The cGAS-STING pathway is a master regulator of OCT4 expression in persistent sarcoma cells and enhances cellular immunotherapy with NK and CIK lymphocytes

The cGAS-STING pathway is a master regulator of OCT4 expression in persistent sarcoma cells and enhances cellular immunotherapy with NK and CIK lymphocytes | 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 Research Article The cGAS-STING pathway is a master regulator of OCT4 expression in persistent sarcoma cells and enhances cellular immunotherapy with NK and CIK lymphocytes Giorgia Giordano, Alessandra Merlini, Federica Capozzi, Giulio Ferrero, and 16 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6463944/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Sep, 2025 Read the published version in Cancer Immunology, Immunotherapy → Version 1 posted 12 You are reading this latest preprint version Abstract Advanced sarcomas have poor prognosis and limited therapeutic options. Disease recurrence is caused by persistent cells that survive drug treatments. The alkylating agent trabectedin, when combined with the poly(ADP-ribose) polymerase 1 (PARP1) inhibitor olaparib, exhibits variable antitumor effects in advanced sarcomas. In this study, we demonstrated that the expression of the transcription factor OCT4 is upregulated in persistent cells surviving trabectedin and olaparib treatment through the cGAS-STING-IRF3-IFNβ pathway. This route also leads to the upregulation of natural killer (NK) and cytokine-induced killer (CIK) lymphocyte activating ligands. This upregulation promoted the antitumor effect of immunotherapy with NK and CIK cells against both bulk and persistent cells that survive drug treatment. In conclusion, activation of the cGAS-STING pathway due to trabectedin + olaparib treatment has a double-edged sword effect, enriching the OCT4 + persistent cell population while increasing the expression of NK/CIK ligands. Therefore, sequential treatment with trabectedin and olaparib followed by NK/CIK immunotherapy is a promising strategy against advanced sarcomas and deserves further investigation. NK/CIK cellular immunotherapy cGAS-STING-IRF3-IFNβ pathway trabectedin olaparib OCT4 persistent sarcoma cells INTRODUCTION Sarcomas are rare, heterogeneous, and malignant tumors of mesenchymal origin. Limited effective therapeutic options are available for the management of advanced, unresectable, or metastatic disease, and their prognosis remains poor [ 1 ]. We have previously demonstrated that the combination of the alkylating agent trabectedin and the poly (ADP-ribose) polymerase-1 (PARP1) inhibitor olaparib is active against a subset of sarcomas in both preclinical and clinical settings [ 2 – 3 ]. In these studies, varying degrees of drug synergism were observed, and half of the treated patients did not benefit from the combination. Resistance to cancer treatments is typically attributed to a subset of cells that survive after treatment [ 4 – 5 ]. The characterization of persistent cells is challenging, and no univocal methods have been established, adding further complexity to the field. Octamer-binding transcription factor 4 (OCT4) is a well-recognized hallmark of pluripotency in embryonic and adult quiescent cells and plays a central role in the maintenance of several cancers, including sarcomas [ 6 – 7 ]. Our group previously reported that sarcoma cells spared by conventional treatment are significantly enriched in their OCT4 + fraction [ 8 ]. In this context, the immune system's activity against persistent cells remains far from fully understood. Adoptive immune cell therapy may represent a promising approach to defeating treatment-resistant cancer cells [ 9 ]. The clinical use of innate immune cells such as natural killer (NK) cells and other human leukocyte antigen (HLA)-independent effectors such as cytokine-induced killer (CIK) lymphocytes has demonstrated a favorable clinical safety profile and represents an encouraging therapeutic alternative [ 10 – 12 ]. We previously investigated the activity of immunotherapy with NK