target specificity in solid tumours, CAR T cell antigens are chosen either due to a specific expression of receptors/surface proteins on tumour cells, tumour-specific integrated viral proteins 55 or by targeting specific mutations (KRAS) 56 , which are presented in a MHC-dependent manner. Our CAR T cell target ROR1 is a transmembrane receptor, which is expressed during embryonic development, but is lost in most adult tissues with the exception of low levels on the parathyroid, a subset of B cell progenitors and alveolar type 1 cells. 57,58 It is re-expressed on several aggressive tumour entities, like lung cancer or triple negative breast cancer 57,59 and on some B cell malignancies. 59–61 By IHC staining, RNA and surface expression we could show that ROR1 is also strongly overexpressed in ATCs, while absent in normal thyroid tissue and expressed on a much lower level in differentiated thyroid carcinoma (DTC). Highest ROR1 levels are associated with the most undifferentiated, stem-cell like ATC variant and are associated with a significantly shorter survival in ATC/PDTC patients implicating ROR1 as a marker for a highly aggressive disease course. ROR1 was not only overexpressed in primary patient tissue, but also on ATC cell lines, independent of the mutation profile, and its expression was sustained in vivo on circulating tumour cells in mouse xenografts. Interestingly, ROR1 expression seems to be higher on BRAF-mutated tumours, which are known to have a more aggressive disease course and an increased metastasis potential compared to RAS-mutated tumours. ROR1 CAR T cells effectively eliminated ATC cell lines in vitro in 2D and 3D cultures and successfully blocked tumour development in vivo , in both KRAS- and BRAF-mutated ATC cell lines. Despite this encouraging results, one-time injected CAR T cells were not able to deplete already established ATC tumours with a tumour volume exceeding 100 mm 3 . The CAR T cell effect could be improved by repeated injection of CAR T cells once every week resulting in a significant tumour size reduction of the already established tumour. The advantage of multiple CAR T injections was previously shown in various models in mice and humans. Multiple CAR T injections can mimic an ongoing immune response in immunodeficient mice with improved efficacy against hematologic malignancies and solid tumours 62–65 , but also enhances CAR T cell efficiency in immunocompetent mice. 66,67 Repeated application of CAR T cells is also a clinically relevant strategy that has already been successfully explored in patients with hematologic malignancies 68,69 and solid tumours like glioblastoma 70–72 and is currently systemically investigated in a clinical trial with different solid tumour entities (NCT05239143). Additionally, manipulation of CAR T cells towards a longer life span, increased activity or better target recognition is surely an alternative to repeated CAR T injections. Besides reduced tumour growth, the repeated CAR T injections also resulted in a more than 2-fold increase in circulating CAR T cells in the peripheral blood and consequently a more efficient reduction of circulating ROR1 + ATC tumour cells. Previous studies have shown that circulating ROR1 high+ tumour cells have a highly invasive potential and are a major source for tumour metastases. 3,73,74 Consequently, the CAR T cell-induced reduction of circulating ROR1 + ATC cells also efficiently blocked tumour metastases into the lung, liver, kidneys and other organs. Lenvatinib treatment is highly effective in DTC and is approved as first-line systemic treatment of this disease. 31,75 In ATC results are controversial with studies showing good responses and others with relatively low or absent clinical benefit. 76,77 In contrast, the combination of lenvatinib with an immune checkpoint inhibitor, like pembrolizumab, in ATCs is highly effective and often induces good and partially durable responses over several years. 9 In this context, lenvatinib not only reduces the vascularisation and growth of the already established tumours, but also improves the functionality of endogenous T cells and therefore enhances the efficacy of immune checkpoint inhibitor treatments in mice and humans. 34 Consequently, lenvatinib and other VEGFR inhibitors could also influence the functionality of CAR T cells. Compared to other VEGFR inhibitors, lenvatinib showed the most improved CAR T cell profile regarding T cell differentiation, proliferation, cytokine secretion, CAR T cell activation and exhaustion. In addition, lenvatinib treatment altered the composition of the TME with elimination of immunosuppressive CAF-S1 fibroblasts. CAF-S1 fibroblasts not only recruit immunosuppressive CD4 + CD25 + regulatory T cells (Tregs) and therefore diminish normal immune responses, but also promote tumour metastases by inducing epithelial-to-mesenchymal transition, migration and invasiveness of tumour cells and by remodelling the extracellular matrix. 78,79 So, the depletion of CAF-S1 fibroblasts by lenvatinib is an important additional step to improve the efficiency of CAR T cell therapies, which can be compromised by similar mechanisms as normal immune responses. Especially important is probably the reduced Treg recruitment to the tumour site, which enables normal CAR T activity and the reduced tightness of the tumour tissue to allow better CAR T cell invasion. As the CAR T cell profile, especially in the interplay of CD4 and CD8 CAR T cells was strongest improved with lenvatinib and due to our excellent experiences of lenvatinib in combination with immune checkpoint inhibitors in patients, lenvatinib was chosen as primary combination partner for ROR1 CAR T cells in vivo . Lenvatinib monotherapy in mice reduced the growth of already established tumours, but the effect was only temporary and the tumour size still increased strongly over time. Furthermore, lenvatinib alone had no protective effect on the quantity of circulating tumour cells and the establishment of tumour metastases. This is a very interesting phenomenon and might be the reason why lenvatinib monotherapies in ATC patients are often short lived and while the primary tumour growth can be stopped, the patient still develops new metastatic lesions at other sites, which results in the death of the patient. The combination of lenvatinib with ROR1 CAR T cells was most effective in all aspects of tumour development and could completely block primary tumour growth, strongly reduced tumour metastases, enhanced CAR T cell functionality and quantity, reduced numbers of circulating tumour cells and also improved the general health condition of treated mice. A previous phase I clinical trial in humans with ROR1 CAR T cells clearly showed the restrictions of the approach in humans and strongly supports the ideas of repeated CAR T cell injections and the search for combination partners. Although CLL and solid tumours had similar levels of ROR1 expression on the cell surface, 2/3 of the CLL patients showed a partial response to the ROR1 CAR T cell treatment. From 18 patients with either lung cancer or triple-negative breast cancer only one had a partial remission, but died from severe pulmonary side effects and hypoxia. Most patients showed no response and lacked infiltration of CAR T cells into the tumour tissue. Furthermore, in solid tumour patients, the CAR T cells often did not expand and showed an exhausted phenotype. 80–82 Our data from this study, but also from patients, show that lenvatinib can improve many of these aspects and has a very good clinical activity in ATC. Therefore we aim to perform a clinical trial, where we combine lenvatinib and ROR1 CAR T cells in ROR1 + ATC patients. 29 Taken together, our experiments identified ROR1 as the first specific CAR T cell target in ATC, which is also expressed on circulating tumour cells and we found lenvatinib as an ideal combination partner to block tumour growth, to improve CAR T cell functionality and to inhibit microenvironment-induced immunosuppression. Material and Methods Experimental Design The objective of the study was to identify a specific and innovative CAR T cell target for the treatment of anaplastic thyroid carcinoma and to find tyrosine kinase inhibitors which enhance CAR T cell activity and anti-tumour efficacy. By using gene expression studies, IHC stainings of ATC patient tissues and flow cytometry analysis, we identified ROR1 to be specifically overexpressed in ATC compared to normal thyroid tissue. Its expression was also conserved in ATC cell lines (ROR1 surface expression by flow cytometry, qPCR). ROR1 CAR T cells were highly efficient in lysing ATC cell lines in 2D and 3D co-cultures (luciferase/SRB-based viability assays and T cell cytokine profile) and in ATC xenograft mouse models, and eliminated circulating tumour cells (measured by flow cytometry) resulting in a significant reduction in tumour metastases (bioluminescence imaging). ROR1-KO cell lines were generated via CRISPR/Cas9 in order to analyse ROR1-CAR T cell specificity. Fast growing already established tumours could not be eliminated by ROR1 CAR T cells only (volume measurements, bioluminescence imaging), due to lack of infiltration of the CAR T cells into the tight tumour tissue (IHC for CD3 and RFP labelled CAR T cells). TKIs (Sorafenib, cabozantinib and lenvatinib) were tested to improve CAR T cell efficacy (RNAseq, proliferation and differentiation profile of CAR T cells in co-cultures treated with kinase inhibitors), and we identified lenvatinib to improve the CAR T cell activation and differentiation profile, to enhance anti-tumour efficiency in mouse models and to reduce immunosuppressive CAF-S1 cells in the tumour microenvironment (in vitro outgrowth of cancer associated fibroblasts from ATC patient tissues and treatment with lenvatinib). In vitro experiments were performed with at least three biological replicates of primary cells and/or cell lines to perform nonparametric statistical analyses (as specified in figure legends). In vivo experiments were performed with previously established numbers of mice by statistical analyses powered to highlight differences in efficacy and accounting for variability observed in previous experiments. Tumour burden was used to randomize mice into treatment groups. All of the treatment groups included at least three, but mostly five experimental mice to perform nonparametric statistical analysis (as specified in figure legends). All continuous variables are graphically represented as median and individual data points. Detailed experiment designs and exact biological replicate numbers are described in the figure legends or Supplementary Materials and Methods. NGS analysis of ATC samples Fresh frozen material from ATC (n = 12) and goiter (n = 8) patients from the Department of Visceral, Vascular and Endocrine Surgery of the University Hospital of Halle (Saale, Germany) was collected after written informed consent (ethic: 2019-037, patients characteristics table S1). Samples were homogenized using the GentleMACS with M tubes (Miltenyi Biotec, Bergisch Gladbach, Germany) and RNA was isolated using the AllPrep DNA/RNA Mini Kit (Qiagen) according to the manufacturers’ instructions. RNA sequencing (RNAseq) was performed by Novogene Co (Munich, Germany). A detailed description for RNAseq is provided in Supplement (S1). Data from gene expression analysis was uploaded on GEO database (GSE298106). ROR1-targeting CAR T cells The CAR construct (ROR1_41BB_CD3zeta_EGFRt) is a lentiviral plasmid vector encoding a second generation anti-ROR1 CAR in cis with the truncated epidermal growth factor receptor (EGFR) under control of EF1α promotor with T2A self-cleaving peptide sequence. 25 The engineered CAR T cells transduced with the ROR1 targeting construct were kindly provided by Prof Dr Michael Hudecek and Dr. Miriam Alb from the University Hospital of Würzburg. Cryopreserved ROR1 CAR T cells were thawed, washed, incubated with DNase I (0.1 mg/mL (Sigma Aldrich) for 10 minutes at room temperature (RT) and then incubated for 48 hours (hrs) in RPMI 1640 with HEPES containing 10% (v/v) human serum (Sigma Aldrich), 1% (v/v) penicillin, streptomycin, 0.1% (v/v) 2-mercaptoethanol (Sigma Aldrich) and with 10 U/mL IL-2 (Miltenyi Biotec) for the first 24 hrs. Then, CAR T cells were washed, counted with trypan blue and used for the in vitro experiments. For the in vivo experiments, the CAR T cells were washed, incubated with DNase I (0.1 mg/mL) for 10 minutes at RT, washed again and resuspended in PBS at the target concentration. In vivo studies All experiments were approved by the Landesverwaltungsamt Sachsen-Anhalt (203.m-42502-2-1725 MLU) and performed according to directive 2010/63/EU. 5–7 weeks old NSG male mice (NOD.Cg-Prkdc scid Il2rg tm1Sug /JicTac) were purchased from Charles River (Germany). 3×10 6 luciferase-expressing 8505C ATC cells were subcutaneously injected into the left flank region or 1×10 5 luciferase-expressing HRAS-mutated C643 ATC cells were injected intravenously into the ophthalmic venous sinus. For early treatment experiments (Fig. 4 ) 5×10 6 CAR T cells (2.5×10 6 CAR T CD4 and 2.5×10 6 CAR T CD8) resuspended in 100 µL sterile PBS were injected intravenously (i.v.) into the tail vein 2 to 7 days later, or for experiments with established tumours on day 18 or later. For lenvatinib/CAR T combinations, lenvatinib was resuspended in 0.5% sterile filtered methylcellulose and administered by oral gavage 18 days after tumour cell inoculation at 5 mg/kg body weight. Body weight and health inspections were performed every day. Tumour volume was measured each week via calliper and calculated as follows: \(\:V=0.5\times\:L\times\:{W}^{2}\) (L = tumour length; W = tumour width). At the end of the experiment, the tumours were excised, weighed and stored in formalin for IHC. Statistics Statistical evaluation was performed using GraphPad Prism statistical software 9.5.1. Comparisons between two groups were analyzed using an unpaired two-tailed Student’s t test. For multiple group comparison one-way analysis of variance (ANOVA) was applied with a Tukey posttest. Results from bar charts are expressed as the mean ± SD. The results were considered significantly different if p values are < 0.05 and are represented as follows: *, p < 0.05; **, p ≤ 0.01; ***, p ≤ 0.001 and ****, p ≤ 0.0001. Non-significant p values are shown as n.s. (p ≥ 0.05). In each graph, the number of individual experimental points are described. Declarations Disclosures: The authors declare no competing financial interests. Author contributions Contribution: O.S. and D.C. performed the experiments. O.S. and D.C. analyzed the data. T.M. and D.B. helped to carry out the in vivo experiments. S.H. and T. B. analyzed the data. B.T. and K.L. provided thyroid carcinoma and goiter samples. M.A. provided the ROR1 CAR T cells. E.W. helped with RNASeq analysis. M.B. and A.W. performed IHC staining. B.S. and M.U. provided help for fibroblast analysis. D.B. reviewed the manuscript. O.S., D.C., B.E., K.M. and C.D. designed the research. O.S., D.C., M.H. and C.D. wrote the paper. All authors approved the final version of this manuscript. Acknowledgements This work was supported by the Wilhelm-Roux program round 32 - NTIEN: Novel Targets and Immunotherapies for Endocrine Neoplasms from the university medicine Halle (Saale). It was further supported by Deutsche Krebshilfe via grants 111025 and 70112614. The work was further supported by the DFG through the Emmy-Noether program from Christine Dierks (DI 1664/1–1), through DFG grants from FOR 2033 DI 1664/2–2 and FOR DI 1664/3 − 1, and FOR5659 517204983 to Christine Dierks. The work was further supported by the José-Carreras grant DJCLS 10 R/2022It was further supported by the “Polyfaces-initative/Land Saxony-Anhalt, EMB, “Vorbereitung Cluster im Rahmen der Exzellenzstrategie des Bundes: Polymere-Nachhaltigkeit” as well as by the European Union and the State Saxony-Anhalt through the Thera4Age project (grant ZS/2023/12/182764). We thank our core facilities, especially Alexander Navarrete Santos for the help with flow cytometry and sorting as well as Nadine Bley for the use of the live cell imaging system. Correspondence : Prof. Dr. Christine Dierks, University of Halle-Wittenberg, Department of Hematology/Oncology, Ernst-Grube-Str. 40, 06120 Halle (Saale), Germany; e-mail: [email protected] References Smallridge RC, Ain KB, Asa SL, et al. American Thyroid Association guidelines for management of patients with anaplastic thyroid cancer. Thyroid . Nov 2012;22(11):1104-39. doi:10.1089/thy.2012.0302 Prasongsook N, Kumar A, Chintakuntlawar AV, et al. Survival in Response to Multimodal Therapy in Anaplastic Thyroid Cancer. J Clin Endocrinol Metab . Dec 1 2017;102(12):4506-4514. doi:10.1210/jc.2017-01180 Cui B, Zhang S, Chen L, et al. Targeting ROR1 inhibits epithelial–mesenchymal transition and metastasis. Cancer research . 2013;73(12):3649-3660. Takano T, Ito Y, Hirokawa M, Yoshida H, Miyauchi A. BRAFV600E mutation in anaplastic thyroid carcinomas and their accompanying differentiated carcinomas. British journal of cancer . 2007;96(10):1549-1553. Xu B, Fuchs T, Dogan S, et al. Dissecting anaplastic thyroid carcinoma: a comprehensive clinical, histologic, immunophenotypic, and molecular study of 360 cases. Thyroid . 2020;30(10):1505-1517. Pozdeyev N, Gay LM, Sokol ES, et al. Genetic analysis of 779 advanced differentiated and anaplastic thyroid cancers. Clinical Cancer Research . 