and CIK in preclinical in vitro and in vivo models of sarcomas [ 13 ], demonstrating their efficacy against the OCT4 + fraction [ 8 ]. NK and CIK kill their target cells without prior immunization or major histocompatibility complex (MHC) restriction. Several tumor histotypes, including sarcomas, express NK/CIK activating ligands, including the NKG2DLs, i.e. the stress-inducible ligands MHC class I chain-related protein A and B (MICA/B) and the UL16 binding proteins (ULBP2/3/5/6) [ 14 – 16 ]. DNA damaging agents, such as trabectedin, may upregulate the expression of NKG2DLs on cancer cells, potentially enhancing NK/CIK activity [ 17 – 18 ]. In addition, olaparib may activate innate cellular immunity by triggering cyclic 2’3’GMP-AMP (2’3’-cGAMP) synthetase (cGAS), stimulator of interferon gene (STING), tank-binding kinase I (TBK1), and interferon regulatory factor 3 (IRF3) ultimately leading to interferon (IFN)-β production [ 19 – 22 ]. In this work, we show that the trabectedin and olaparib combination has a double-edged effect in sarcoma cells: it increases the OCT4 + fraction on sarcoma cells by triggering the DNA-sensing cGAS-STING pathway, and in the same way, it increases and the expression of NK/CIK activating ligands eventually enhancing NK/CIK antitumor activity. NK/CIK cells kill OCT4 + persistent sarcoma cells that survived after trabectedin and olaparib treatment. Hence, trabectedin and olaparib show immunomodulatory activity in a preclinical experimental setting, paving the way for sequential immunotherapeutic strategies with NK/CIK-based adoptive cell therapy. MATERIALS AND METHODS Cell lines , ex vivo expansion of CIK cells, and DMR xenografts Bone and soft tissue sarcoma primary and commercially available cell lines (chondrosarcoma HT-1080 [ 23 – 24 ], leiomyosarcoma DMR [ 2 ], and undifferentiated pleomorphic sarcoma S018 [ 13 ]) were obtained from surgical resection or provided and cultured as previously described [ 2 , 13 ]. The NK-92 cell line was purchased from ATCC and cultured in Alpha Minimum Essential Medium with 12.5% horse serum (Gibco) and 12.5% FBS, supplemented with 0.2 mM inositol, 2-0.1 mM 2-mercaptoethanol, 0.02 mM folic acid, 100–200 IU/ml recombinant interleukin (IL)-2 (Chiron Corporation). All the cell lines were maintained at 37°C in a humidified atmosphere containing 5% CO 2 . CIK cells were obtained from the expansion of peripheral blood mononuclear cells (PBMCs) collected as previously described from patients affected by histologically confirmed bone and soft tissue sarcomas [ 13 ]. All participants provided written informed consent in accordance with a protocol that adheres to the Declaration of Helsinki, and the study was approved by the local Institutional Review Board and Ethics Committee (approval number 296–2020/14-OCT-2020). DMR xenograft models were obtained by orthotopic injection of DMR cells into the uterine wall of non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice (Charles River), as previously described [ 2 ]. This protocol was approved by the Institutional Animal Welfare Organization (OPBA) of the Candiolo Cancer Institute according to Italian Legislative Decree no. 26 of 03.14.2014 and by the Italian Ministry of Health (Aut. Min. 178/2015-PR). Pharmacological treatments Trabectedin (PharmaMar) and olaparib (Carbosynth) were dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich), stored at -80°C, and diluted in fresh media immediately before use at a final concentration of 0.5 nM trabectedin and 5 µM olaparib for 24 or 72-hour treatment. IFNβ (PHC4244, Thermo Fisher Scientific) was stored at -80°C and diluted to a final concentration of 10-100-1000 IU/mL in the culture medium for 24-hour treatments. Itacitinib (INCB39110, Selleck Chemicals) was stored at -80°C and diluted in fresh media immediately before use at a final concentration of 1 µM for 24-hour treatments. 2’3’-cGAMP (InvivoGen) was diluted to a final concentration of 10 µM and transfected into cells for 6 hours using Lipofectamine®RNAiMAX (Thermo Fisher Scientific) according to the manufacturer’s protocol. DMR xenografts were treated with trabectedin and olaparib as previously described [ 2 ]. Co-culture with NK-92 or CIK cells, and cell viability assays After 72 hours of drug treatment, tumor cells were co-cultured for additional 72 hours with NK or CIK cells at different effector:target ratios. Cell viability was measured by the CellTiter-Glo® Luminescent Cell Viability Assay (Promega) using a GloMax® Discover Microplate Reader (Promega). The killing activity was calculated as the mortality of each effector:target ratio normalized to the mortality in the absence of NK/CIK (effecor:target = 0:1). In dedicated experiments, viable and total cell counts were performed by 0.1% Trypan blue dye exclusion (Thermo Fisher Scientific) and OCT4 + cells were evaluated by flow cytometry, as described below. Cells were subjected to IRF3 silencing (siIRF3) for 48 hours, incubated for 24 hours with trabectedin and olaparib as single agents and in combination, and co-cultured for additional 24 hours with NK-92 (1:1) or CIK (10:1) cells to test cell viability in comparison with silencer negative control (siCTRL)-treated cells. RNA Silencing Tumor cells were grown to 60% confluence in complete medium and then incubated with 10 nM siRNAs (listed in Supplementary Table S1 ) and Lipofectamine®RNAiMAX (Thermo Fisher Scientific) diluted in Opti-MEM® (Gibco) without antibiotics, as recommended by the manufacturer. RT-qPCR and western blotting were performed to check silencing efficacy after 24 and 48 hours (methods described below). RNA extraction and RT-qPCR Total RNA was extracted from sarcoma cell lines and xenografts using the Maxwell® RSC miRNA Tissue Kit and the RSC Maxwell Instrument (Promega), following the manufacturer’s instructions. RNA quality and concentration were checked using a DS 11 + spectrophotometer (Denovix Inc). Starting with 500 ng of total RNA, Superscript VILO IV Master Mix (Thermo Fisher Scientific) was used to obtain cDNA. RT-qPCR was performed using TaqMan Fast Advanced Mastermix (Thermo Fisher Scientific) and TaqMan probes ( Supplementary Table S2 ) using the ABI PRISM 7900HT System (Thermo Fisher Scientific). RT-qPCR for IFNAR1/2 was performed using SYBR Green Master mix (Thermo Fisher Scientific) and an appropriate set of primers ( Supplementary Table S3 ). The expression data were normalized to housekeeping genes, and the fold change for each group of treatments in comparison with untreated controls was calculated using the 2 −ΔΔct formula. Spheroid-formation Assay HT-1080 cells were seeded in ultra-low-attachment round-bottom 96-well plates (Corning) at a density of 1000 viable cells/well in DMEM-F12 (Sigma Aldrich), B27 (Gibco), 10 ng/mL h-EGF, 10 ng/mL h-FGF, 4 µg/mL insulin (Thermo Fisher Scientific) and penicillin/streptomycin, with or without 2’3’-cGAMP (10 µM), centrifuged at 1000 rpm for 10 minutes, and cultured for 14 days, replacing half of the medium every 72 hours. Spheroid-forming ability was monitored by taking photographs at different time points (1, 3, 6, 10, and 14 days) using an inverted phase-contrast microscope at 10× magnification (Leica). Digital images of the cultured spheroids were analyzed using ImageJ software [ 25 ] to calculate their area (µm 2 ). The number of viable cells was counted by trypan blue dye exclusion, and spheroid growth was quantified at the end of the experiment (day 14) using the Cell Titer-Glo® assay. Chromatin immunoprecipitation (ChIP) ChIP was conducted using the Zymo-Spin ChIP kit (D5210, Zymo Research), following the manufacturer’s instructions. Briefly, 10 6 cells were used per 1 ml of ChIP reaction, and sonication was conducted using Bioruptor Pico (Diagenode) for five cycles, with 30 seconds ON and 30 seconds OFF at 40% amplitude. For each ChIP reaction, 2.5 µg of primary antibody (listed in Supplementary Table S4 ) was added. qPCR analysis was performed on immunoprecipitated samples or input controls in each reaction mixed with SYBR Green Master mix (Thermo Fisher Scientific) and an appropriate set of