2018;24(13):3059-3068. Landa I, Ibrahimpasic T, Boucai L, et al. Genomic and transcriptomic hallmarks of poorly differentiated and anaplastic thyroid cancers. The Journal of clinical investigation . 2016;126(3):1052-1066. Adam P, Kircher S, Sbiera I, et al. FGF-Receptors and PD-L1 in Anaplastic and Poorly Differentiated Thyroid Cancer: Evaluation of the Preclinical Rationale. Front Endocrinol (Lausanne) . 2021;12:712107. doi:10.3389/fendo.2021.712107 Dierks C, Seufert J, Aumann K, et al. Combination of Lenvatinib and Pembrolizumab Is an Effective Treatment Option for Anaplastic and Poorly Differentiated Thyroid Carcinoma. Thyroid . Jul 2021;31(7):1076-1085. doi:10.1089/thy.2020.0322 Prasad V. Tisagenlecleucel—the first approved CAR-T-cell therapy: implications for payers and policy makers. Nature Reviews Clinical Oncology . 2018;15(1):11-12. Wang V, Gauthier M, Decot V, Reppel L, Bensoussan D. Systematic Review on CAR-T Cell Clinical Trials Up to 2022: Academic Center Input. Cancers (Basel) . Feb 4 2023;15(4)doi:10.3390/cancers15041003 Daei Sorkhabi A, Mohamed Khosroshahi L, Sarkesh A, et al. The current landscape of CAR T-cell therapy for solid tumors: Mechanisms, research progress, challenges, and counterstrategies. Frontiers in immunology . 2023;14:1113882. Li H, Zhou X, Wang G, et al. CAR-T Cells Targeting TSHR Demonstrate Safety and Potent Preclinical Activity Against Differentiated Thyroid Cancer. J Clin Endocrinol Metab . Mar 24 2022;107(4):1110-1126. doi:10.1210/clinem/dgab819 Hasan MK, Rassenti L, Widhopf GF, 2nd, Yu J, Kipps TJ. Wnt5a causes ROR1 to complex and activate cortactin to enhance migration of chronic lymphocytic leukemia cells. Leukemia . Mar 2019;33(3):653-661. doi:10.1038/s41375-018-0306-7 Zhang S, Chen L, Cui B, et al. ROR1 is expressed in human breast cancer and associated with enhanced tumor-cell growth. PloS one . 2012;7(3):e31127. Rebagay G, Yan S, Liu C, Cheung N-K. ROR1 and ROR2 in human malignancies: potentials for targeted therapy. Frontiers in oncology . 2012;2:34. Hudecek M, Schmitt TM, Baskar S, et al. The B-cell tumor–associated antigen ROR1 can be targeted with T cells modified to express a ROR1-specific chimeric antigen receptor. Blood, The Journal of the American Society of Hematology . 2010;116(22):4532-4541. Borcherding N, Kusner D, Liu G-H, Zhang W. ROR1, an embryonic protein with an emerging role in cancer biology. Protein & Cell . 2014;5(7):496-502. doi:10.1007/s13238-014-0059-7 Katoh M, Katoh M. WNT signaling pathway and stem cell signaling network. Clinical cancer research . 2007;13(14):4042-4045. Parsons MJ, Tammela T, Dow LE. WNT as a driver and dependency in cancer. Cancer discovery . 2021;11(10):2413-2429. Menck K, Heinrichs S, Baden C, Bleckmann A. The WNT/ROR pathway in cancer: from signaling to therapeutic intervention. Cells . 2021;10(1):142. Masoumi J, Jafarzadeh A, Abdolalizadeh J, et al. Cancer stem cell-targeted chimeric antigen receptor (CAR)-T cell therapy: Challenges and prospects. Acta Pharm Sin B . Jul 2021;11(7):1721-1739. doi:10.1016/j.apsb.2020.12.015 June CH, O’Connor RS, Kawalekar OU, Ghassemi S, Milone MC. CAR T cell immunotherapy for human cancer. Science . 2018;359(6382):1361-1365. Wallstabe L, Göttlich C, Nelke LC, et al. ROR1-CAR T cells are effective against lung and breast cancer in advanced microphysiologic 3D tumor models. JCI insight . 2019;4(18) Hudecek M, Lupo-Stanghellini M-T, Kosasih PL, et al. Receptor affinity and extracellular domain modifications affect tumor recognition by ROR1-specific chimeric antigen receptor T cells. Clinical cancer research . 2013;19(12):3153-3164. Stüber T, Monjezi R, Wallstabe L, et al. Inhibition of TGF-β-receptor signaling augments the antitumor function of ROR1-specific CAR T-cells against triple-negative breast cancer. Journal for immunotherapy of cancer . 2020;8(1) Berger C, Sommermeyer D, Hudecek M, et al. Safety of targeting ROR1 in primates with chimeric antigen receptor-modified T cells. Cancer immunology research . 2015;3(2):206-216. doi:10.1158/2326-6066.CIR-14-0163 Maulana TI, Teufel C, Cipriano M, et al. Breast cancer-on-chip for patient-specific efficacy and safety testing of CAR-T cells. Cell Stem Cell . 2024;31(7):989-1002. e9. Jaeger-Ruckstuhl CA, Specht JM, Voutsinas JM, et al. Phase I Study of ROR1-Specific CAR-T Cells in Advanced Hematopoietic and Epithelial Malignancies. Clin Cancer Res . Feb 3 2025;31(3):503-514. doi:10.1158/1078-0432.CCR-24-2172 Cabanillas ME, Brose MS, Holland J, Ferguson KC, Sherman SI. A phase I study of cabozantinib (XL184) in patients with differentiated thyroid cancer. Thyroid . Oct 2014;24(10):1508-14. doi:10.1089/thy.2014.0125 Schlumberger M, Tahara M, Wirth LJ, et al. Lenvatinib versus placebo in radioiodine-refractory thyroid cancer. N Engl J Med . Feb 12 2015;372(7):621-30. doi:10.1056/NEJMoa1406470 Stjepanovic N, Capdevila J. Multikinase inhibitors in the treatment of thyroid cancer: specific role of lenvatinib. Biologics . 2014;8:129-39. doi:10.2147/BTT.S39381 Thomas L, Lai SY, Dong W, et al. Sorafenib in metastatic thyroid cancer: a systematic review. Oncologist . Mar 2014;19(3):251-8. doi:10.1634/theoncologist.2013-0362 Gunda V, Gigliotti B, Ashry T, et al. Anti-PD-1/PD-L1 therapy augments lenvatinib's efficacy by favorably altering the immune microenvironment of murine anaplastic thyroid cancer. Int J Cancer . May 1 2019;144(9):2266-2278. doi:10.1002/ijc.32041 Shabani M, Naseri J, Shokri F. Receptor tyrosine kinase-like orphan receptor 1: a novel target for cancer immunotherapy. Expert opinion on therapeutic targets . 2015;19(7):941-955. Zhao Y, Zhang D, Guo Y, et al. Tyrosine kinase ROR1 as a target for anti-cancer therapies. Frontiers in Oncology . 2021;11:680834. Hudecek M, Schmitt TM, Baskar S, et al. The B-cell tumor-associated antigen ROR1 can be targeted with T cells modified to express a ROR1-specific chimeric antigen receptor. Blood . Nov 25 2010;116(22):4532-41. doi:10.1182/blood-2010-05-283309 Wallstabe L, Gottlich C, Nelke LC, et al. ROR1-CAR T cells are effective against lung and breast cancer in advanced microphysiologic 3D tumor models. JCI Insight . Sep 19 2019;4(18)doi:10.1172/jci.insight.126345 Dierks C, Ruf J, Seufert J, et al. 1646MO Phase II ATLEP trial: Final results for lenvatinib/pembrolizumab in metastasized anaplastic and poorly differentiated thyroid carcinoma. Annals of Oncology . 2022;33:S1295. doi:10.1016/j.annonc.2022.07.1726 Chen Y, Dai S, Cheng C-s, Chen L. Lenvatinib and immune-checkpoint inhibitors in hepatocellular carcinoma: mechanistic insights, clinical efficacy, and future perspectives. Journal of Hematology & Oncology . 2024/12/21 2024;17(1):130. doi:10.1186/s13045-024-01647-1 Mei Z, Gao X, Pan C, et al. Lenvatinib enhances antitumor immunity by promoting the infiltration of TCF1(+) CD8(+) T cells in HCC via blocking VEGFR2. Cancer Sci . Apr 2023;114(4):1284-1296. doi:10.1111/cas.15719 Kato Y, Tabata K, Kimura T, et al. Lenvatinib plus anti-PD-1 antibody combination treatment activates CD8+ T cells through reduction of tumor-associated macrophage and activation of the interferon pathway. PloS one . 2019;14(2):e0212513. Lu M, Zhang X, Gao X, et al. Lenvatinib enhances T cell immunity and the efficacy of adoptive chimeric antigen receptor-modified T cells by decreasing myeloid-derived suppressor cells in cancer. Pharmacological Research . 2021;174:105829. Subramanian A, Tamayo P, Mootha VK, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A . Oct 25 2005;102(43):15545-50. doi:10.1073/pnas.0506580102 Mhaidly R, Mechta-Grigoriou F. Fibroblast heterogeneity in tumor micro-environment: Role in immunosuppression and new therapies. Elsevier; 2020:101417. Chen R, Li Q, Xu S, et al. Modulation of the tumour microenvironment in hepatocellular carcinoma by tyrosine kinase inhibitors: from modulation to combination therapy targeting the microenvironment. Cancer Cell International . 2022/02/11 2022;22(1):73. doi:10.1186/s12935-021-02435-4 Desbois M, Wang Y. Cancer‐associated fibroblasts: key players in shaping the tumor immune microenvironment. Immunological reviews . 2021;302(1):241-258. McLellan AD, Ali Hosseini Rad SM. Chimeric antigen receptor T cell persistence and memory cell formation. Immunol Cell Biol . Aug 2019;97(7):664-674. doi:10.1111/imcb.12254 Doan AE, Mueller KP, Chen AY, et al. FOXO1 is a master regulator of memory programming in CAR T cells. Nature . May 2024;629(8010):211-218. doi:10.1038/s41586-024-07300-8 Brown CE, Hibbard JC, Alizadeh D, et al. Locoregional delivery of IL-13Ralpha2-targeting CAR-T cells in recurrent high-grade glioma: a phase 1 trial. Nat Med . Apr 2024;30(4):1001-1012. doi:10.1038/s41591-024-02875-1 Stevanovic S, Draper LM, Langhan MM, et al. Complete regression of metastatic cervical cancer after treatment with human papillomavirus-targeted tumor-infiltrating T cells. J Clin Oncol . May 10 2015;33(14):1543-50. doi:10.1200/JCO.2014.58.9093 Stevanovic S, Helman SR, Wunderlich JR, et al. A Phase II Study of Tumor-infiltrating Lymphocyte Therapy for Human Papillomavirus-associated Epithelial Cancers. Clin Cancer Res . Mar 1 2019;25(5):1486-1493. doi:10.1158/1078-0432.CCR-18-2722 Creelan BC, Wang C, Teer JK, et al. Tumor-infiltrating lymphocyte treatment for anti-PD-1-resistant metastatic lung cancer: a phase 1 trial. Nat Med . Aug 2021;27(8):1410-1418. doi:10.1038/s41591-021-01462-y Zacharakis N, Chinnasamy H, Black M, et al. Immune recognition of somatic mutations leading to complete durable regression in metastatic breast cancer. Nat Med . Jun 2018;24(6):724-730. doi:10.1038/s41591-018-0040-8 Comoli P, Pedrazzoli P, Maccario R, et al. Cell therapy of stage IV nasopharyngeal carcinoma with autologous Epstein-Barr virus-targeted cytotoxic T lymphocytes. J Clin Oncol . Dec 10 2005;23(35):8942-9. doi:10.1200/JCO.2005.02.6195 Tran E, Robbins PF, Lu YC, et al. T-Cell Transfer Therapy Targeting Mutant KRAS in Cancer. N Engl J Med . Dec 8 2016;375(23):2255-2262. doi:10.1056/NEJMoa1609279 Balakrishnan A, Goodpaster T, Randolph-Habecker J, et al. Analysis of ROR1 Protein Expression in Human Cancer and Normal Tissues. Clin Cancer Res . Jun 15 2017;23(12):3061-3071. doi:10.1158/1078-0432.CCR-16-2083 Al-Shawi R, Ashton SV, Underwood C, Simons JP. Expression of the Ror1 and Ror2 receptor tyrosine kinase genes during mouse development. Dev Genes Evol . Apr 2001;211(4):161-71. doi:10.1007/s004270100140 Baskar S, Kwong KY, Hofer T, et al. Unique cell surface expression of receptor tyrosine kinase ROR1 in human B-cell chronic lymphocytic leukemia. Clin Cancer Res . Jan 15 2008;14(2):396-404. doi:10.1158/1078-0432.CCR-07-1823 Cui B, Ghia EM, Chen L, et al. High-level ROR1 associates with accelerated disease progression in chronic lymphocytic leukemia. Blood . Dec 22 2016;128(25):2931-2940. doi:10.1182/blood-2016-04-712562 Broome HE, Rassenti LZ, Wang HY, Meyer LM, Kipps TJ. ROR1 is expressed on hematogones (non-neoplastic human B-lymphocyte precursors) and a minority of precursor-B acute lymphoblastic leukemia. Leuk Res . Oct 2011;35(10):1390-4. doi:10.1016/j.leukres.2011.06.021 Zhao Y, Moon E, Carpenito C, et al. Multiple injections of electroporated autologous T cells expressing a chimeric antigen receptor mediate regression of human disseminated tumor. Cancer Res . Nov 15 2010;70(22):9053-61. doi:10.1158/0008-5472.CAN-10-2880 Liu M, Wang X, Li W, et al. Targeting PD-L1 in non-small cell lung cancer using CAR T cells. Oncogenesis . Aug 13 2020;9(8):72. doi:10.1038/s41389-020-00257-z Luo Y, Gadd ME, Qie Y, et al. Solid cancer-directed CAR T cell therapy that attacks both tumor and immunosuppressive cells via targeting PD-L1. Mol Ther Oncol . Dec 19 2024;32(4):200891. doi:10.1016/j.omton.2024.200891 Altvater B, Kailayangiri S, Spurny C, et al. CAR T cells as micropharmacies against solid cancers: Combining effector T-cell mediated cell death with vascular targeting in a one-step engineering process. Cancer Gene Ther . Oct 2023;30(10):1355-1368. doi:10.1038/s41417-023-00642-x Klampatsa A, Leibowitz MS, Sun J, Liousia M, Arguiri E, Albelda SM. Analysis and Augmentation of the Immunologic Bystander Effects of CAR T Cell Therapy in a Syngeneic Mouse Cancer Model. Mol Ther Oncolytics . Sep 25 2020;18:360-371. doi:10.1016/j.omto.2020.07.005 Xie YJ, Dougan M, Jailkhani N, et al. Nanobody-based CAR T cells that target the tumor microenvironment inhibit the growth of solid tumors in immunocompetent mice. Proc Natl Acad Sci U S A . Apr 16 2019;116(16):7624-7631. doi:10.1073/pnas.1817147116 Deng L, Xiaolin Y, Wu Q, et al. Multiple CAR-T cell therapy for acute B-cell lymphoblastic leukemia after hematopoietic stem cell transplantation: A case report. Front Immunol . 2022;13:1039929. doi:10.3389/fimmu.2022.1039929 Jiao C, Zvonkov E, Lai X, et al. 4SCAR2.0: a multi-CAR-T therapy regimen for the treatment of relapsed/refractory B cell lymphomas. Blood Cancer J . Mar 17 2021;11(3):59. doi:10.1038/s41408-021-00455-x Brown CE, Alizadeh D, Starr R, et al. Regression of Glioblastoma after Chimeric Antigen Receptor T-Cell Therapy. N Engl J Med . Dec 29 2016;375(26):2561-9. doi:10.1056/NEJMoa1610497 Bagley SJ, Binder ZA, Lamrani L, et al. Repeated peripheral infusions of anti-EGFRvIII CAR T cells in combination with pembrolizumab show no efficacy in glioblastoma: a phase 1 trial. Nat Cancer . Mar 2024;5(3):517-531. doi:10.1038/s43018-023-00709-6 Vitanza NA, Johnson AJ, Wilson AL, et al. Locoregional infusion of HER2-specific CAR T cells in children and young adults with recurrent or refractory CNS tumors: an interim analysis. Nat Med . Sep 2021;27(9):1544-1552. doi:10.1038/s41591-021-01404-8 Xu GL, Shen J, Xu YH, Wang WS, Ni CF. ROR1 is highly expressed in circulating tumor cells and promotes invasion of pancreatic cancer. Mol Med Rep . Dec 2018;18(6):5087-5094. doi:10.3892/mmr.2018.9500 Hasan MK, Widhopf GF, 2nd, Zhang S, et al. Wnt5a induces ROR1 to recruit cortactin to promote breast-cancer migration and metastasis. NPJ Breast Cancer . 2019;5:35. doi:10.1038/s41523-019-0131-9 Tohyama O, Matsui J, Kodama K, et al. Antitumor activity of lenvatinib (e7080): an angiogenesis inhibitor that targets multiple receptor tyrosine kinases in preclinical human thyroid cancer models. J Thyroid Res . 2014;2014:638747. doi:10.1155/2014/638747 Tahara M, Kiyota N, Yamazaki T, et al. Lenvatinib for Anaplastic Thyroid Cancer. Front Oncol . 2017;7:25. doi:10.3389/fonc.2017.00025 Kiyota N, Schlumberger M, Muro K, et al. Subgroup analysis of Japanese patients in a phase 3 study of lenvatinib in radioiodine-refractory differentiated thyroid cancer. Cancer Sci . Dec 2015;106(12):1714-21. doi:10.1111/cas.12826 Costa A, Kieffer Y, Scholer-Dahirel A, et al. Fibroblast Heterogeneity and Immunosuppressive Environment in Human Breast Cancer. Cancer Cell . Mar 12 2018;33(3):463-479 e10. doi:10.1016/j.ccell.2018.01.011 Pelon F, Bourachot B, Kieffer Y, et al. Cancer-associated fibroblast heterogeneity in axillary lymph nodes drives metastases in breast cancer through complementary mechanisms. Nat Commun . Jan 21 2020;11(1):404. doi:10.1038/s41467-019-14134-w Chen T, Wang M, Chen Y, Liu Y. Current challenges and therapeutic advances of CAR-T cell therapy for solid tumors. Cancer Cell International . 2024;24(1):133. Hou AJ, Chen LC, Chen YY. Navigating CAR-T cells through the solid-tumour microenvironment. Nature reviews Drug discovery . 2021;20(7):531-550. Schurich A, Magalhaes I, Mattsson J. Metabolic regulation of CAR T cell function by the hypoxic microenvironment in solid tumors. Immunotherapy . 2019;11(4):335-345. Additional Declarations There is NO Competing Interest. Supplementary Files ManuscriptROR1inATCSupplements.docx Supplementary Dataset 1 Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8690827","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":582001769,"identity":"097bbf24-92e2-4b17-b233-973c573840cb","order_by":0,"name":"Christine 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07:25:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8690827/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8690827/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101384531,"identity":"97959524-0066-4cf3-a19c-c11a92c3c003","added_by":"auto","created_at":"2026-01-29 07:06:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":524027,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eROR1 is upregulated in anaplastic thyroid carcinoma\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e \u003cem\u003eROR1\u003c/em\u003e transcript levels of fresh-frozen tissue samples from patients with ATC (n = 12) compared to goiter (n = 8); mean±SD, unpaired t-test (*** p ≤ 0.0001). Patient data is available in table S1. \u003cstrong\u003e(B)\u003c/strong\u003e NGS analysis of \u003cem\u003eROR1\u003c/em\u003e expression in ATC (n = 18 ●) versus PDTC (n = 17 ●) using the GSE76039 dataset \u003csup\u003e7\u003c/sup\u003e; mean±SD, unpaired t-test (*** p ≤ 0.0001). \u003cstrong\u003e(C)\u003c/strong\u003e Overall survival (OS) of ATC and PDTC patients with high (above median) and low (below median) \u003cem\u003eROR1\u003c/em\u003e-expression from the GSE76039 dataset \u003csup\u003e7\u003c/sup\u003e; log-rank (Mantel-Cox) test (**** p ≤ 0.0001). \u003cstrong\u003e(D) \u003c/strong\u003ePearson’s correlation of \u003cem\u003eROR1\u003c/em\u003e and \u003cem\u003eTSHR\u003c/em\u003e RNA expression in the Halle patient cohort (Table S1). \u003cstrong\u003e(E) \u003c/strong\u003ePrinciple component analysis and cluster analysis of the NGS data of goiter patient samples (n=8) and ATC patient samples (n=12). \u003cstrong\u003e(F) \u003c/strong\u003eHeatmap of the expression of the thyroid-tissue specific markers and \u003cem\u003eROR1\u003c/em\u003e. \u003cem\u003eTG\u003c/em\u003e – Thyroglobulin, \u003cem\u003eIYD\u003c/em\u003e – Iodotyrosine Deiodinase, \u003cem\u003eTSHR\u003c/em\u003e – Thyroid Stimulating Hormone Receptor, \u003cem\u003eNKX2-1\u003c/em\u003e – NK2 Homeobox or Thyroid Transcription Factor-1. \u003cstrong\u003e(G, I)\u003c/strong\u003e\u0026nbsp;Representative examples for ROR1 and TSHR IHC DAB-staining in healthy thyroid tissue (HTT) and ATC within the same patient (n = 20); black scale bar corresponds to 200 µm, red – to 50 µm. \u003cstrong\u003e(H, J)\u003c/strong\u003e H Score of the ROR1 and TSHR in ATC tissue samples (ATC) ● and healthy thyroid tissue samples (HTT) ○ from the same patient (n=20); paired t-test (*** p ≤ 0.001; **** p ≤ 0.0001). \u003cstrong\u003e(K) \u003c/strong\u003eROR1 surface expression as measured by flow cytometry in 3 different ATC cell lines (8505C, C643, SW1736) and one ovarian cancer cell line (A2780). The values in red represent median of ROR1-PE expression of each cell line. Experiments were performed in triplicates.