primers designed for the promoter region of POUF5F1 ( Supplementary Table S3 ). Briefly, the genomic region from − 2000 to + 400 bp of the OCT4 gene ( POU5F1 ) Transcription Start Site (TSS) was explored for candidate IRF3 and IRF9 binding motifs using the Eukaryotic Promoter Database (EPD), Expasy [ 26 ] and Multiple Em for Motif Elicitation (MEME) tool [ 27 ] considering the positional weight matrices annotated in JASPAR CORE 2024 [ 28 ]. Specifically, IRF3 and IRF9 binding motifs annotated in this database were analyzed and only those associated with a p-value < 0.05 were considered statistically significant. IRF3 and IRF9 consensus sequences in the POU5F1 promoter region were identified at -1598 bp (p < 0.001) and − 1596 bp (p < 0.01) from the TSS, respectively ( Supplementary Figure S1 A-C ). Western blot Cells were lysed in Lysis Buffer 6 (R&D Systems) to obtain protein extracts, as previously described [ 29 ]. Protein concentrations were determined using the BCA Protein Assay (Thermo Fisher Scientific) and Glomax Discover System (Promega). After gel electrophoresis and blotting, nonspecific binding sites were blocked with 10% bovine serum albumin for 1 hour. Primary antibodies (listed in Supplementary Table S4 ) were incubated overnight at 4°C, followed by 1-hour incubation of with HRP-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories). Imaging and quantification were performed using the Bio-Rad Chemidoc™ Touch Imaging System (Bio-Rad Laboratories), and intensity was quantified using QuantityOne (Bio-Rad Laboratories), using Vinculin or ACTB as a normalizer. Flow cytometry Tumor cells were incubated for one hour at + 4°C in the dark with fluorochrome-conjugated primary antibodies against NKG2DLs ( Supplementary Table S5 ). The fluorescent signal was detected by ADP Cyan (Beckman Coulter Srl) and analyzed using Summit v4.3 software (Beckman Coulter Srl). In dedicated experiments, cells were permeabilized with a fix-and-perm kit (Thermo Fisher) and incubated with OCT4 antibody ( Supplementary Table S5 ). NK and CIK cell phenotypes were evaluated using fluorochrome-conjugated monoclonal antibodies against CD3, CD8, CD56, CD134, and CD226 ( Supplementary Table S5 ). RNA sequencing and Bioinformatic analysis After RNA extraction, RNA concentration was determined using the Qubit RNA BR (broad range) assay kit (Thermo Fisher Scientific) and a Qubit® 3.0 Fluorometer (Thermo Fisher Scientific). RNA fragmentation was assessed using the 2100 Bioanalyzer High-Sensitivity RNA Assay Kit (Agilent Technologies). The total RNA was processed for RNA-sequencing analysis with TruSeq Stranded Total RNA Gold (Illumina) following the manufacturer’s instructions and sequenced on a NovaSeq6000 (Illumina). Sequencing reads were preprocessed and filtered using fastp software v0.23.1 [ 30 ]. Filtered reads were aligned to transcript annotations using Salmon v1.4.0 in default settings with seqBias and gcBias options to correct for hexamer priming and GC-content biases, respectively [ 31 ]. Gencode v36 was used as a reference transcriptome. Read alignments were preprocessed with the tximport v1.18.0 R package [ 32 ] and expression levels were normalized to transcripts per million (TPM). DESeq2 v1.30.1 [ 33 ] was used to perform the differential expression analysis. A gene was considered differentially expressed if it was associated with an adjusted p-value 1 in all samples. Metascape v3.5 [ 34 ] was used in the default settings for functional enrichment analysis. Immunofluorescence Cytosolic dsDNA was visualized in drug-treated cells and formalin-fixed paraffin-embedded (FFPE) DMR xenografts. Cells were seeded in an 8-well chamber (Ibidi), treated with trabectedin, olaparib, or their combination for 24 h, and fixed with cold 4% paraformaldehyde (#sc-281692; Santa Cruz Biotechnology) for 10 min at room temperature. FFPE-DMR xenografts were 2-µm sectioned using an RM 2125 microtome (Leica Biosystems), mounted on polarized glass slides (Leica Biosystem), and dried at 40°C for 30 min. Sections were deparaffinized, subjected to antigen retrieval with sodium citrate buffer pH 6 (#C9999, Sigma Aldrich) at 96°C for 30 min, and permeabilized in 0.01% Triton-X100, 0.1% Tween-20, and phosphate-buffer saline (PBST) for 10 min. Nonspecific binding sites were blocked for 30 min in blocking buffer (1% BSA, 2.25% Glycine, PBST). Finally, the sections were incubated overnight at 4°C with dsDNA antibody ( Supplementary Table S4 ), followed by a 1-hour incubation with Alexa Fluor 488 secondary antibody (Thermo Fisher Scientific), DAPI (Thermo Fisher Scientific), and phalloidin (PHN-Alexa 647, Sigma Aldrich). Imaging was performed using an SPE II confocal microscope (Leica) with an oil immersion objective (63x). The number of intracellular dsDNA spots was quantified using ImageJ software as the total occupied area within the cell. Crystal violet growth assay Cells were seeded in 12-well plates in complete medium. After 24 h, cells were treated with trabectedin, olaparib, or their combination for 72 h. The medium was then replaced, and surviving cells were cultured for another 72 h in the presence or absence of NK-92 (effector:target ratio = 1:12) or CIK (effector:target ratio = 1:1) cells. The cells were treated with 2’3’-cGAMP for 72 h. At the end of the experiment, the cells were stained with 0.1% crystal violet (Sigma-Aldrich), and images were captured using the high-throughput imaging platform LIPSI. ELISA assay Cell supernatants were collected and analyzed using the Human IFNβ Beta ELISA Kit (DIFNB0, R&D Systems) according to the manufacturer’s instructions. Absorbance was measured at 450 nM, with wavelength correction at 570 nM, using the Glomax Discover System (Promega). Study design and Statistical analysis No prior sample-size calculations were performed. In vitro experiments were performed with three cell lines and validation of specific molecular mechanisms was conducted with one cell line, as specified in the results section. No inclusion or exclusion criteria were applied. No randomization, blinding, or confounding controls were applied. Differences between treatment groups (including untreated controls) were analyzed with two-way ANOVA (Tukey’s test with Bonferroni’s correction for multiple comparisons) using Prism 8 GraphPad Software. The results are reported as mean ± standard deviation of at least three replicates. No replicate exclusions were made. Results with a p-value < 0.05 were deemed to be statistically significant. RESULTS Persistent sarcoma cells spared by trabectedin and olaparib combination express OCT4 in vitro and in vivo Based on the previous observation of the increase in OCT4 + cell population fraction upon targeted and conventional chemotherapeutic treatments [ 8 ], we investigated its expression in persistent sarcoma cells treated with trabectedin and olaparib. In three out of three sarcoma cell lines (DMR, HT-1080, and S018), OCT4 mRNA expression levels were significantly higher in persistent cells surviving trabectedin compared to untreated controls, as well as in the olaparib and combination treatments. Moreover, the combination treatment significantly increased OCT4 mRNA expression compared to that in the untreated control (Fig. 1 A). In vivo , DMR xenografts that survived trabectedin treatment exhibited higher levels of OCT4 mRNA compared to untreated controls, as well as olaparib and combination treatments (Fig. 1 A). Furthermore, we confirmed the modulation of OCT4 expression at the protein level by flow cytometry, showing that trabectedin as a single agent significantly increased the percentage of OCT4 + cells in three out of three cell lines compared to untreated controls. Olaparib, a single agent, did not modify OCT4 + population fraction. Furthermore, in cells treated with the combination, the percentage of OCT4 + cells was significantly higher than that in untreated cells, but lower than that in trabecular-treated cells (Fig. 1 B). The combination of trabectedin and olaparib upregulates the genotoxicity pathway and inhibits the cellular response to DNA damage The transcriptomic