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8690827/v1/cc1177fc678d8e662f017911.png"},{"id":101384528,"identity":"62ada026-e5cd-42fd-8c26-3a9e397d7f9f","added_by":"auto","created_at":"2026-01-29 07:06:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":578196,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffective and specific lysis of ROR1 targeted CAR T cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Schematic representation of the targeting principle using ROR1 CAR T cells with ROR1\u003csup\u003e+\u003c/sup\u003e ATC (anaplastic thyroid carcinoma) cell lines. Right picture shows the CAR construct with the single chain variable fragment (scFv) domain, the CD28 transmembrane domain (TMD), the 4-1BB costimulatory domain and the CD3ζ intracellular domain (ICD).\u003csup\u003e25,37,38\u003c/sup\u003e\u003cstrong\u003e (B)\u0026nbsp;\u003c/strong\u003eRepresentative fluorescence and HD phase image (Incucyte) of the ROR1-expressing ATC-cell line 8505C co-incubated with untransduced (UTD) T cells (upper image) or ROR1-targeting CAR T cells for 24 hours. Propidium iodide (PI) staining in red clusters represents dead cancer cells (scale bar 400 µm). \u003cstrong\u003e(C)\u003c/strong\u003e Relative number of living cells using a SRB assay (measurement of relative cellular protein content) for ROR1-expressing ATC cell lines (8505C, SW1736, C643) and the ROR1-negative ovarian cancer cell line A2780 after co-incubation with two- and fivefold numbers of UTD or ROR1 CAR T cells; n = 3, mean ± SD, multiple t-tests ( *** p ≤ 0.001, ** p ≤ 0.01; *\u0026nbsp;p\u0026nbsp;\u0026lt;\u0026nbsp;0.05; ns ≥\u003cstrong\u003e \u003c/strong\u003e0.05). \u003cstrong\u003e(D)\u003c/strong\u003e\u0026nbsp;Quantification of cytokines like interferon-γ using the MACSPlex Cytokine 12 Kit from Miltenyi Biotec. Cytokine levels were assessed in the supernatant of co-cultures of ROR1+ ATC cell lines (8505C, SW1736, C643) or ROR1- ovarian cancer cells (A2780) with control, UTD (untransduced T cells) or ROR1 CAR T cells. N = 3, mean ± SD, multiple t-tests, (*** p ≤ 0.001, ** p ≤ 0.01, * p \u0026lt; 0.05; ns ≥ 0.05). \u003cstrong\u003e(E)\u003c/strong\u003e Flow cytometry analysis of ROR1 expression in wild type ATC cell lines (8505C, C643, SW1736, red graph) and the respective ROR1-KO ATC cell line (blue graph) generated with Crispr-Cas9 against the ROR1 sequence. The values in red represent median fluorescence intensity (MFI) of the ROR1-PE staining of each cell line. \u003cstrong\u003e(F)\u003c/strong\u003e SRB-mediated relative viability assay measuring the cellular protein content in ATC cell monolayers and treated with either mock, UTD or ROR1 CAR T cells. n=3, mean±SD, One-Way ANOVA. (***\u0026nbsp;p\u0026nbsp;≤\u0026nbsp;0.001, ns ≥ 0.05) \u003cstrong\u003e(G)\u003c/strong\u003e Luminescence-based viability assay of the ROR1-expressing ATC model cell line C643 and SW1736 in 3D culture as spheroids and its ROR1-KO counterpart (3D) co-incubated with mock, untransduced T cells (UTD T) and ROR1-targeting CAR T cells (CAR T). Luminescence was measured with Tecan Spark® Multimode Mikroplate Reader. N = 3, mean ± SD, two-Way ANOVA. (*** p ≤ 0.001, ns ≥\u003cstrong\u003e \u003c/strong\u003e0.05). \u003cstrong\u003e(H)\u003c/strong\u003e Visualisation of the co-incubation of C643 spheroids as well as C643 with ROR1‑KO with UTD control T cells or CAR T cells (scale bar 200\u0026nbsp;µm). The experimental setup is identical to that described in (G). For the visual analysis and video generation (Supplemental video 1)\u003cstrong\u003e \u003c/strong\u003ewith the Incucyte, the T cells were labelled with CellTracker™ Red CMTPX according to the manufacturer's instructions.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8690827/v1/da27a5eccdce84bb2e1f980b.png"},{"id":101384527,"identity":"39c2f961-501e-450f-a97b-0f1a6ff49673","added_by":"auto","created_at":"2026-01-29 07:06:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":534748,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eROR1\u003c/strong\u003e \u003cstrong\u003eCAR T-monotherapy is efficient in vivo only at the early time points of the tumor development and has low efficiency with established tumors.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Experimental scheme for NSG mice subcutaneously injected with BRAF V600E\u003csup\u003e+\u003c/sup\u003e mutated 8505C ATC cells (3×10⁶ 8505C cells transduced with a luciferase vector) and treated with intravenously (i.v.) applied ROR1 CAR T cells 2 or 7 days after tumour cell injection. The administration of 100 µL of CAR T cells (5×10⁶ CAR T cells per NSG mouse with a CD4 to CD8 ratio of 1:1) or vehicle (PBS) was conducted via the tail vein. \u003cstrong\u003e(B, C)\u003c/strong\u003e Exemplary imaging analysis (luminescence imaging, IVIS Spectrum) before (experimental day 2) and 26 days after CAR T injection (day 28) and tumour volume analysis with a digital caliper gauge are shown in Figures (B) and (C), respectively. \u003cstrong\u003e(D)\u003c/strong\u003e Experimental scheme for NSG mice retroorbitally injected with RAS-mutated C643 ATC cells (1×10⁵ in 100 µL PBS) and treated with control (n = 3) or ROR1 CAR T cells (5×10⁶, CD4 to CD8 ratio of 1:1, retroorbital opposite site, n = 3) two days after tumour cell injection. \u003cstrong\u003e(E, F)\u003c/strong\u003e Representative imaging analysis (luminescence imaging, IVIS Spectrum) before (experimental day 2) and 12 days after CAR T cell injection (day 14) and tumour volume analysis with a digital caliper gauge are shown in figures (E) and (F), respectively. N = 3, mean ± SD, two-way ANOVA (**** p ≤ 0.0001). \u003cstrong\u003e(G)\u003c/strong\u003e Experimental design for CAR T cell treatment in already established 8505C tumours. 3×10⁶ 8505C cells were injected in the left flank of NSG mice and grew over 25 days (mean volume 163.7 ± 53.7 mm\u003csup\u003e2\u003c/sup\u003e), then mock (n = 7) or 5×10⁶ ROR1 CAR T cells were injected intravenously in the tail vein once (n = 8) or 3-times weekly (n = 7). \u003cstrong\u003e(H)\u003c/strong\u003e Mean tumour volume over time; \u003cstrong\u003e(I)\u003c/strong\u003e mean tumour volume on day 42 (end of control group) and \u003cstrong\u003e(J)\u003c/strong\u003e mean tumour weight at the end of the experiment 42 days after tumor cell injection and 17 days after first CAR T cell injection; n = 7-8, mean ± SD, unpaired t-test (* p ≤ 0.05, *** p ≤ 0.001). \u003cstrong\u003e(K)\u003c/strong\u003e Fluorescent images of tumours treated either with control of IVISense 680 Fluorescent Cell labelled ROR1 CAR T cellss. Images show either front view (surface) or cross sections.\u003cstrong\u003e (L)\u003c/strong\u003e Percentage of ROR1 CAR T cells within tumour tissue analysed by IHC = 7-8, mean ± SD, unpaired t-test (*** p ≤ 0.001).\u003cstrong\u003e (M)\u003c/strong\u003e Images of tumour sections stained with a CD5-antibody marking the infiltrating ROR1 CAR T cells. The IHC slides were analysed using the QuPath 0.4.3 software, red-marked cells represent CD5+ CAR T cells, blue-marked cells represent CD5- tumor and other cell types. \u003cstrong\u003e(N)\u003c/strong\u003e Percentage of ROR1 CAR T cells within tumour tissue analysed by flow cytometry of mouse peripheral blood; n = 7-8, mean ± SD, unpaired t-test (*** p ≤ 0.001).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8690827/v1/7721d40689792917e9940bb6.png"},{"id":101384532,"identity":"8fc7c68a-b011-4201-a6d4-f7eb75a6a6b9","added_by":"auto","created_at":"2026-01-29 07:06:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":537943,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLenvatinib enhances ROR1 CAR T cell activation and proliferation and does not affect ROR1 target expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Schematic overview of the experimental setup to analyse the influence of TKIs on activated CAR T cells. 8505C WT cells (0.01×10⁶) were seeded in a 24-well plate and incubated for 24 hours before treatment with 0.02% DMSO (mock) or 1 µM lenvatinib, 1 µM cabozantinib, or 1\u0026nbsp;µM sorafenib, respectively, CAR T cells (1.2–1.5×10⁶) were added 24 hrs later for another 24 hrs, followed by RNA isolation from CAR T cells and subsequent RNA sequencing. \u003cstrong\u003e(B)\u003c/strong\u003e\u0026nbsp;Heatmap of upregulated and downregulated genes in activated CAR T cells (left CD4\u003csup\u003e+\u003c/sup\u003e T cells only and right CD8\u003csup\u003e+\u003c/sup\u003e T cells only) after treatment with different TKIs in comparison to mock from the experiment described in (A). (https://software.broadinstitute.org/morpheus), L\u0026nbsp;=\u0026nbsp;Lenvatinib, C = Cabozantinib, S = Sorafenib. \u003cstrong\u003e(C)\u003c/strong\u003e Radar charts illustrating RNA expression in key biochemical pathways for the T cell immune response from experiment (A). Charts were generated using the radar chart function from the fsmb package. T\u0026nbsp;cell-related GO terms were selected from Gene Set Enrichment Analysis (GSEA)\u003csup\u003e44\u003c/sup\u003e, and mean gene expression was calculated for each CAR T cell treatment group, with mock samples as the baseline. Percent expression differences relative to the mock group are shown as coloured lines in the radar charts. \u003cstrong\u003e(D) \u003c/strong\u003eRelative numbers of viable cells measured with an SRB-based viability assay in which ROR-1 expressing ATC cell lines were co-incubated with different TKIs (n = 3, mean ± SD, t-test, ** p \u0026lt; 0.01) \u003cstrong\u003e(E) \u003c/strong\u003eFlow cytometric analysis for ROR1 expression of ATC cell lines treated with vehicle or lenvatinib at 1 or 2 µM concentration for 72 hrs, respectively. \u003cstrong\u003e(F) \u003c/strong\u003eSRB-based viability assay was performed using the ROR1-expressing 8505C ATC cell line co-incubated with UTD or CAR T cells at 1:2 and 1:5 ratios with or without additional lenvatinib treatment for 24 hrs. N = 4, mean ± SD, t-test (ns ≥ 0.05).\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8690827/v1/990815d70c9599de21de8228.png"},{"id":101384529,"identity":"428cc027-f245-46f8-a05f-40f5cc2e7cac","added_by":"auto","created_at":"2026-01-29 07:06:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":958853,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLenvatinib reduces immunosuppressive CAF-S1 microenvironmental cells in patient-derived ATC and PDTC samples.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eScheme presenting the different phenotypes of cancer-associated fibroblasts (CAFs) including their proposed function according to the literature.\u003csup\u003e45-47\u003c/sup\u003e \u003cstrong\u003e(B)\u003c/strong\u003e FACS gating strategy for CAF subpopulations of ATC patient material. \u003cstrong\u003e(C)\u003c/strong\u003e Images showing patient- derived fibroblasts from thyroid preparations from ATC/PDTC patients. \u003cstrong\u003e(D, E)\u003c/strong\u003e Quantification of total CAFs (D) or\u0026nbsp; total immunosuppressive CAF-S1 (E) with and without lenvatinib treatment over seven days (7d, 1 µM), n = 6, unpaired t-test (** p ≤ 0.01, ns p ≥ 0.05). \u003cstrong\u003e(F)\u003c/strong\u003e Flow cytometric images showing CAF-S1 cells (EpCAM-CD45-CD31-FAP\u003csup\u003ehigh\u003c/sup\u003eCD29\u003csup\u003ehigh\u003c/sup\u003e) with and without lenvatinib treatment. Phenotypic changes in ATC and PDTC patient-derived fibroblasts after treatment \u003cem\u003ein vitro\u003c/em\u003e with 1 µM lenvatinib for one week.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8690827/v1/0f15b4e8e3a19522f701c302.png"},{"id":101384530,"identity":"916a4a45-5b96-498e-a764-494ec9da7337","added_by":"auto","created_at":"2026-01-29 07:06:09","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":576253,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eROR1 CAR T cell efficiency in combination with lenvatinib in vivo.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Experimental scheme for the \u003cem\u003ein vivo\u003c/em\u003e treatment of NSG mice with the lenvatinib/ROR1 CAR T cell combination. 8505C cancer cells (3×10⁶ cells/mouse) were injected into the right flank of NSG mice and grew until a median volume of 177.8 mm³ was reached. Then lenvatinib (5 mg /kg BW) or vehicle treatment was started for one week, followed by ROR1 CAR T injections (5×10⁶, CD4:CD8 = 1:1) once or weekly. \u003cstrong\u003e(B)\u003c/strong\u003e Mean tumour volume as measured by calliper gauche over time from 4 - 6 mice per group. \u003cstrong\u003e(C)\u003c/strong\u003e Mean tumour volume on experimental day 42 (end of control group). \u003cstrong\u003e(D)\u003c/strong\u003eMean tumour volume on day 46 (end of lenvatinib treatment groups); n = 4 – 6, mean ± SD, two-way ANOVA for (B), one-way ANOVA for (C), t-test for (D), **** p ≤ 0.0001, ** p ≤ 0.01, * p \u0026lt; 0.05; ns ≥ 0.05. \u003cstrong\u003e(E)\u003c/strong\u003e Scheme for lenvatinib/ROR1 CAR T cell treatments with multiple CAR T cell injections. 8505C cancer cells (3×10⁶ cells/mouse) were injected into the right flank of NSG mice and grew until a median volume of 118.2 mm³ was reached. \u003cstrong\u003e(F)\u003c/strong\u003eMean tumour volume over time (n = 4-5 per group). \u003cstrong\u003e(G)\u003c/strong\u003e Mean tumour volume on day 46 (end of control group), n = 4-5, mean ± SD, (** p ≤ 0.01, * p \u0026lt; 0.05).\u003cstrong\u003e (H)\u003c/strong\u003e Mean tumour volume on day 49 (end of lenvatinib group); n = 4 - 5, mean ± SD, (* p \u0026lt; 0.05). \u003cstrong\u003e(I)\u003c/strong\u003eExperimental scheme to monitor tumour metastasis into different organs using the luciferase IVIS imaging system. Lung imaging results are shown in \u003cstrong\u003e(J)\u003c/strong\u003e and liver results in \u003cstrong\u003e(K)\u003c/strong\u003e from all treatment groups, other organs are depicted in Figure S15. N = 3–4, mean ± SD, one-way ANOVA (* p \u0026lt; 0.05, ns ≥ 0.05). \u003cstrong\u003e(L) \u003c/strong\u003eQuantified lung metastasis results are shown for day 46 (end of control groups) and day 49 (end of lenvatinib treatment groups); n = 3–4, mean ± SD, One-Way ANOVA (* p \u0026lt; 0.05). \u003cstrong\u003e(M)\u003c/strong\u003e Analysis of circulating tumour cells by flow cytometry in the peripheral blood of NSG mice; n = 3-4, unpaired t-test (** p ≤ 0.01).\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-8690827/v1/ad3095eda001ba7314812914.png"},{"id":101384534,"identity":"de7f3cea-e943-4206-8c3b-32ab40292187","added_by":"auto","created_at":"2026-01-29 07:06:09","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":407514,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLenvatinib increases quantity of circulating CAR T cells and alters CAR T cell activation and differentiation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Gating strategy for circulating ROR1 CAR T cells in the peripheral blood of ATC mice. \u003cstrong\u003e(B)\u003c/strong\u003e Quantification of circulating ROR1 CAR T cells in the peripheral blood, n = 7 per group, mean ± SD, unpaired t-test (*** p ≤ 0.001). \u003cstrong\u003e(C)\u003c/strong\u003e Surface markers determining activation and exhaustion profiles of circulating CAR T cells. \u003cstrong\u003e(D-G)\u003c/strong\u003e Analysis of activation and exhaustion surface markers of circulating ROR1 CAR T cells in mice with or without lenvatinib treatment; n = 7, mean ± SD, unpaired t-test (**** p ≤ 0.0001, ** p ≤ 0.01, ns p ≥ 0.05). \u003cstrong\u003e(H)\u003c/strong\u003e Differentiation profile of circulating CAR T cells determined by different surface markers. T\u003csub\u003eN\u003c/sub\u003e –naïve T cells; T\u003csub\u003eE\u003c/sub\u003e – effector T cells; T\u003csub\u003eEM\u003c/sub\u003e – effector memory T cells; T\u003csub\u003eCM\u003c/sub\u003e – central memory T cells. \u003cstrong\u003e(I-K)\u003c/strong\u003e Quantity of circulating CAR T cells in different differentiation stages with and without lenvatinib treatment; n = 7, mean ± SD, unpaired t-test (**** p ≤ 0.0001, *** p ≤ 0.001).