profiles of persistent sarcoma cells treated with trabectedin and olaparib were analyzed in cell lines and xenograft models. Total RNA sequencing revealed that 437 genes were differentially expressed between treated versus untreated cells (adj. p 1) ( Fig. 2 A ) . Specifically, trabectedin, olaparib, and their combination significantly modulated 23, 1, and 434 genes, respectively. Remarkably, 21 genes were modulated by both trabectedin and its combination, whereas 413 genes were significantly modulated only by the combination ( Fig. 2 A ) . Notably, S018 cells showed the most relevant gene expression variation between untreated and combination-treated samples, accounting for 347 differentially expressed genes (adj. p < 0.05, Supplementary Table S6 ). As shown in Fig. 2 B, the fold-change in expression was consistent among DMR, HT-1080, and S018, with a prevalence of gene downregulation. These expression patterns were also observed in vivo (Fig. 2 B ) . Functional enrichment analysis of both cell lines and xenografts showed that the Genotoxicity pathway (WP4286) was the most enriched term for upregulated genes, and the Cellular response to DNA damage stimulus (GO:0006974) was the most downregulated (Fig. 2 C and Supplementary Table S7 ). Trabectedin and olaparib combination increases cytosolic dsDNA and activates the cGAS-STING-IRF3-IFNβ pathway The activation of genotoxic stress and inhibition of the DNA damage response may induce dsDNA leakage into the cytosol. Indeed, we observed by confocal microscopy the accumulation of cytosolic dsDNA after 24 h of treatment, and the effect of the combination was significantly higher than that of both single agents and controls ( Fig. 3 A-B ) , which was further confirmed in vivo. Consistently, DMR xenografts treated with trabectedin and olaparib, and their combination, showed a significant increase in cytosolic dsDNA content compared to untreated controls. Notably, the effect induced by trabectedin + olaparib was significantly higher than that induced by either agent (Fig. 3 A-B). The increment of cytosolic dsDNA triggers the cGAS-STING pathway [ 19 ]. First, we measured the expression of cGAS-STING pathway transducers (STING, TBK1, IRF3) in cell lines and DMR xenografts, showing that trabectedin and olaparib combination induced a significant increase of their mRNA expression if compared to untreated controls (Fig. 3 C). Moreover, to evaluate the cGAS-STING pathway activation, we performed western blot analyses of the phosphorylated TBK1 (S172) and IRF3 (S396) and the corresponding total protein in all cell lines and observed a significant increase in both phosphorylation in trabectedin- and combination-treated cells, but not in olaparib-treated cells, as compared to controls without differences between trabectedin as a single agent or in combination (Fig. 3 D-E). Moreover, the final product of cGAS-pathway activation, IFNβ, was significantly increased in all cell lines after combination treatment compared to untreated controls ( Fig. 3 F). The cGAS-STING-IRF3-IFNβ pathway controls OCT4 gene expression To understand whether cGAS-STING pathway activation plays a role in persistent cells spared by drug treatment, we investigated its effect on OCT4 expression. Treatment with 2'3'-cGAMP induced a significant upregulation of both IFNβ and OCT4 mRNA expression levels (Fig. 4 A). The functional role of this upregulation was investigated using sphere-forming and growth assays in the presence or absence of 2’3’-cGAMP. DMR and S018 cells were unable to grow in sphere-forming conditions (data not shown), otherwise, HT-1080 cells grown as spheroids and the addition of 2’3’-cGAMP significantly increased this growth, if compared to untreated controls ( Fig. 4 B-D ). To further confirm this evidence, we tested the colony growth ability of HT-1080 cells treated with 2’3’-cGAMP and found that the number of colonies was significantly higher than that in the untreated control ( Fig. 4 E ). In addition, after verifying the expression of IFNβ receptors (IFNAR1/2) in sarcoma cells