\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-8690827/v1/5bc23d84a9ab8142924a9ee1.png"},{"id":101398116,"identity":"6e98d5d7-e755-4216-a0bf-9649acf224ed","added_by":"auto","created_at":"2026-01-29 09:39:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5526432,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8690827/v1/cffadfc6-fb42-447b-aecb-3738a600eeb8.pdf"},{"id":101384533,"identity":"c240f9a7-01bc-44bf-bb48-d229491c4368","added_by":"auto","created_at":"2026-01-29 07:06:09","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":30070571,"visible":true,"origin":"","legend":"Supplementary Dataset 1","description":"","filename":"ManuscriptROR1inATCSupplements.docx","url":"https://assets-eu.researchsquare.com/files/rs-8690827/v1/48b684a04f8db943ec1dc84f.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"ROR1 CAR T cells and lenvatinib cooperatively target anaplastic thyroid carcinoma","fulltext":[{"header":"Key points","content":"\u003cp\u003eROR1 is specifically overexpressed in ATC and functions as a prime target for CAR T cell therapy.\u003c/p\u003e\u003cp\u003eWhile ROR1 CAR T cells efficiently target circulating tumour cells and tumour metastases, lenvatinib blocks primary tumour growth and enhances CAR T cell functionality and activation.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eAnaplastic thyroid carcinoma (ATC) is a devastating disease with a median survival of 4 months despite multimodal therapy including surgery, irradiation and chemotherapy.\u003csup\u003e1\u0026ndash;3\u003c/sup\u003e Although ATC accounts for only 1% of thyroid carcinomas (TC), it is responsible for 50% of the deaths caused by thyroid cancer. ATCs arise from the follicular epithelium of the thyroid gland, with 50% developing de novo and 50% with a pre-existing differentiated TC. Tumours are high proliferative and show a very fast and infiltrative growth into local structures like oesophagus, trachea or the carotid artery, with 50% having distant metastasis already at diagnosis. Genetic alterations include mutations in TP53, HRAS, KRAS, PIK3CA, PTEN and only 20\u0026ndash;30% carry targetable mutations in BRAF or have NTRK or RET fusions.\u003csup\u003e4\u0026ndash;7\u003c/sup\u003e Since ATCs usually have high PD-L1 levels and a strong inflammatory tumour microenvironment (TME),\u003csup\u003e8\u003c/sup\u003e we and others have developed novel treatment strategies combining kinase inhibitors with immune checkpoint inhibitors (lenvatinib/pembrolizumab), which achieve improved responses and an increased median survival compared to chemotherapy.\u003csup\u003e9\u003c/sup\u003e Despite some progress, the majority of patients relapse and urgently need alternative treatment options.\u003c/p\u003e \u003cp\u003eCAR T cells are engineered T cells which carry a synthetic receptor, which is directed against a tumour antigen. In 2017, the first CAR T cells against CD19 were approved for the treatment of B cell lymphoma and B cell acute lymphoblastic leukaemia.\u003csup\u003e10,11\u003c/sup\u003e While CAR T cells are highly effective in hematologic malignancies, several obstacles exist for CAR T cells in solid tumours that must be overcome. These include the identification of specific tumour antigens, the hypoxic and acidic TME, immunosuppressive mechanisms and the compactness and desmoplasia of many carcinoma entities, which prevent CAR T cell access to its target cells.\u003csup\u003e12\u003c/sup\u003e Previous efforts in differentiated TCs include CAR T cell targeting of the TSH receptor\u003csup\u003e13\u003c/sup\u003e, but this option cannot be used for ATCs as its expression is lost during the dedifferentiation process.\u003c/p\u003e \u003cp\u003eThe tyrosine protein kinase transmembrane orphan receptor 1, ROR1, is part of the non-canonical Wnt signalling pathway and can be activated via Wnt5a.\u003csup\u003e14\u003c/sup\u003e It is mainly expressed during embryonic development and reactivated in various neoplasms.\u003csup\u003e15\u0026ndash;17\u003c/sup\u003e In this context, ROR1 can influence several intracellular signalling cascades like pro-apoptotic signalling pathways, MAP kinase signalling, phosphoinositide 3-kinase or NF-κB leading to an elevated cell survival, migration and proliferation promoting tumour growth and resistance to apoptosis.\u003csup\u003e18\u0026ndash;20\u003c/sup\u003e Furthermore, ROR1 was shown to be overexpressed on cancer stem cells and to be predominantly expressed in undifferentiated tumour types, which positively correlates with occurrence of tumour metastasis and relapse.\u003csup\u003e21,22\u003c/sup\u003e Due to its specificity especially for un- and dedifferentiated malignant neoplasias, while sparing most normal adult tissues, ROR1 represents an interesting target for an adoptive immunotherapy based on chimeric antigen receptor T cells (ROR1 CAR T cells).\u003csup\u003e23\u003c/sup\u003e ROR1 CAR T cells were shown to specifically target some hematological malignancies, such as e.g. CLL and solid tumours.\u003csup\u003e17,24\u0026ndash;26\u003c/sup\u003e Furthermore, their safety was evaluated in various preclinical models \u003csup\u003e27,28\u003c/sup\u003e and in a phase I clinical trial in hematologic malignancies and solid tumours.\u003csup\u003e29\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eKinase inhibitors, like lenvatinib, sorafenib and cabozantinib, are approved for the treatment of radioiodine refractory differentiated TCs.\u003csup\u003e30\u0026ndash;33\u003c/sup\u003e Their common feature is targeting the VEGFR signalling axis and therefore preventing neoangiogenesis and thus reducing tumour growth. Besides neoangiogenesis, VEGFR inhibitors can also improve T cell function in immune checkpoint inhibitor therapies and can transform the immunosuppressive microenvironment in several tumour entities.\u003csup\u003e34\u003c/sup\u003e Based on these data, we hypothesized that VEGFR inhibitors might also support CAR T cell function.\u003c/p\u003e \u003cp\u003eIn the study shown here, we identified ROR1 as a prime target for anaplastic thyroid carcinoma and identified lenvatinib as an ideal combination partner, which blocks tumour growth, improves CAR T cell functionality and reduces local immunosuppression.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eROR1 is overexpressed in ATC compared to normal thyroid tissue and high expression levels are associated with reduced patient survival\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eROR1\u003c/em\u003e expression was determined via transcriptome analysis, IHC and ROR1 surface expression. By using RNAseq data from fresh-frozen own ATC/PDTC patient samples (n\u0026thinsp;=\u0026thinsp;20) compared to goiter, we found \u003cem\u003eROR1\u003c/em\u003e RNA to be overexpressed in anaplastic and poorly differentiated TC compared to goiter (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Through an NCBI dataset (GSE76039)\u003csup\u003e7\u003c/sup\u003e which includes transcriptome data (Affymetrix U133 plus 2.0 array) from fresh frozen and histologically confirmed ATC (n\u0026thinsp;=\u0026thinsp;18) and PDTC (n\u0026thinsp;=\u0026thinsp;17) samples compared to PTCs we could determine that \u003cem\u003eROR1\u003c/em\u003e was significantly higher expressed in ATCs (n\u0026thinsp;=\u0026thinsp;18) compared to PDTCs (n\u0026thinsp;=\u0026thinsp;17) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, Figure S5 A-C). TCs with a BRAF-like signature or BRAF mutations (n\u0026thinsp;=\u0026thinsp;275) had significantly higher \u003cem\u003eROR1\u003c/em\u003e levels than RAS-like or RAS mutated thyroid tumours (n\u0026thinsp;=\u0026thinsp;118) respectively (Figure S5 A, B). High expression levels for \u003cem\u003eROR1\u003c/em\u003e were associated with a significantly shorter survival (median survival ROR1\u003csup\u003ehigh\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;5.9 month, ROR1\u003csup\u003elow\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;102.1 month, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). \u003cem\u003eROR1\u003c/em\u003e expression was inversely correlated with the expression of the \u003cem\u003eTSH receptor\u003c/em\u003e, indicating it as a marker for undifferentiated thyroid cancer (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, Figure S5 C). Also gene clustering analysis on primary frozen ATC/PDTC and goiter showed that the clusters with the most undifferentiated tumours (GSEA cluster undifferentiated cancer) within ATCs (ATC cluster II) are associated with the highest \u003cem\u003eROR1\u003c/em\u003e transcript levels, while losing differentiation markers like thyreotropin stimulating hormone receptor \u003cem\u003e(TSHR)\u003c/em\u003e, thyreoperoxidase (\u003cem\u003eTPO)\u003c/em\u003e, thyreoglobulin \u003cem\u003e(TG)\u003c/em\u003e and others (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, F, Figure S6 D-F). Histologically, the ATC areas displayed high ROR1 levels, while adjacent normal thyroid tissue from the same patient sample showed low to absent ROR1 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG, H). Conversely, ATC areas were negative for TSHR, which was always expressed on normal thyroid tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI, J). ATC cell lines, which were either BRAF V600E mutated (8505C, SW1736) or HRAS G13R mutated (C643) completely recapitulated the features of primary ATC patient tissues and showed high ROR1 surface expression and absent TSHR expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK, Figure S7 A-D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e(A)\u003c/b\u003e \u003cem\u003eROR1\u003c/em\u003e transcript levels of fresh-frozen tissue samples from patients with ATC (n\u0026thinsp;=\u0026thinsp;12) compared to goiter (n\u0026thinsp;=\u0026thinsp;8); mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, unpaired t-test (*** p\u0026thinsp;\u0026le;\u0026thinsp;0.0001). Patient data is available in table S1. \u003cb\u003e(B)\u003c/b\u003e NGS analysis of \u003cem\u003eROR1\u003c/em\u003e expression in ATC (n\u0026thinsp;=\u0026thinsp;18 ●) versus PDTC (n\u0026thinsp;=\u0026thinsp;17 ●) using the GSE76039 dataset \u003csup\u003e7\u003c/sup\u003e; mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, unpaired t-test (*** p\u0026thinsp;\u0026le;\u0026thinsp;0.0001). \u003cb\u003e(C)\u003c/b\u003e Overall survival (OS) of ATC and PDTC patients with high (above median) and low (below median) \u003cem\u003eROR1\u003c/em\u003e-expression from the GSE76039 dataset \u003csup\u003e7\u003c/sup\u003e; log-rank (Mantel-Cox) test (**** p\u0026thinsp;\u0026le;\u0026thinsp;0.0001). \u003cb\u003e(D)\u003c/b\u003e Pearson\u0026rsquo;s correlation of \u003cem\u003eROR1\u003c/em\u003e and \u003cem\u003eTSHR\u003c/em\u003e RNA expression in the Halle patient cohort (Table S1). \u003cb\u003e(E)\u003c/b\u003e Principle component analysis and cluster analysis of the NGS data of goiter patient samples (n\u0026thinsp;=\u0026thinsp;8) and ATC patient samples (n\u0026thinsp;=\u0026thinsp;12). \u003cb\u003e(F)\u003c/b\u003e Heatmap of the expression of the thyroid-tissue specific markers and \u003cem\u003eROR1\u003c/em\u003e. \u003cem\u003eTG\u003c/em\u003e \u0026ndash; Thyroglobulin, \u003cem\u003eIYD\u003c/em\u003e \u0026ndash; Iodotyrosine Deiodinase, \u003cem\u003eTSHR\u003c/em\u003e \u0026ndash; Thyroid Stimulating Hormone Receptor, \u003cem\u003eNKX2-1\u003c/em\u003e \u0026ndash; NK2 Homeobox or Thyroid Transcription Factor-1. \u003cb\u003e(G, I)\u003c/b\u003e Representative examples for ROR1 and TSHR IHC DAB-staining in healthy thyroid tissue (HTT) and ATC within the same patient (n\u0026thinsp;=\u0026thinsp;20); black scale bar corresponds to 200 \u0026micro;m, red \u0026ndash; to 50 \u0026micro;m. \u003cb\u003e(H, J)\u003c/b\u003e H Score of the ROR1 and TSHR in ATC tissue samples (ATC) ● and healthy thyroid tissue samples (HTT) ○ from the same patient (n\u0026thinsp;=\u0026thinsp;20); paired t-test (*** p\u0026thinsp;\u0026le;\u0026thinsp;0.001; **** p\u0026thinsp;\u0026le;\u0026thinsp;0.0001). \u003cb\u003e(K)\u003c/b\u003e ROR1 surface expression as measured by flow cytometry in 3 different ATC cell lines (8505C, C643, SW1736) and one ovarian cancer cell line (A2780). The values in red represent median of ROR1-PE expression of each cell line. Experiments were performed in triplicates.\u003c/p\u003e \u003cp\u003eTaken together, ROR1 is specifically overexpressed at the RNA, protein and surface level in both, primary ATC tissues and ATC cell lines and high ROR1 expression levels are associated with a reduced patients\u0026rsquo; survival. Since previous studies have shown absence or low levels of ROR1 expression in healthy tissues, ROR1 represents an interesting and specific novel CAR T cell target for ATCs.\u003csup\u003e3,17,27,35,36\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eROR1-directed CAR T cells effectively and specifically lyse ROR1-positive ATC cells\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSecond-generation ROR1 CAR T cells were developed by the laboratory of Prof. M. Hudecek and the ROR1 antigen targeting CAR is expressed in the lentiviral targeting vector R12 4-1BB CD3 zeta (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). ROR1 CAR T cells were previously shown to be effective against their target; a high target specificity and can be safely applied to animals and humans.\u003csup\u003e17,27\u003c/sup\u003e Confirming these results in ATC, co-culture of ROR1 CAR T cells with any of the 3 different ROR1\u0026thinsp;+\u0026thinsp;ATC cell lines induced efficient and fast tumour cell lysis (PI staining for dead cells and SRB-based viability assay; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C), while ROR1-negative ovarian cancer cells were not affected and also untransduced control T cells (UTD) did not induce tumour cell lysis. In addition, ROR1\u0026thinsp;+\u0026thinsp;ATC cell lines induced specific activation of ROR1 CAR T cells including production of inflammatory cytokines, like INF-α and GM-CSF (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, Figure S8). Target specificity was tested via CRISPR-Cas9-induced knockout (KO) of ROR1 in all 3 ATC cell lines, which was verified via sequence analysis and flow cytometry for ROR1 surface expression (blue graph; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). The ROR1 KO completely abolished tumour cell lysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, right columns), indicating target specificity of the ROR1 CAR T cells in ATCs. Next, ROR1 CAR T cell efficacy was tested in 3D spheroid cultures, where T cells need to invade into a semisolid structure under hypoxic and acidic conditions. ROR1 CAR T cells were able to efficiently infiltrate the spheroids formed by C643 and SW1736 cells, destroyed the spheroid structure including the spheroid barriers and efficiently lysed ROR1\u0026thinsp;+\u0026thinsp;tumour cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG, H), which was documented via live cell imaging over 24 hrs (supplementary video 1). ROR1 KO ATC cells were also able to form spheroids, but were not affected by ROR1 CAR T cells confirming target specificity also in 3D cultures.\u003c/p\u003e \u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u003cb\u003e(A)\u003c/b\u003e Schematic representation of the targeting principle using ROR1 CAR T cells with ROR1\u003csup\u003e+\u003c/sup\u003e ATC (anaplastic thyroid carcinoma) cell lines. Right picture shows the CAR construct with the single chain variable fragment (scFv) domain, the CD28 transmembrane domain (TMD), the 4-1BB costimulatory domain and the CD3ζ intracellular domain (ICD).\u003csup\u003e25,37,38\u003c/sup\u003e\u003cb\u003e(B)\u003c/b\u003e Representative fluorescence and HD phase image (Incucyte) of the ROR1-expressing ATC-cell line 8505C co-incubated with untransduced (UTD) T cells (upper image) or ROR1-targeting CAR T cells for 24 hours. Propidium iodide (PI) staining in red clusters represents dead cancer cells (scale bar 400 \u0026micro;m). \u003cb\u003e(C)\u003c/b\u003e Relative number of living cells using a SRB assay (measurement of relative cellular protein content) for ROR1-expressing ATC cell lines (8505C, SW1736, C643) and the ROR1-negative ovarian cancer cell line A2780 after co-incubation with two- and fivefold numbers of UTD or ROR1 CAR T cells; n\u0026thinsp;=\u0026thinsp;3, mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, multiple t-tests ( *** p\u0026thinsp;\u0026le;\u0026thinsp;0.001, ** p\u0026thinsp;\u0026le;\u0026thinsp;0.01; * p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; ns\u0026thinsp;\u0026ge;\u0026thinsp;0.05). \u003cb\u003e(D)\u003c/b\u003e Quantification of cytokines like interferon-γ using the MACSPlex Cytokine 12 Kit from Miltenyi Biotec. Cytokine levels were assessed in the supernatant of co-cultures of ROR1\u0026thinsp;+\u0026thinsp;ATC cell lines (8505C, SW1736, C643) or ROR1- ovarian cancer cells (A2780) with control, UTD (untransduced T cells) or ROR1 CAR T cells. N\u0026thinsp;=\u0026thinsp;3, mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, multiple t-tests, (*** p\u0026thinsp;\u0026le;\u0026thinsp;0.001, ** p\u0026thinsp;\u0026le;\u0026thinsp;0.01, * p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; ns\u0026thinsp;\u0026ge;\u0026thinsp;0.05). \u003cb\u003e(E)\u003c/b\u003e Flow cytometry analysis of ROR1 expression in wild type ATC cell lines (8505C, C643, SW1736, red graph) and the respective ROR1-KO ATC cell line (blue graph) generated with Crispr-Cas9 against the ROR1 sequence. The values in red represent median fluorescence intensity (MFI) of the ROR1-PE staining of each cell line. \u003cb\u003e(F)\u003c/b\u003e SRB-mediated relative viability assay measuring the cellular protein content in ATC cell monolayers and treated with either mock, UTD or ROR1 CAR T cells. n\u0026thinsp;=\u0026thinsp;3, mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, One-Way ANOVA. (*** p\u0026thinsp;\u0026le;\u0026thinsp;0.001, ns\u0026thinsp;\u0026ge;\u0026thinsp;0.05) \u003cb\u003e(G)\u003c/b\u003e Luminescence-based viability assay of the ROR1-expressing ATC model cell line C643 and SW1736 in 3D culture as spheroids and its ROR1-KO counterpart (3D) co-incubated with mock, untransduced T cells (UTD T) and ROR1-targeting CAR T cells (CAR T). Luminescence was measured with Tecan Spark\u0026reg; Multimode Mikroplate Reader. N\u0026thinsp;=\u0026thinsp;3, mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, two-Way ANOVA. (*** p\u0026thinsp;\u0026le;\u0026thinsp;0.001, ns\u0026thinsp;\u0026ge;\u0026thinsp;0.05). \u003cb\u003e(H)\u003c/b\u003e Visualisation of the co-incubation of C643 spheroids as well as C643 with ROR1-KO with UTD control T cells or CAR T cells (scale bar 200 \u0026micro;m). The experimental setup is identical to that described in (G). For the visual analysis and video generation (Supplemental video 1) with the Incucyte, the T cells were labelled with CellTracker\u0026trade; Red CMTPX according to the manufacturer's instructions.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eROR1 CAR T cells eliminate small ATC xenograft tumours in vivo\u003c/h2\u003e \u003cp\u003eNext, ROR1 CAR T cell efficacy was tested in different ATC xenograft models \u003cem\u003ein vivo\u003c/em\u003e. While the ROR1-expressing BRAF-mutated ATC cell line 8505C was subcutaneously (s.c.) injected into the left flank of immunocompromised NSG (NOD.Cg-Prkdcscid Il2rgtm1Sug/JicTac) mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-C), the RAS mutated ATC cell line C643 was injected retroorbitally and cells were mainly detected at the injection site and within the lungs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-F). After 2 or 7 days 5 *10\u003csup\u003e6\u003c/sup\u003e ROR1 CAR T cells (CD4/CD8 mixed 1:1) or vehicle were injected intravenously (i.v.) and tumour cell expansion was monitored via volume determination (s.c. model) and luminescence imaging (i.v. model). ROR1 CAR T cells efficiently blocked tumour development independent of the cell line, mode of engraftment and detection method (volume and luminescence detection), injection time point and mutation status (BRAF- or RAS mutated) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-F).\u003c/p\u003e \u003cp\u003eNext, we aimed to establish a more physiological situation, like in patients, where CAR T cells are used when tumours are already established with sizes at least beyond 100 mm\u0026sup3;. Therefore, tumour cells were injected and tumours were allowed to develop for 25 days until a medium start volume of 165.9 mm\u003csup\u003e3\u003c/sup\u003e was reached (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG, H). At day 25, mice received either a control injection with PBS (n\u0026thinsp;=\u0026thinsp;7), a one-time ROR1 CAR T cell injection (CD4:CD8 ratio 1:1) (n\u0026thinsp;=\u0026thinsp;8) or weekly ROR1 CAR T cell injections (n\u0026thinsp;=\u0026thinsp;7), respectively. In contrast to the previous experiments, the one-time injection of ROR1 CAR T cells was not able to significantly reduce the tumour mass of these already established, fast growing ATC tumours (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). In contrast, repeated CAR T cell injections (3 x once weekly) showed improved effects, i.e. tumour volumes were reduced by one third (control mean\u0026thinsp;=\u0026thinsp;957 mm\u0026sup3;, 3 x CAR T cells\u0026thinsp;=\u0026thinsp;630 mm\u0026sup3;, p\u0026thinsp;=\u0026thinsp;0.0254) and tumour weight to about half (control mean\u0026thinsp;=\u0026thinsp;1.0 g, 3 x CAR Ts\u0026thinsp;=\u0026thinsp;0.58 g; p\u0026thinsp;=\u0026thinsp;0.0002, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI, J). To visualize the infiltration into the tumour, CAR T cell, were labeled with IVISense 680 Fluorescent Cell Labeling for further experiments prior to injection into mice. Extracted tumours (40 days after injection) showed CAR T cell fluorescence signals on the tumour surface and also within the tumour indicating that CAR T cells can indeed penetrate the tumour (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK), which was confirmed by immunohistological stainings (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eL, M).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003e \u003cb\u003e(A)\u003c/b\u003e Experimental scheme for NSG mice subcutaneously injected with BRAF V600E\u003csup\u003e+\u003c/sup\u003e mutated 8505C ATC cells (3\u0026times;10⁶ 8505C cells transduced with a luciferase vector) and treated with intravenously (i.v.) applied ROR1 CAR T cells 2 or 7 days after tumour cell injection. The administration of 100 \u0026micro;L of CAR T cells (5\u0026times;10⁶ CAR T cells per NSG mouse with a CD4 to CD8 ratio of 1:1) or vehicle (PBS) was conducted via the tail vein. \u003cb\u003e(B, C)\u003c/b\u003e Exemplary imaging analysis (luminescence imaging, IVIS Spectrum) before (experimental day 2) and 26 days after CAR T injection (day 28) and tumour volume analysis with a digital caliper gauge are shown in Figures (B) and (C), respectively. \u003cb\u003e(D)\u003c/b\u003e Experimental scheme for NSG mice retroorbitally injected with RAS-mutated C643 ATC cells (1\u0026times;10⁵ in 100 \u0026micro;L PBS) and treated with control (n\u0026thinsp;=\u0026thinsp;3) or ROR1 CAR T cells (5\u0026times;10⁶, CD4 to CD8 ratio of 1:1, retroorbital opposite site, n\u0026thinsp;=\u0026thinsp;3) two days after tumour cell injection. \u003cb\u003e(E, F)\u003c/b\u003e Representative imaging analysis (luminescence imaging, IVIS Spectrum) before (experimental day 2) and 12 days after CAR T cell injection (day 14) and tumour volume analysis with a digital caliper gauge are shown in figures (E) and (F), respectively. N\u0026thinsp;=\u0026thinsp;3, mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, two-way ANOVA (**** p\u0026thinsp;\u0026le;\u0026thinsp;0.0001). \u003cb\u003e(G)\u003c/b\u003e Experimental design for CAR T cell treatment in already established 8505C tumours. 3\u0026times;10⁶ 8505C cells were injected in the left flank of NSG mice and grew over 25 days (mean volume 163.7\u0026thinsp;\u0026plusmn;\u0026thinsp;53.7 mm\u003csup\u003e2\u003c/sup\u003e), then mock (n\u0026thinsp;=\u0026thinsp;7) or 5\u0026times;10⁶ ROR1 CAR T cells were injected intravenously in the tail vein once (n\u0026thinsp;=\u0026thinsp;8) or 3-times weekly (n\u0026thinsp;=\u0026thinsp;7). \u003cb\u003e(H)\u003c/b\u003e Mean tumour volume over time; \u003cb\u003e(I)\u003c/b\u003e mean tumour volume on day 42 (end of control group) and \u003cb\u003e(J)\u003c/b\u003e mean tumour weight at the end of the experiment 42 days after tumor cell injection and 17 days after first CAR T cell injection; n\u0026thinsp;=\u0026thinsp;7\u0026ndash;8, mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, unpaired t-test (* p\u0026thinsp;\u0026le;\u0026thinsp;0.05, *** p\u0026thinsp;\u0026le;\u0026thinsp;0.001). \u003cb\u003e(K)\u003c/b\u003e Fluorescent images of tumours treated either with control of IVISense 680 Fluorescent Cell labelled ROR1 CAR T cellss. Images show either front view (surface) or cross sections. \u003cb\u003e(L)\u003c/b\u003e Percentage of ROR1 CAR T cells within tumour tissue analysed by IHC\u0026thinsp;=\u0026thinsp;7\u0026ndash;8, mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, unpaired t-test (*** p\u0026thinsp;\u0026le;\u0026thinsp;0.001). \u003cb\u003e(M)\u003c/b\u003e Images of tumour sections stained with a CD5-antibody marking the infiltrating ROR1 CAR T cells. The IHC slides were analysed using the QuPath 0.4.3 software, red-marked cells represent CD5\u0026thinsp;+\u0026thinsp;CAR T cells, blue-marked cells represent CD5- tumor and other cell types. \u003cb\u003e(N)\u003c/b\u003e Percentage of ROR1 CAR T cells within tumour tissue analysed by flow cytometry of mouse peripheral blood; n\u0026thinsp;=\u0026thinsp;7\u0026ndash;8, mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, unpaired t-test (*** p\u0026thinsp;\u0026le;\u0026thinsp;0.001).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eM, ROR1 CAR T cells (in red) are located mainly around the tumour blood vessels, but are not equally distributed over the complete tumour mass. Quantification shows that CAR T cells account for about 2\u0026ndash;3% of total cells within a tumour (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eL). In addition, circulating CAR T cells are found in the peripheral blood of mice 19d after CAR T cell treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eN).\u003c/p\u003e \u003cp\u003e \u003cb\u003eLenvatinib and other kinase inhibitors can improve CAR T cell functionality\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFrom previous preclinical work and from the clinical ATLEP trial in ATC and PDTC patients, we know that multikinase/VGFR inhibitors, like lenvatinib, enhance the efficacy of endogenous T cells in the context of immune checkpoint inhibitor therapies and can change the microenvironment.\u003csup\u003e39\u0026ndash;43\u003c/sup\u003e Based on these results, it was hypothesized that multikinase inhibitors might also improve CAR T cell functionality and tumour invasion.\u003c/p\u003e \u003cp\u003eTo test this, co-cultures of tumour cells and ROR1 CAR T cells were treated with the multikinase inhibitors lenvatinib, sorafenib and cabozantinib for 24 hrs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Gene expression analysis of the CAR T cells in co-culture demonstrated strong effects of the kinase inhibitors on CD4\u0026thinsp;+\u0026thinsp;CAR T cells demonstrated by enhanced expression of genes related to T cell activation, T cell proliferation, T cell differentiation, adaptive immune response, TCR signalling, T cell-mediated toxicity and T cell migration, while CD8\u0026thinsp;+\u0026thinsp;CAR T cells were less affected (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, Figure S10-11). Lenvatinib showed a distinct cluster profile from sorafenib and cabozantinib (Figure S12) with strongest effects in the most physiological setting with CD4/CD8-mixed CAR T cell cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). From all 3 inhibitors tested, lenvatinib had the strongest outcome on major T cell functions, like T cell proliferation, TCR pathway activation, T cell mediated cytotoxicity, T cell cytokine production and T cell migration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, blue). The VEGFR inhibitors lenvatinib and cabozantinib neither directly influenced the viability of tumour cells nor altered ROR1 receptor expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, E; Figure S13). High sorafenib concentrations reduced the viability of the RAS-mutated cell line C643. None of the inhibitors negatively affected tumour cell killing of ROR1 CAR T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF; Figure S14 A-C)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e(A)\u003c/b\u003e Schematic overview of the experimental setup to analyse the influence of TKIs on activated CAR T cells. 8505C WT cells (0.01\u0026times;10⁶) were seeded in a 24-well plate and incubated for 24 hours before treatment with 0.02% DMSO (mock) or 1 \u0026micro;M lenvatinib, 1 \u0026micro;M cabozantinib, or 1 \u0026micro;M sorafenib, respectively, CAR T cells (1.2\u0026ndash;1.5\u0026times;10⁶) were added 24 hrs later for another 24 hrs, followed by RNA isolation from CAR T cells and subsequent RNA sequencing. \u003cb\u003e(B)\u003c/b\u003e Heatmap of upregulated and downregulated genes in activated CAR T cells (left CD4\u003csup\u003e+\u003c/sup\u003e T cells only and right CD8\u003csup\u003e+\u003c/sup\u003e T cells only) after treatment with different TKIs in comparison to mock from the experiment described in (A). (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://software.broadinstitute.org/morpheus\u003c/span\u003e\u003cspan address=\"https://software.broadinstitute.org/morpheus\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), L\u0026thinsp;=\u0026thinsp;Lenvatinib, C\u0026thinsp;=\u0026thinsp;Cabozantinib, S\u0026thinsp;=\u0026thinsp;Sorafenib. \u003cb\u003e(C)\u003c/b\u003e Radar charts illustrating RNA expression in key biochemical pathways for the T cell immune response from experiment (A). Charts were generated using the radar chart function from the fsmb package. T cell-related GO terms were selected from Gene Set Enrichment Analysis (GSEA)\u003csup\u003e44\u003c/sup\u003e, and mean gene expression was calculated for each CAR T cell treatment group, with mock samples as the baseline. Percent expression differences relative to the mock group are shown as coloured lines in the radar charts. \u003cb\u003e(D)\u003c/b\u003e Relative numbers of viable cells measured with an SRB-based viability assay in which ROR-1 expressing ATC cell lines were co-incubated with different TKIs (n\u0026thinsp;=\u0026thinsp;3, mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, t-test, ** p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) \u003cb\u003e(E)\u003c/b\u003e Flow cytometric analysis for ROR1 expression of ATC cell lines treated with vehicle or lenvatinib at 1 or 2 \u0026micro;M concentration for 72 hrs, respectively. \u003cb\u003e(F)\u003c/b\u003e SRB-based viability assay was performed using the ROR1-expressing 8505C ATC cell line co-incubated with UTD or CAR T cells at 1:2 and 1:5 ratios with or without additional lenvatinib treatment for 24 hrs. N\u0026thinsp;=\u0026thinsp;4, mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, t-test (ns\u0026thinsp;\u0026ge;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eLenvatinib reduces the quantity of immunosuppressive CAFs S1 in ATC fibroblast cultures\u003c/h3\u003e\n\u003cp\u003eIn order to understand whether lenvatinib not only alters CAR T cell differentiation, but also changes the immunosuppressive TME, cancer-associated fibroblasts (CAFs) from 6 different patients with ATC or PDTC were isolated and their subtypes determined. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B, the patient-derived CAFs comprise of all different CAF subtypes, such as normal fibroblast-like (CAF-S2 and CAF-S3), immunosuppressive CAF-S1 and CAF-S4 known to drive tumour metastasis. Treatment of patient-derived ATC fibroblasts with lenvatinib did not alter CAF density or phenotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), and did not reduce total quantity of CAFs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD), but strongly reduced the immunosuppressive CAF-S1 subtype (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e(A)\u003c/b\u003e Scheme presenting the different phenotypes of cancer-associated fibroblasts (CAFs) including their proposed function according to the literature.\u003csup\u003e45\u0026ndash;47\u003c/sup\u003e \u003cb\u003e(B)\u003c/b\u003e FACS gating strategy for CAF subpopulations of ATC patient material. \u003cb\u003e(C)\u003c/b\u003e Images showing patient- derived fibroblasts from thyroid preparations from ATC/PDTC patients. \u003cb\u003e(D, E)\u003c/b\u003e Quantification of total CAFs (D) or total immunosuppressive CAF-S1 (E) with and without lenvatinib treatment over seven days (7d, 1 \u0026micro;M), n\u0026thinsp;=\u0026thinsp;6, unpaired t-test (** p\u0026thinsp;\u0026le;\u0026thinsp;0.01, ns p\u0026thinsp;\u0026ge;\u0026thinsp;0.05). \u003cb\u003e(F)\u003c/b\u003e Flow cytometric images showing CAF-S1 cells (EpCAM-CD45-CD31-FAP\u003csup\u003ehigh\u003c/sup\u003eCD29\u003csup\u003ehigh\u003c/sup\u003e) with and without lenvatinib treatment. Phenotypic changes in ATC and PDTC patient-derived fibroblasts after treatment \u003cem\u003ein vitro\u003c/em\u003e with 1 \u0026micro;M lenvatinib for one week.\u003c/p\u003e \u003cp\u003e \u003cb\u003eLenvatinib improves ROR1 CAR T cell efficacy\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo test, if lenvatinib can indeed improve the efficacy of ROR1 CAR T cells \u003cem\u003ein vivo\u003c/em\u003e, we combined ROR1 CAR T cell with lenvatinib in the 8505C xenograft mouse model (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Again, ROR1 CAR T cells alone were not able to stop tumour growth in the exponential growth phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, red line) compared to vehicle control (grey line). Lenvatinib alone (blue line) significantly reduced tumour volume and delayed disease development, but did not completely inhibit tumour growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, C). In contrast, the combination of lenvatinib with ROR1 CAR T cells (pink line) not only efficiently blocked tumour development, but was also able to induce regression of already established tumours (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-D). Results were further improved by combination of lenvatinib treatment with repeated ROR1 CAR T cell injections (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE-H, violet lane) resulting in about one-third tumour volumes (control\u0026thinsp;=\u0026thinsp;1458.3 mm\u003csup\u003e3\u003c/sup\u003e, lenvatinib alone\u0026thinsp;=\u0026thinsp;750.3 mm\u0026sup3;, lenvatinib\u0026thinsp;+\u0026thinsp;1 x CAR T cells\u0026thinsp;=\u0026thinsp;522.4 mm\u0026sup3;, lenvatinib\u0026thinsp;+\u0026thinsp;weekly CAR T cells\u0026thinsp;=\u0026thinsp;390.3 mm\u0026sup3;).\u003c/p\u003e \u003cp\u003eNext, we investigated the effect of the combination treatment on tumour metastasis. Tumour metastasis was measured by bioluminescence imaging in different organs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI). Interestingly, lenvatinib treatment alone did not influence tumour metastasis and could not prevent spreading of cancer cells into the different organs like lungs, liver or spleen (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ, K; Figure S15). In contrast, ROR1 CAR T cell treatments were able to nearly completely block tumour metastases, both as single treatments and even better as weekly repeated treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ-L; Figure S15). In concordance, ROR1 CAR T cells significantly reduced the numbers of circulating tumour cells by around 70% determined in the peripheral blood of treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eM). The combination of ROR1 CAR T cells with lenvatinib could further enhance the anti-metastasis effects and reduced the quantitiy of metastasis in lung and liver (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ, K). Our results indicate that lenvatinib is an ideal combination partner for ROR1 CAR T cells supporting their effect on reducing primary tumour growth and tumour metastases.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e(A)\u003c/b\u003e Experimental scheme for the \u003cem\u003ein vivo\u003c/em\u003e treatment of NSG mice with the lenvatinib/ROR1 CAR T cell combination. 8505C cancer cells (3\u0026times;10⁶ cells/mouse) were injected into the right flank of NSG mice and grew until a median volume of 177.8 mm\u0026sup3; was reached. Then lenvatinib (5 mg /kg BW) or vehicle treatment was started for one week, followed by ROR1 CAR T injections (5\u0026times;10⁶, CD4:CD8\u0026thinsp;=\u0026thinsp;1:1) once or weekly. \u003cb\u003e(B)\u003c/b\u003e Mean tumour volume as measured by calliper gauche over time from 4\u0026ndash;6 mice per group. \u003cb\u003e(C)\u003c/b\u003e Mean tumour volume on experimental day 42 (end of control group). \u003cb\u003e(D)\u003c/b\u003e Mean tumour volume on day 46 (end of lenvatinib treatment groups); n\u0026thinsp;=\u0026thinsp;4\u0026ndash;6, mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, two-way ANOVA for (B), one-way ANOVA for (C), t-test for (D), **** p\u0026thinsp;\u0026le;\u0026thinsp;0.0001, ** p\u0026thinsp;\u0026le;\u0026thinsp;0.01, * p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; ns\u0026thinsp;\u0026ge;\u0026thinsp;0.05. \u003cb\u003e(E)\u003c/b\u003e Scheme for lenvatinib/ROR1 CAR T cell treatments with multiple CAR T cell injections. 8505C cancer cells (3\u0026times;10⁶ cells/mouse) were injected into the right flank of NSG mice and grew until a median volume of 118.2 mm\u0026sup3; was reached. \u003cb\u003e(F)\u003c/b\u003e Mean tumour volume over time (n\u0026thinsp;=\u0026thinsp;4\u0026ndash;5 per group). \u003cb\u003e(G)\u003c/b\u003e Mean tumour volume on day 46 (end of control group), n\u0026thinsp;=\u0026thinsp;4\u0026ndash;5, mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, (** p\u0026thinsp;\u0026le;\u0026thinsp;0.01, * p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). \u003cb\u003e(H)\u003c/b\u003e Mean tumour volume on day 49 (end of lenvatinib group); n\u0026thinsp;=\u0026thinsp;4\u0026ndash;5, mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, (* p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). \u003cb\u003e(I)\u003c/b\u003e Experimental scheme to monitor tumour metastasis into different organs using the luciferase IVIS imaging system. Lung imaging results are shown in \u003cb\u003e(J)\u003c/b\u003e and liver results in \u003cb\u003e(K)\u003c/b\u003e from all treatment groups, other organs are depicted in Figure S15. N\u0026thinsp;=\u0026thinsp;3\u0026ndash;4, mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, one-way ANOVA (* p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ns\u0026thinsp;\u0026ge;\u0026thinsp;0.05). \u003cb\u003e(L)\u003c/b\u003e Quantified lung metastasis results are shown for day 46 (end of control groups) and day 49 (end of lenvatinib treatment groups); n\u0026thinsp;=\u0026thinsp;3\u0026ndash;4, mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, One-Way ANOVA (* p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). \u003cb\u003e(M)\u003c/b\u003e Analysis of circulating tumour cells by flow cytometry in the peripheral blood of NSG mice; n\u0026thinsp;=\u0026thinsp;3\u0026ndash;4, unpaired t-test (** p\u0026thinsp;\u0026le;\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003e \u003cb\u003eLenvatinib enhances CAR T cell quantity and activity\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo understand, how lenvatinib affects ROR1 CAR T cell activity \u003cem\u003ein vivo\u003c/em\u003e in our ATC mouse models, we monitored human ROR1 CAR T cells in the peripheral blood of NSG mice via flow cytometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Lenvatinib significantly enhanced the quantity of ROR1 CAR T cells circulating in the peripheral blood of xenografted mice compared to ROR1 CAR T cell treatment alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Then, we investigated the activation and differentiation profile of the circulating CAR T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). In concordance with the \u003cem\u003ein vitro\u003c/em\u003e data, CAR T cells in the lenvatinib treated group showed enhanced expression of activation markers (CD25, CD137) and reduced expression of exhaustion markers (CD279, CD366) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD-G). Furthermore, compared to the CAR T cells only group the frequency of circulating CAR T cells in the lenvatinib group shifted towards increased numbers of more differentiated CAR T cells like effector T cells (from 0.8*10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e% to 6*10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e%, 7 fold increase), effector memory T cells (from 2*10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e% to 4.3*10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e%, 2 fold increase) and central memory (from 0.7*10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e% to 5.9*10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e%, 8 fold increase) T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH-K).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e(A)\u003c/b\u003e Gating strategy for circulating ROR1 CAR T cells in the peripheral blood of ATC mice. \u003cb\u003e(B)\u003c/b\u003e Quantification of circulating ROR1 CAR T cells in the peripheral blood, n\u0026thinsp;=\u0026thinsp;7 per group, mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, unpaired t-test (*** p\u0026thinsp;\u0026le;\u0026thinsp;0.001). \u003cb\u003e(C)\u003c/b\u003e Surface markers determining activation and exhaustion profiles of circulating CAR T cells. \u003cb\u003e(D-G)\u003c/b\u003e Analysis of activation and exhaustion surface markers of circulating ROR1 CAR T cells in mice with or without lenvatinib treatment; n\u0026thinsp;=\u0026thinsp;7, mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, unpaired t-test (**** p\u0026thinsp;\u0026le;\u0026thinsp;0.0001, ** p\u0026thinsp;\u0026le;\u0026thinsp;0.01, ns p\u0026thinsp;\u0026ge;\u0026thinsp;0.05). \u003cb\u003e(H)\u003c/b\u003e Differentiation profile of circulating CAR T cells determined by different surface markers. T\u003csub\u003eN\u003c/sub\u003e \u0026ndash;na\u0026iuml;ve T cells; T\u003csub\u003eE\u003c/sub\u003e \u0026ndash; effector T cells; T\u003csub\u003eEM\u003c/sub\u003e \u0026ndash; effector memory T cells; T\u003csub\u003eCM\u003c/sub\u003e \u0026ndash; central memory T cells. \u003cb\u003e(I-K)\u003c/b\u003e Quantity of circulating CAR T cells in different differentiation stages with and without lenvatinib treatment; n\u0026thinsp;=\u0026thinsp;7, mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, unpaired t-test (**** p\u0026thinsp;\u0026le;\u0026thinsp;0.0001, *** p\u0026thinsp;\u0026le;\u0026thinsp;0.001).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eATC is a devastating disease and despite great progress with immune-kinase inhibitor therapies the majority of patients die due to cervical tumour progression or metastasis into critical organs. CAR T cell therapies at least in hematologic malignancies hold the promise to induce complete tumour regression in part of the patients and can prevent tumour relapse over a long time period due to the formation of memory CAR T cells.\u003csup\u003e48,49\u003c/sup\u003e In contrast to hematologic malignancies, the effect of CAR T cells in solid tumours is quite limited. Best responses are seen in glioblastoma \u003csup\u003e50\u003c/sup\u003e, papillomavirus-associated cervical cancer \u003csup\u003e51,52\u003c/sup\u003e, lung cancer \u003csup\u003e53\u003c/sup\u003e, breast cancer \u003csup\u003e54\u003c/sup\u003e and several other entities, but effects are often short-lived and more of preventive nature. To achieve target specificity in solid tumours, CAR T cell antigens are chosen either due to a specific expression of receptors/surface proteins on tumour cells, tumour-specific integrated viral proteins \u003csup\u003e55\u003c/sup\u003e or by targeting specific mutations (KRAS) \u003csup\u003e56\u003c/sup\u003e, which are presented in a MHC-dependent manner.\u003c/p\u003e \u003cp\u003eOur CAR T cell target ROR1 is a transmembrane receptor, which is expressed during embryonic development, but is lost in most adult tissues with the exception of low levels on the parathyroid, a subset of B cell progenitors and alveolar type 1 cells.\u003csup\u003e57,58\u003c/sup\u003e It is re-expressed on several aggressive tumour entities, like lung cancer or triple negative breast cancer\u003csup\u003e57,59\u003c/sup\u003e and on some B cell malignancies.\u003csup\u003e59\u0026ndash;61\u003c/sup\u003e By IHC staining, RNA and surface expression we could show that ROR1 is also strongly overexpressed in ATCs, while absent in normal thyroid tissue and expressed on a much lower level in differentiated thyroid carcinoma (DTC). Highest ROR1 levels are associated with the most undifferentiated, stem-cell like ATC variant and are associated with a significantly shorter survival in ATC/PDTC patients implicating ROR1 as a marker for a highly aggressive disease course. ROR1 was not only overexpressed in primary patient tissue, but also on ATC cell lines, independent of the mutation profile, and its expression was sustained \u003cem\u003ein vivo\u003c/em\u003e on circulating tumour cells in mouse xenografts. Interestingly, ROR1 expression seems to be higher on BRAF-mutated tumours, which are known to have a more aggressive disease course and an increased metastasis potential compared to RAS-mutated tumours. ROR1 CAR T cells effectively eliminated ATC cell lines \u003cem\u003ein vitro\u003c/em\u003e in 2D and 3D cultures and successfully blocked tumour development \u003cem\u003ein vivo\u003c/em\u003e, in both KRAS- and BRAF-mutated ATC cell lines. Despite this encouraging results, one-time injected CAR T cells were not able to deplete already established ATC tumours with a tumour volume exceeding 100 mm\u003csup\u003e3\u003c/sup\u003e. The CAR T cell effect could be improved by repeated injection of CAR T cells once every week resulting in a significant tumour size reduction of the already established tumour. The advantage of multiple CAR T injections was previously shown in various models in mice and humans. Multiple CAR T injections can mimic an ongoing immune response in immunodeficient mice with improved efficacy against hematologic malignancies and solid tumours \u003csup\u003e62\u0026ndash;65\u003c/sup\u003e, but also enhances CAR T cell efficiency in immunocompetent mice.\u003csup\u003e66,67\u003c/sup\u003e Repeated application of CAR T cells is also a clinically relevant strategy that has already been successfully explored in patients with hematologic malignancies \u003csup\u003e68,69\u003c/sup\u003e and solid tumours like glioblastoma \u003csup\u003e70\u0026ndash;72\u003c/sup\u003e and is currently systemically investigated in a clinical trial with different solid tumour entities (NCT05239143). Additionally, manipulation of CAR T cells towards a longer life span, increased activity or better target recognition is surely an alternative to repeated CAR T injections.\u003c/p\u003e \u003cp\u003eBesides reduced tumour growth, the repeated CAR T injections also resulted in a more than 2-fold increase in circulating CAR T cells in the peripheral blood and consequently a more efficient reduction of circulating ROR1\u0026thinsp;+\u0026thinsp;ATC tumour cells. Previous studies have shown that circulating ROR1 high+ tumour cells have a highly invasive potential and are a major source for tumour metastases.\u003csup\u003e3,73,74\u003c/sup\u003e Consequently, the CAR T cell-induced reduction of circulating ROR1\u0026thinsp;+\u0026thinsp;ATC cells also efficiently blocked tumour metastases into the lung, liver, kidneys and other organs.\u003c/p\u003e \u003cp\u003eLenvatinib treatment is highly effective in DTC and is approved as first-line systemic treatment of this disease.\u003csup\u003e31,75\u003c/sup\u003e In ATC results are controversial with studies showing good responses and others with relatively low or absent clinical benefit.\u003csup\u003e76,77\u003c/sup\u003e In contrast, the combination of lenvatinib with an immune checkpoint inhibitor, like pembrolizumab, in ATCs is highly effective and often induces good and partially durable responses over several years.\u003csup\u003e9\u003c/sup\u003e In this context, lenvatinib not only reduces the vascularisation and growth of the already established tumours, but also improves the functionality of endogenous T cells and therefore enhances the efficacy of immune checkpoint inhibitor treatments in mice and humans.\u003csup\u003e34\u003c/sup\u003e Consequently, lenvatinib and other VEGFR inhibitors could also influence the functionality of CAR T cells. Compared to other VEGFR inhibitors, lenvatinib showed the most improved CAR T cell profile regarding T cell differentiation, proliferation, cytokine secretion, CAR T cell activation and exhaustion. In addition, lenvatinib treatment altered the composition of the TME with elimination of immunosuppressive CAF-S1 fibroblasts. CAF-S1 fibroblasts not only recruit immunosuppressive CD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e+\u003c/sup\u003e regulatory T cells (Tregs) and therefore diminish normal immune responses, but also promote tumour metastases by inducing epithelial-to-mesenchymal transition, migration and invasiveness of tumour cells and by remodelling the extracellular matrix.\u003csup\u003e78,79\u003c/sup\u003e So, the depletion of CAF-S1 fibroblasts by lenvatinib is an important additional step to improve the efficiency of CAR T cell therapies, which can be compromised by similar mechanisms as normal immune responses. Especially important is probably the reduced Treg recruitment to the tumour site, which enables normal CAR T activity and the reduced tightness of the tumour tissue to allow better CAR T cell invasion.\u003c/p\u003e \u003cp\u003eAs the CAR T cell profile, especially in the interplay of CD4 and CD8 CAR T cells was strongest improved with lenvatinib and due to our excellent experiences of lenvatinib in combination with immune checkpoint inhibitors in patients, lenvatinib was chosen as primary combination partner for ROR1 CAR T cells \u003cem\u003ein vivo\u003c/em\u003e. Lenvatinib monotherapy in mice reduced the growth of already established tumours, but the effect was only temporary and the tumour size still increased strongly over time. Furthermore, lenvatinib alone had no protective effect on the quantity of circulating tumour cells and the establishment of tumour metastases. This is a very interesting phenomenon and might be the reason why lenvatinib monotherapies in ATC patients are often short lived and while the primary tumour growth can be stopped, the patient still develops new metastatic lesions at other sites, which results in the death of the patient. The combination of lenvatinib with ROR1 CAR T cells was most effective in all aspects of tumour development and could completely block primary tumour growth, strongly reduced tumour metastases, enhanced CAR T cell functionality and quantity, reduced numbers of circulating tumour cells and also improved the general health condition of treated mice.\u003c/p\u003e \u003cp\u003eA previous phase I clinical trial in humans with ROR1 CAR T cells clearly showed the restrictions of the approach in humans and strongly supports the ideas of repeated CAR T cell injections and the search for combination partners. Although CLL and solid tumours had similar levels of ROR1 expression on the cell surface, 2/3 of the CLL patients showed a partial response to the ROR1 CAR T cell treatment. From 18 patients with either lung cancer or triple-negative breast cancer only one had a partial remission, but died from severe pulmonary side effects and hypoxia. Most patients showed no response and lacked infiltration of CAR T cells into the tumour tissue. Furthermore, in solid tumour patients, the CAR T cells often did not expand and showed an exhausted phenotype.\u003csup\u003e80\u0026ndash;82\u003c/sup\u003e Our data from this study, but also from patients, show that lenvatinib can improve many of these aspects and has a very good clinical activity in ATC. Therefore we aim to perform a clinical trial, where we combine lenvatinib and ROR1 CAR T cells in ROR1\u0026thinsp;+\u0026thinsp;ATC patients.\u003csup\u003e29\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eTaken together, our experiments identified ROR1 as the first specific CAR T cell target in ATC, which is also expressed on circulating tumour cells and we found lenvatinib as an ideal combination partner to block tumour growth, to improve CAR T cell functionality and to inhibit microenvironment-induced immunosuppression.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eExperimental Design\u003c/h2\u003e \u003cp\u003eThe objective of the study was to identify a specific and innovative CAR T cell target for the treatment of anaplastic thyroid carcinoma and to find tyrosine kinase inhibitors which enhance CAR T cell activity and anti-tumour efficacy. By using gene expression studies, IHC stainings of ATC patient tissues and flow cytometry analysis, we identified ROR1 to be specifically overexpressed in ATC compared to normal thyroid tissue. Its expression was also conserved in ATC cell lines (ROR1 surface expression by flow cytometry, qPCR). ROR1 CAR T cells were highly efficient in lysing ATC cell lines in 2D and 3D co-cultures (luciferase/SRB-based viability assays and T cell cytokine profile) and in ATC xenograft mouse models, and eliminated circulating tumour cells (measured by flow cytometry) resulting in a significant reduction in tumour metastases (bioluminescence imaging). ROR1-KO cell lines were generated via CRISPR/Cas9 in order to analyse ROR1-CAR T cell specificity. Fast growing already established tumours could not be eliminated by ROR1 CAR T cells only (volume measurements, bioluminescence imaging), due to lack of infiltration of the CAR T cells into the tight tumour tissue (IHC for CD3 and RFP labelled CAR T cells). TKIs (Sorafenib, cabozantinib and lenvatinib) were tested to improve CAR T cell efficacy (RNAseq, proliferation and differentiation profile of CAR T cells in co-cultures treated with kinase inhibitors), and we identified lenvatinib to improve the CAR T cell activation and differentiation profile, to enhance anti-tumour efficiency in mouse models and to reduce immunosuppressive CAF-S1 cells in the tumour microenvironment (in vitro outgrowth of cancer associated fibroblasts from ATC patient tissues and treatment with lenvatinib).\u003c/p\u003e \u003cp\u003eIn vitro experiments were performed with at least three biological replicates of primary cells and/or cell lines to perform nonparametric statistical analyses (as specified in figure legends). In vivo experiments were performed with previously established numbers of mice by statistical analyses powered to highlight differences in efficacy and accounting for variability observed in previous experiments. Tumour burden was used to randomize mice into treatment groups. All of the treatment groups included at least three, but mostly five experimental mice to perform nonparametric statistical analysis (as specified in figure legends). All continuous variables are graphically represented as median and individual data points.\u003c/p\u003e \u003cp\u003eDetailed experiment designs and exact biological replicate numbers are described in the figure legends or Supplementary Materials and Methods.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eNGS analysis of ATC samples\u003c/h2\u003e \u003cp\u003e Fresh frozen material from ATC (n\u0026thinsp;=\u0026thinsp;12) and goiter (n\u0026thinsp;=\u0026thinsp;8) patients from the Department of Visceral, Vascular and Endocrine Surgery of the University Hospital of Halle (Saale, Germany) was collected after written informed consent (ethic: 2019-037, patients characteristics table S1). Samples were homogenized using the GentleMACS with M tubes (Miltenyi Biotec, Bergisch Gladbach, Germany) and RNA was isolated using the AllPrep DNA/RNA Mini Kit (Qiagen) according to the manufacturers\u0026rsquo; instructions. RNA sequencing (RNAseq) was performed by Novogene Co (Munich, Germany). A detailed description for RNAseq is provided in Supplement (S1). Data from gene expression analysis was uploaded on GEO database (GSE298106).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eROR1-targeting CAR T cells\u003c/h3\u003e\n\u003cp\u003eThe CAR construct (ROR1_41BB_CD3zeta_EGFRt) is a lentiviral plasmid vector encoding a second generation anti-ROR1 CAR in cis with the truncated epidermal growth factor receptor (EGFR) under control of EF1α promotor with T2A self-cleaving peptide sequence.\u003csup\u003e25\u003c/sup\u003e The engineered CAR T cells transduced with the ROR1 targeting construct were kindly provided by Prof Dr Michael Hudecek and Dr. Miriam Alb from the University Hospital of W\u0026uuml;rzburg. Cryopreserved ROR1 CAR T cells were thawed, washed, incubated with DNase I (0.1 mg/mL (Sigma Aldrich) for 10 minutes at room temperature (RT) and then incubated for 48 hours (hrs) in RPMI 1640 with HEPES containing 10% (v/v) human serum (Sigma Aldrich), 1% (v/v) penicillin, streptomycin, 0.1% (v/v) 2-mercaptoethanol (Sigma Aldrich) and with 10 U/mL IL-2 (Miltenyi Biotec) for the first 24 hrs. Then, CAR T cells were washed, counted with trypan blue and used for the \u003cem\u003ein vitro\u003c/em\u003e experiments.\u003c/p\u003e \u003cp\u003eFor the \u003cem\u003ein vivo\u003c/em\u003e experiments, the CAR T cells were washed, incubated with DNase I (0.1 mg/mL) for 10 minutes at RT, washed again and resuspended in PBS at the target concentration.\u003c/p\u003e\n\u003ch3\u003eIn vivo studies\u003c/h3\u003e\n\u003cp\u003eAll experiments were approved by the Landesverwaltungsamt Sachsen-Anhalt (203.m-42502-2-1725 MLU) and performed according to directive 2010/63/EU. 5\u0026ndash;7 weeks old NSG male mice (NOD.Cg-Prkdc\u003csup\u003escid\u003c/sup\u003e Il2rg\u003csup\u003etm1Sug\u003c/sup\u003e/JicTac) were purchased from Charles River (Germany). 3\u0026times;10\u003csup\u003e6\u003c/sup\u003e luciferase-expressing 8505C ATC cells were subcutaneously injected into the left flank region or 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e luciferase-expressing HRAS-mutated C643 ATC cells were injected intravenously into the ophthalmic venous sinus. For early treatment experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) 5\u0026times;10\u003csup\u003e6\u003c/sup\u003e CAR T cells (2.5\u0026times;10\u003csup\u003e6\u003c/sup\u003e CAR T CD4 and 2.5\u0026times;10\u003csup\u003e6\u003c/sup\u003e CAR T CD8) resuspended in 100 \u0026micro;L sterile PBS were injected intravenously (i.v.) into the tail vein 2 to 7 days later, or for experiments with established tumours on day 18 or later. For lenvatinib/CAR T combinations, lenvatinib was resuspended in 0.5% sterile filtered methylcellulose and administered by oral gavage 18 days after tumour cell inoculation at 5 mg/kg body weight. Body weight and health inspections were performed every day. Tumour volume was measured each week via calliper and calculated as follows: \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:V=0.5\\times\\:L\\times\\:{W}^{2}\\)\u003c/span\u003e\u003c/span\u003e (L\u0026thinsp;=\u0026thinsp;tumour length; W\u0026thinsp;=\u0026thinsp;tumour width). At the end of the experiment, the tumours were excised, weighed and stored in formalin for IHC.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eStatistical evaluation was performed using GraphPad Prism statistical software 9.5.1. Comparisons between two groups were analyzed using an unpaired two-tailed Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e test. For multiple group comparison one-way analysis of variance (ANOVA) was applied with a Tukey posttest. Results from bar charts are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. The results were considered significantly different if p values are \u0026lt;\u0026thinsp;0.05 and are represented as follows: *, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.01; ***, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.001 and ****, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.0001. Non-significant p values are shown as n.s. (p\u0026thinsp;\u0026ge;\u0026thinsp;0.05). In each graph, the number of individual experimental points are described.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eDisclosures:\u003c/h2\u003e \u003cp\u003eThe authors declare no competing financial interests.\u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eContribution: O.S. and D.C. performed the experiments. O.S. and D.C. analyzed the data. T.M. and D.B. helped to carry out the \u003cem\u003ein vivo\u003c/em\u003e experiments. S.H. and T. B. analyzed the data. B.T. and K.L. provided thyroid carcinoma and goiter samples. M.A. provided the ROR1 CAR T cells. E.W. helped with RNASeq analysis. M.B. and A.W. performed IHC staining. B.S. and M.U. provided help for fibroblast analysis. D.B. reviewed the manuscript. O.S., D.C., B.E., K.M. and C.D. designed the research. O.S., D.C., M.H. and C.D. wrote the paper. All authors approved the final version of this manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis work was supported by the Wilhelm-Roux program round 32 - NTIEN: Novel Targets and Immunotherapies for Endocrine Neoplasms from the university medicine Halle (Saale). It was further supported by Deutsche Krebshilfe via grants 111025 and 70112614. The work was further supported by the DFG through the Emmy-Noether program from Christine Dierks (DI 1664/1\u0026ndash;1), through DFG grants from FOR 2033 DI 1664/2\u0026ndash;2 and FOR DI 1664/3\u0026thinsp;\u0026minus;\u0026thinsp;1, and FOR5659 517204983 to Christine Dierks. The work was further supported by the Jos\u0026eacute;-Carreras grant DJCLS 10 R/2022It was further supported by the \u0026ldquo;Polyfaces-initative/Land Saxony-Anhalt, EMB, \u0026ldquo;Vorbereitung Cluster im Rahmen der Exzellenzstrategie des Bundes: Polymere-Nachhaltigkeit\u0026rdquo; as well as by the European Union and the State Saxony-Anhalt through the Thera4Age project (grant ZS/2023/12/182764). We thank our core facilities, especially Alexander Navarrete Santos for the help with flow cytometry and sorting as well as Nadine Bley for the use of the live cell imaging system.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrespondence\u003c/strong\u003e: Prof. Dr. Christine Dierks, University of Halle-Wittenberg, Department of Hematology/Oncology, Ernst-Grube-Str. 40, 06120 Halle (Saale), Germany; e-mail: [email protected]\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSmallridge RC, Ain KB, Asa SL, et al. American Thyroid Association guidelines for management of patients with anaplastic thyroid cancer. \u003cem\u003eThyroid\u003c/em\u003e. Nov 2012;22(11):1104-39. doi:10.1089/thy.2012.0302\u003c/li\u003e\n\u003cli\u003ePrasongsook N, Kumar A, Chintakuntlawar AV, et al. Survival in Response to Multimodal Therapy in Anaplastic Thyroid Cancer. \u003cem\u003eJ Clin Endocrinol Metab\u003c/em\u003e. Dec 1 2017;102(12):4506-4514. doi:10.1210/jc.2017-01180\u003c/li\u003e\n\u003cli\u003eCui B, Zhang S, Chen L, et al. Targeting ROR1 inhibits epithelial\u0026ndash;mesenchymal transition and metastasis. \u003cem\u003eCancer research\u003c/em\u003e. 2013;73(12):3649-3660.\u003c/li\u003e\n\u003cli\u003eTakano T, Ito Y, Hirokawa M, Yoshida H, Miyauchi A. BRAFV600E mutation in anaplastic thyroid carcinomas and their accompanying differentiated carcinomas. \u003cem\u003eBritish journal of cancer\u003c/em\u003e. 2007;96(10):1549-1553.\u003c/li\u003e\n\u003cli\u003eXu B, Fuchs T, Dogan S, et al. Dissecting anaplastic thyroid carcinoma: a comprehensive clinical, histologic, immunophenotypic, and molecular study of 360 cases. \u003cem\u003eThyroid\u003c/em\u003e. 2020;30(10):1505-1517.\u003c/li\u003e\n\u003cli\u003ePozdeyev N, Gay LM, Sokol ES, et al. Genetic analysis of 779 advanced differentiated and anaplastic thyroid cancers. \u003cem\u003eClinical Cancer Research\u003c/em\u003e. 2018;24(13):3059-3068.\u003c/li\u003e\n\u003cli\u003eLanda I, Ibrahimpasic T, Boucai L, et al. Genomic and transcriptomic hallmarks of poorly differentiated and anaplastic thyroid cancers. \u003cem\u003eThe Journal of clinical investigation\u003c/em\u003e. 2016;126(3):1052-1066.\u003c/li\u003e\n\u003cli\u003eAdam P, Kircher S, Sbiera I, et al. FGF-Receptors and PD-L1 in Anaplastic and Poorly Differentiated Thyroid Cancer: Evaluation of the Preclinical Rationale. \u003cem\u003eFront Endocrinol (Lausanne)\u003c/em\u003e. 2021;12:712107. doi:10.3389/fendo.2021.712107\u003c/li\u003e\n\u003cli\u003eDierks C, Seufert J, Aumann K, et al. Combination of Lenvatinib and Pembrolizumab Is an Effective Treatment Option for Anaplastic and Poorly Differentiated Thyroid Carcinoma. \u003cem\u003eThyroid\u003c/em\u003e. Jul 2021;31(7):1076-1085. doi:10.1089/thy.2020.0322\u003c/li\u003e\n\u003cli\u003ePrasad V. Tisagenlecleucel\u0026mdash;the first approved CAR-T-cell therapy: implications for payers and policy makers. \u003cem\u003eNature Reviews Clinical Oncology\u003c/em\u003e. 2018;15(1):11-12.\u003c/li\u003e\n\u003cli\u003eWang V, Gauthier M, Decot V, Reppel L, Bensoussan D. Systematic Review on CAR-T Cell Clinical Trials Up to 2022: Academic Center Input. \u003cem\u003eCancers (Basel)\u003c/em\u003e. Feb 4 2023;15(4)doi:10.3390/cancers15041003\u003c/li\u003e\n\u003cli\u003eDaei Sorkhabi A, Mohamed Khosroshahi L, Sarkesh A, et al. The current landscape of CAR T-cell therapy for solid tumors: Mechanisms, research progress, challenges, and counterstrategies. \u003cem\u003eFrontiers in immunology\u003c/em\u003e. 2023;14:1113882.\u003c/li\u003e\n\u003cli\u003eLi H, Zhou X, Wang G, et al. CAR-T Cells Targeting TSHR Demonstrate Safety and Potent Preclinical Activity Against Differentiated Thyroid Cancer. \u003cem\u003eJ Clin Endocrinol Metab\u003c/em\u003e. Mar 24 2022;107(4):1110-1126. doi:10.1210/clinem/dgab819\u003c/li\u003e\n\u003cli\u003eHasan MK, Rassenti L, Widhopf GF, 2nd, Yu J, Kipps TJ. Wnt5a causes ROR1 to complex and activate cortactin to enhance migration of chronic lymphocytic leukemia cells. \u003cem\u003eLeukemia\u003c/em\u003e. Mar 2019;33(3):653-661. doi:10.1038/s41375-018-0306-7\u003c/li\u003e\n\u003cli\u003eZhang S, Chen L, Cui B, et al. ROR1 is expressed in human breast cancer and associated with enhanced tumor-cell growth. \u003cem\u003ePloS one\u003c/em\u003e. 2012;7(3):e31127.\u003c/li\u003e\n\u003cli\u003eRebagay G, Yan S, Liu C, Cheung N-K. ROR1 and ROR2 in human malignancies: potentials for targeted therapy. \u003cem\u003eFrontiers in oncology\u003c/em\u003e. 2012;2:34.\u003c/li\u003e\n\u003cli\u003eHudecek M, Schmitt TM, Baskar S, et al. The B-cell tumor\u0026ndash;associated antigen ROR1 can be targeted with T cells modified to express a ROR1-specific chimeric antigen receptor. \u003cem\u003eBlood, The Journal of the American Society of Hematology\u003c/em\u003e. 2010;116(22):4532-4541.\u003c/li\u003e\n\u003cli\u003eBorcherding N, Kusner D, Liu G-H, Zhang W. ROR1, an embryonic protein with an emerging role in cancer biology. \u003cem\u003eProtein \u0026amp; Cell\u003c/em\u003e. 2014;5(7):496-502. doi:10.1007/s13238-014-0059-7\u003c/li\u003e\n\u003cli\u003eKatoh M, Katoh M. WNT signaling pathway and stem cell signaling network. \u003cem\u003eClinical cancer research\u003c/em\u003e. 2007;13(14):4042-4045.\u003c/li\u003e\n\u003cli\u003eParsons MJ, Tammela T, Dow LE. WNT as a driver and dependency in cancer. \u003cem\u003eCancer discovery\u003c/em\u003e. 2021;11(10):2413-2429.\u003c/li\u003e\n\u003cli\u003eMenck K, Heinrichs S, Baden C, Bleckmann A. The WNT/ROR pathway in cancer: from signaling to therapeutic intervention. \u003cem\u003eCells\u003c/em\u003e. 2021;10(1):142.\u003c/li\u003e\n\u003cli\u003eMasoumi J, Jafarzadeh A, Abdolalizadeh J, et al. Cancer stem cell-targeted chimeric antigen receptor (CAR)-T cell therapy: Challenges and prospects. \u003cem\u003eActa Pharm Sin B\u003c/em\u003e. Jul 2021;11(7):1721-1739. doi:10.1016/j.apsb.2020.12.015\u003c/li\u003e\n\u003cli\u003eJune CH, O\u0026rsquo;Connor RS, Kawalekar OU, Ghassemi S, Milone MC. CAR T cell immunotherapy for human cancer. \u003cem\u003eScience\u003c/em\u003e. 2018;359(6382):1361-1365.\u003c/li\u003e\n\u003cli\u003eWallstabe L, G\u0026ouml;ttlich C, Nelke LC, et al. ROR1-CAR T cells are effective against lung and breast cancer in advanced microphysiologic 3D tumor models. \u003cem\u003eJCI insight\u003c/em\u003e. 2019;4(18)\u003c/li\u003e\n\u003cli\u003eHudecek M, Lupo-Stanghellini M-T, Kosasih PL, et al. Receptor affinity and extracellular domain modifications affect tumor recognition by ROR1-specific chimeric antigen receptor T cells. \u003cem\u003eClinical cancer research\u003c/em\u003e. 2013;19(12):3153-3164.\u003c/li\u003e\n\u003cli\u003eSt\u0026uuml;ber T, Monjezi R, Wallstabe L, et al. Inhibition of TGF-\u0026beta;-receptor signaling augments the antitumor function of ROR1-specific CAR T-cells against triple-negative breast cancer. \u003cem\u003eJournal for immunotherapy of cancer\u003c/em\u003e. 2020;8(1)\u003c/li\u003e\n\u003cli\u003eBerger C, Sommermeyer D, Hudecek M, et al. Safety of targeting ROR1 in primates with chimeric antigen receptor-modified T cells. \u003cem\u003eCancer immunology research\u003c/em\u003e. 2015;3(2):206-216. doi:10.1158/2326-6066.CIR-14-0163\u003c/li\u003e\n\u003cli\u003eMaulana TI, Teufel C, Cipriano M, et al. Breast cancer-on-chip for patient-specific efficacy and safety testing of CAR-T cells. \u003cem\u003eCell Stem Cell\u003c/em\u003e. 2024;31(7):989-1002. e9.\u003c/li\u003e\n\u003cli\u003eJaeger-Ruckstuhl CA, Specht JM, Voutsinas JM, et al. Phase I Study of ROR1-Specific CAR-T Cells in Advanced Hematopoietic and Epithelial Malignancies. \u003cem\u003eClin Cancer Res\u003c/em\u003e. Feb 3 2025;31(3):503-514. doi:10.1158/1078-0432.CCR-24-2172\u003c/li\u003e\n\u003cli\u003eCabanillas ME, Brose MS, Holland J, Ferguson KC, Sherman SI. A phase I study of cabozantinib (XL184) in patients with differentiated thyroid cancer. \u003cem\u003eThyroid\u003c/em\u003e. Oct 2014;24(10):1508-14. doi:10.1089/thy.2014.0125\u003c/li\u003e\n\u003cli\u003eSchlumberger M, Tahara M, Wirth LJ, et al. Lenvatinib versus placebo in radioiodine-refractory thyroid cancer. \u003cem\u003eN Engl J Med\u003c/em\u003e. Feb 12 2015;372(7):621-30. doi:10.1056/NEJMoa1406470\u003c/li\u003e\n\u003cli\u003eStjepanovic N, Capdevila J. Multikinase inhibitors in the treatment of thyroid cancer: specific role of lenvatinib. \u003cem\u003eBiologics\u003c/em\u003e. 2014;8:129-39. doi:10.2147/BTT.S39381\u003c/li\u003e\n\u003cli\u003eThomas L, Lai SY, Dong W, et al. Sorafenib in metastatic thyroid cancer: a systematic review. \u003cem\u003eOncologist\u003c/em\u003e. Mar 2014;19(3):251-8. doi:10.1634/theoncologist.2013-0362\u003c/li\u003e\n\u003cli\u003eGunda V, Gigliotti B, Ashry T, et al. Anti-PD-1/PD-L1 therapy augments lenvatinib\u0026apos;s efficacy by favorably altering the immune microenvironment of murine anaplastic thyroid cancer. \u003cem\u003eInt J Cancer\u003c/em\u003e. May 1 2019;144(9):2266-2278. doi:10.1002/ijc.32041\u003c/li\u003e\n\u003cli\u003eShabani M, Naseri J, Shokri F. Receptor tyrosine kinase-like orphan receptor 1: a novel target for cancer immunotherapy. \u003cem\u003eExpert opinion on therapeutic targets\u003c/em\u003e. 2015;19(7):941-955.\u003c/li\u003e\n\u003cli\u003eZhao Y, Zhang D, Guo Y, et al. Tyrosine kinase ROR1 as a target for anti-cancer therapies. \u003cem\u003eFrontiers in Oncology\u003c/em\u003e. 2021;11:680834.\u003c/li\u003e\n\u003cli\u003eHudecek M, Schmitt TM, Baskar S, et al. The B-cell tumor-associated antigen ROR1 can be targeted with T cells modified to express a ROR1-specific chimeric antigen receptor. \u003cem\u003eBlood\u003c/em\u003e. Nov 25 2010;116(22):4532-41. doi:10.1182/blood-2010-05-283309\u003c/li\u003e\n\u003cli\u003eWallstabe L, Gottlich C, Nelke LC, et al. ROR1-CAR T cells are effective against lung and breast cancer in advanced microphysiologic 3D tumor models. \u003cem\u003eJCI Insight\u003c/em\u003e. Sep 19 2019;4(18)doi:10.1172/jci.insight.126345\u003c/li\u003e\n\u003cli\u003eDierks C, Ruf J, Seufert J, et al. 1646MO Phase II ATLEP trial: Final results for lenvatinib/pembrolizumab in metastasized anaplastic and poorly differentiated thyroid carcinoma. \u003cem\u003eAnnals of Oncology\u003c/em\u003e. 2022;33:S1295. doi:10.1016/j.annonc.2022.07.1726\u003c/li\u003e\n\u003cli\u003eChen Y, Dai S, Cheng C-s, Chen L. Lenvatinib and immune-checkpoint inhibitors in hepatocellular carcinoma: mechanistic insights, clinical efficacy, and future perspectives. \u003cem\u003eJournal of Hematology \u0026amp; Oncology\u003c/em\u003e. 2024/12/21 2024;17(1):130. doi:10.1186/s13045-024-01647-1\u003c/li\u003e\n\u003cli\u003eMei Z, Gao X, Pan C, et al. Lenvatinib enhances antitumor immunity by promoting the infiltration of TCF1(+) CD8(+) T cells in HCC via blocking VEGFR2. \u003cem\u003eCancer Sci\u003c/em\u003e. Apr 2023;114(4):1284-1296. doi:10.1111/cas.15719\u003c/li\u003e\n\u003cli\u003eKato Y, Tabata K, Kimura T, et al. Lenvatinib plus anti-PD-1 antibody combination treatment activates CD8+ T cells through reduction of tumor-associated macrophage and activation of the interferon pathway. \u003cem\u003ePloS one\u003c/em\u003e. 2019;14(2):e0212513. \u003c/li\u003e\n\u003cli\u003eLu M, Zhang X, Gao X, et al. Lenvatinib enhances T cell immunity and the efficacy of adoptive chimeric antigen receptor-modified T cells by decreasing myeloid-derived suppressor cells in cancer. \u003cem\u003ePharmacological Research\u003c/em\u003e. 2021;174:105829.\u003c/li\u003e\n\u003cli\u003eSubramanian A, Tamayo P, Mootha VK, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e. Oct 25 2005;102(43):15545-50. doi:10.1073/pnas.0506580102\u003c/li\u003e\n\u003cli\u003eMhaidly R, Mechta-Grigoriou F. Fibroblast heterogeneity in tumor micro-environment: Role in immunosuppression and new therapies. Elsevier; 2020:101417.\u003c/li\u003e\n\u003cli\u003eChen R, Li Q, Xu S, et al. Modulation of the tumour microenvironment in hepatocellular carcinoma by tyrosine kinase inhibitors: from modulation to combination therapy targeting the microenvironment. \u003cem\u003eCancer Cell International\u003c/em\u003e. 2022/02/11 2022;22(1):73. doi:10.1186/s12935-021-02435-4\u003c/li\u003e\n\u003cli\u003eDesbois M, Wang Y. Cancer‐associated fibroblasts: key players in shaping the tumor immune microenvironment. \u003cem\u003eImmunological reviews\u003c/em\u003e. 2021;302(1):241-258.\u003c/li\u003e\n\u003cli\u003eMcLellan AD, Ali Hosseini Rad SM. Chimeric antigen receptor T cell persistence and memory cell formation. \u003cem\u003eImmunol Cell Biol\u003c/em\u003e. Aug 2019;97(7):664-674. doi:10.1111/imcb.12254\u003c/li\u003e\n\u003cli\u003eDoan AE, Mueller KP, Chen AY, et al. FOXO1 is a master regulator of memory programming in CAR T cells. \u003cem\u003eNature\u003c/em\u003e. May 2024;629(8010):211-218. doi:10.1038/s41586-024-07300-8\u003c/li\u003e\n\u003cli\u003eBrown CE, Hibbard JC, Alizadeh D, et al. Locoregional delivery of IL-13Ralpha2-targeting CAR-T cells in recurrent high-grade glioma: a phase 1 trial. \u003cem\u003eNat Med\u003c/em\u003e. Apr 2024;30(4):1001-1012. doi:10.1038/s41591-024-02875-1\u003c/li\u003e\n\u003cli\u003eStevanovic S, Draper LM, Langhan MM, et al. Complete regression of metastatic cervical cancer after treatment with human papillomavirus-targeted tumor-infiltrating T cells. \u003cem\u003eJ Clin Oncol\u003c/em\u003e. May 10 2015;33(14):1543-50. doi:10.1200/JCO.2014.58.9093\u003c/li\u003e\n\u003cli\u003eStevanovic S, Helman SR, Wunderlich JR, et al. A Phase II Study of Tumor-infiltrating Lymphocyte Therapy for Human Papillomavirus-associated Epithelial Cancers. \u003cem\u003eClin Cancer Res\u003c/em\u003e. Mar 1 2019;25(5):1486-1493. doi:10.1158/1078-0432.CCR-18-2722\u003c/li\u003e\n\u003cli\u003eCreelan BC, Wang C, Teer JK, et al. Tumor-infiltrating lymphocyte treatment for anti-PD-1-resistant metastatic lung cancer: a phase 1 trial. \u003cem\u003eNat Med\u003c/em\u003e. Aug 2021;27(8):1410-1418. doi:10.1038/s41591-021-01462-y\u003c/li\u003e\n\u003cli\u003eZacharakis N, Chinnasamy H, Black M, et al. Immune recognition of somatic mutations leading to complete durable regression in metastatic breast cancer. \u003cem\u003eNat Med\u003c/em\u003e. Jun 2018;24(6):724-730. doi:10.1038/s41591-018-0040-8\u003c/li\u003e\n\u003cli\u003eComoli P, Pedrazzoli P, Maccario R, et al. Cell therapy of stage IV nasopharyngeal carcinoma with autologous Epstein-Barr virus-targeted cytotoxic T lymphocytes. \u003cem\u003eJ Clin Oncol\u003c/em\u003e. Dec 10 2005;23(35):8942-9. doi:10.1200/JCO.2005.02.6195\u003c/li\u003e\n\u003cli\u003eTran E, Robbins PF, Lu YC, et al. T-Cell Transfer Therapy Targeting Mutant KRAS in Cancer. \u003cem\u003eN Engl J Med\u003c/em\u003e. Dec 8 2016;375(23):2255-2262. doi:10.1056/NEJMoa1609279\u003c/li\u003e\n\u003cli\u003eBalakrishnan A, Goodpaster T, Randolph-Habecker J, et al. Analysis of ROR1 Protein Expression in Human Cancer and Normal Tissues. \u003cem\u003eClin Cancer Res\u003c/em\u003e. Jun 15 2017;23(12):3061-3071. doi:10.1158/1078-0432.CCR-16-2083\u003c/li\u003e\n\u003cli\u003eAl-Shawi R, Ashton SV, Underwood C, Simons JP. Expression of the Ror1 and Ror2 receptor tyrosine kinase genes during mouse development. \u003cem\u003eDev Genes Evol\u003c/em\u003e. Apr 2001;211(4):161-71. doi:10.1007/s004270100140\u003c/li\u003e\n\u003cli\u003eBaskar S, Kwong KY, Hofer T, et al. Unique cell surface expression of receptor tyrosine kinase ROR1 in human B-cell chronic lymphocytic leukemia. \u003cem\u003eClin Cancer Res\u003c/em\u003e. Jan 15 2008;14(2):396-404. doi:10.1158/1078-0432.CCR-07-1823\u003c/li\u003e\n\u003cli\u003eCui B, Ghia EM, Chen L, et al. High-level ROR1 associates with accelerated disease progression in chronic lymphocytic leukemia. \u003cem\u003eBlood\u003c/em\u003e. Dec 22 2016;128(25):2931-2940. doi:10.1182/blood-2016-04-712562\u003c/li\u003e\n\u003cli\u003eBroome HE, Rassenti LZ, Wang HY, Meyer LM, Kipps TJ. ROR1 is expressed on hematogones (non-neoplastic human B-lymphocyte precursors) and a minority of precursor-B acute lymphoblastic leukemia. \u003cem\u003eLeuk Res\u003c/em\u003e. Oct 2011;35(10):1390-4. doi:10.1016/j.leukres.2011.06.021\u003c/li\u003e\n\u003cli\u003eZhao Y, Moon E, Carpenito C, et al. Multiple injections of electroporated autologous T cells expressing a chimeric antigen receptor mediate regression of human disseminated tumor. \u003cem\u003eCancer Res\u003c/em\u003e. Nov 15 2010;70(22):9053-61. doi:10.1158/0008-5472.CAN-10-2880\u003c/li\u003e\n\u003cli\u003eLiu M, Wang X, Li W, et al. Targeting PD-L1 in non-small cell lung cancer using CAR T cells. \u003cem\u003eOncogenesis\u003c/em\u003e. Aug 13 2020;9(8):72. doi:10.1038/s41389-020-00257-z\u003c/li\u003e\n\u003cli\u003eLuo Y, Gadd ME, Qie Y, et al. Solid cancer-directed CAR T cell therapy that attacks both tumor and immunosuppressive cells via targeting PD-L1. \u003cem\u003eMol Ther Oncol\u003c/em\u003e. Dec 19 2024;32(4):200891. doi:10.1016/j.omton.2024.200891\u003c/li\u003e\n\u003cli\u003eAltvater B, Kailayangiri S, Spurny C, et al. CAR T cells as micropharmacies against solid cancers: Combining effector T-cell mediated cell death with vascular targeting in a one-step engineering process. \u003cem\u003eCancer Gene Ther\u003c/em\u003e. Oct 2023;30(10):1355-1368. doi:10.1038/s41417-023-00642-x\u003c/li\u003e\n\u003cli\u003eKlampatsa A, Leibowitz MS, Sun J, Liousia M, Arguiri E, Albelda SM. Analysis and Augmentation of the Immunologic Bystander Effects of CAR T Cell Therapy in a Syngeneic Mouse Cancer Model. \u003cem\u003eMol Ther Oncolytics\u003c/em\u003e. Sep 25 2020;18:360-371. doi:10.1016/j.omto.2020.07.005\u003c/li\u003e\n\u003cli\u003eXie YJ, Dougan M, Jailkhani N, et al. Nanobody-based CAR T cells that target the tumor microenvironment inhibit the growth of solid tumors in immunocompetent mice. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e. Apr 16 2019;116(16):7624-7631. doi:10.1073/pnas.1817147116\u003c/li\u003e\n\u003cli\u003eDeng L, Xiaolin Y, Wu Q, et al. Multiple CAR-T cell therapy for acute B-cell lymphoblastic leukemia after hematopoietic stem cell transplantation: A case report. \u003cem\u003eFront Immunol\u003c/em\u003e. 2022;13:1039929. doi:10.3389/fimmu.2022.1039929\u003c/li\u003e\n\u003cli\u003eJiao C, Zvonkov E, Lai X, et al. 4SCAR2.0: a multi-CAR-T therapy regimen for the treatment of relapsed/refractory B cell lymphomas. \u003cem\u003eBlood Cancer J\u003c/em\u003e. Mar 17 2021;11(3):59. doi:10.1038/s41408-021-00455-x\u003c/li\u003e\n\u003cli\u003eBrown CE, Alizadeh D, Starr R, et al. Regression of Glioblastoma after Chimeric Antigen Receptor T-Cell Therapy. \u003cem\u003eN Engl J Med\u003c/em\u003e. Dec 29 2016;375(26):2561-9. doi:10.1056/NEJMoa1610497\u003c/li\u003e\n\u003cli\u003eBagley SJ, Binder ZA, Lamrani L, et al. Repeated peripheral infusions of anti-EGFRvIII CAR T cells in combination with pembrolizumab show no efficacy in glioblastoma: a phase 1 trial. \u003cem\u003eNat Cancer\u003c/em\u003e. Mar 2024;5(3):517-531. doi:10.1038/s43018-023-00709-6\u003c/li\u003e\n\u003cli\u003eVitanza NA, Johnson AJ, Wilson AL, et al. Locoregional infusion of HER2-specific CAR T cells in children and young adults with recurrent or refractory CNS tumors: an interim analysis. \u003cem\u003eNat Med\u003c/em\u003e. Sep 2021;27(9):1544-1552. doi:10.1038/s41591-021-01404-8\u003c/li\u003e\n\u003cli\u003eXu GL, Shen J, Xu YH, Wang WS, Ni CF. ROR1 is highly expressed in circulating tumor cells and promotes invasion of pancreatic cancer. \u003cem\u003eMol Med Rep\u003c/em\u003e. Dec 2018;18(6):5087-5094. doi:10.3892/mmr.2018.9500\u003c/li\u003e\n\u003cli\u003eHasan MK, Widhopf GF, 2nd, Zhang S, et al. Wnt5a induces ROR1 to recruit cortactin to promote breast-cancer migration and metastasis. \u003cem\u003eNPJ Breast Cancer\u003c/em\u003e. 2019;5:35. doi:10.1038/s41523-019-0131-9\u003c/li\u003e\n\u003cli\u003eTohyama O, Matsui J, Kodama K, et al. Antitumor activity of lenvatinib (e7080): an angiogenesis inhibitor that targets multiple receptor tyrosine kinases in preclinical human thyroid cancer models. \u003cem\u003eJ Thyroid Res\u003c/em\u003e. 2014;2014:638747. doi:10.1155/2014/638747\u003c/li\u003e\n\u003cli\u003eTahara M, Kiyota N, Yamazaki T, et al. Lenvatinib for Anaplastic Thyroid Cancer. \u003cem\u003eFront Oncol\u003c/em\u003e. 2017;7:25. doi:10.3389/fonc.2017.00025\u003c/li\u003e\n\u003cli\u003eKiyota N, Schlumberger M, Muro K, et al. Subgroup analysis of Japanese patients in a phase 3 study of lenvatinib in radioiodine-refractory differentiated thyroid cancer. \u003cem\u003eCancer Sci\u003c/em\u003e. Dec 2015;106(12):1714-21. doi:10.1111/cas.12826\u003c/li\u003e\n\u003cli\u003eCosta A, Kieffer Y, Scholer-Dahirel A, et al. Fibroblast Heterogeneity and Immunosuppressive Environment in Human Breast Cancer. \u003cem\u003eCancer Cell\u003c/em\u003e. Mar 12 2018;33(3):463-479 e10. doi:10.1016/j.ccell.2018.01.011\u003c/li\u003e\n\u003cli\u003ePelon F, Bourachot B, Kieffer Y, et al. Cancer-associated fibroblast heterogeneity in axillary lymph nodes drives metastases in breast cancer through complementary mechanisms. \u003cem\u003eNat Commun\u003c/em\u003e. Jan 21 2020;11(1):404. doi:10.1038/s41467-019-14134-w\u003c/li\u003e\n\u003cli\u003eChen T, Wang M, Chen Y, Liu Y. Current challenges and therapeutic advances of CAR-T cell therapy for solid tumors. \u003cem\u003eCancer Cell International\u003c/em\u003e. 2024;24(1):133.\u003c/li\u003e\n\u003cli\u003eHou AJ, Chen LC, Chen YY. Navigating CAR-T cells through the solid-tumour microenvironment. \u003cem\u003eNature reviews Drug discovery\u003c/em\u003e. 2021;20(7):531-550.\u003c/li\u003e\n\u003cli\u003eSchurich A, Magalhaes I, Mattsson J. Metabolic regulation of CAR T cell function by the hypoxic microenvironment in solid tumors. \u003cem\u003eImmunotherapy\u003c/em\u003e. 2019;11(4):335-345.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8690827/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8690827/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAnaplastic thyroid carcinoma (ATC) is a rare thyroid malignancy with poor prognosis and very limited treatment options. Therefore, the development of novel therapies is urgently needed.\u003c/p\u003e \u003cp\u003eHere, we identified the Receptor Tyrosine Kinase like Orphan Receptor 1 (ROR1) protein as a specific CAR T cell target for ATC. ROR1 is part of the Wnt signalling pathway, mainly expressed during embryogenesis, but mostly absent in differentiated adult tissue. In ATCs, ROR1 is strongly overexpressed (RNA/protein level, surface expression) and high expression levels are associated with reduced survival. ROR1 CAR Ts specifically target ATC cell lines in 2D and 3D spheroid cultures, reduce the quantity of circulating tumour cells and block tumour metastases in different ATC mouse models. While small tumours were completely eliminated by ROR1 CAR T cells alone, larger tumours required the combination of ROR1 CAR T cells with the multikinase inhibitor lenvatinib. Lenvatinib blocked primary tumour growth, reduced the quantity of immunosuppressive cancer associated fibroblasts (CAF-S1) in the microenvironment and enhanced CAR T cell functionality and activation.\u003c/p\u003e \u003cp\u003eOverall, we validated ROR1 as a prime target for CAR T cell therapies in ATC and identified lenvatinib as highly valuable combination partner, which is able to improve CAR T cell functionality.\u003c/p\u003e","manuscriptTitle":"ROR1 CAR T cells and lenvatinib cooperatively target anaplastic thyroid carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-29 07:06:04","doi":"10.21203/rs.3.rs-8690827/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"01a3b7d9-27a3-4631-9a06-515f3118f4d9","owner":[],"postedDate":"January 29th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":61921414,"name":"Health sciences/Oncology/Cancer/Cancer therapy/Cancer immunotherapy"},{"id":61921415,"name":"Health sciences/Endocrinology/Endocrine system and metabolic diseases/Endocrine cancer"}],"tags":[],"updatedAt":"2026-03-03T11:00:50+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-29 07:06:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8690827","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8690827","identity":"rs-8690827","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}