at both the mRNA and protein levels ( Supplementary Figure S2 A-B ), we directly treated sarcoma cells with IFNβ, showing a dose-dependent OCT4 upregulation (Fig. 4 F-G). To further validate the role of IFNβ in the control of OCT4 expression, we inhibited its production by silencing IFNB1 expression in three cell lines. HT-1080 and S018 cells died after IFNβ silencing (data not shown), but not after scramble treatment (siCTRL), suggesting a potential role of IFNβ in maintaining cell viability. In contrast, DMR cells survived IFNβ silencing, and a significant decrease in OCT4 mRNA expression was observed in IFNβ-silenced cells compared to siCTRL cells (Fig. 4 H). In addition, we investigated the role of the IFNβ downstream pathway through JAK/STAT signaling in the control of OCT4 expression. Treatment with the JAK1 inhibitor itacitinib blocked 2’3’-cGAMP and IFNβ-induced STAT1 phosphorylation (Y701) ( Supplementary Figure S2 C-D) and OCT4 upregulation ( Supplementary Figure S2 E-F). To further elucidate the regulation of OCT4 expression, we explored whether OCT4 gene transcription could be directly activated by the transcription factors IRF3 and IRF9 as key upstream and downstream components of the IFNβ signaling pathway, respectively. ChIP assay showed that both IRF3 and IRF9 bind to the OCT4 promoter region. Moreover, treatment with 2'3'-cGAMP significantly increased this binding compared with that in the untreated controls (Fig. 4 I ). To validate their role in OCT4 expression modulation after drug treatment, we silenced IRF3 and IRF9 in the three cell lines. siIRF3 significantly reduced IRF3 expression under all treatment conditions, whereas siIRF9 did not properly silence IRF9 ( Supplementary S2G-H) . Notably, OCT4 mRNA expression in IRF3-silenced cells was significantly reduced compared with that in siCTRL mock-treated cells. Moreover, silencing of IRF3 also reduced OCT4 expression in drug-treated cells (trabectedin single-agent, olaparib single-agent, and trabectedin + olaparib combination) (Fig. 4 J). OCT4 upregulation in combination-treated cells was significantly lower than that in trabecular-treated cells (Fig. 1 ), even though cGAS-STING was activated (Fig. 3 ). To explain this observation, we assessed OCT4 expression in the presence of 2’3’-cGAMP and olaparib. Notably, PARP1 inhibition counteracted 2’3’-cGAMP-induced OCT4 mRNA upregulation (Fig. 4 K), evoking the effects observed in trabectedin-olaparib combination-treated cells. The cGAS-STING-IRF3-IFNβ pathway activation increases the NKG2DLs Based on previous observations regarding the induction of NK/CIK-activating ligand expression through the cGAS-STING pathway [ 35 – 36 ], we hypothesized the potential application of adoptive cell immunotherapy to target persistent tumor cells spared by drug treatment. We tested whether the combination of trabectedin and olaparib boosts NK/CIK antitumor activity against sarcoma cells by upregulating NK/CIK activating ligands. The expression of these ligands was measured at both the mRNA and protein levels, and it was observed that the combination upregulated the expression of MICA/B and ULBPs in three out of three sarcoma cell lines, compared to untreated controls and single agents (Fig. 5 A-B). Similar results were obtained in vivo in combination-treated DMR xenografts (Fig. 5 A). To evaluate the involvement of the cGAS-STING pathway in drug-induced NKG2DL upregulation, we blocked its activation by silencing the crucial component, IRF3. In IRF3-silenced cells, IFNβ production ( Supplementary Figure S3 A ) and drug-induced NKG2DL upregulation were impaired in three of the three cell lines (Fig. 5 C-E). These results were confirmed at the protein level by western blot analysis. As shown in Supplementary Figure S3 B-C , IRF3 silencing significantly impeded the upregulation of all NKG2DLs induced by trabectedin and combination treatment. To further validate the direct role of cGAS-STING-IRF3-IFNβ in the upregulation of NKG2DLs, we treated sarcoma cells with increasing concentrations of IFNβ, showing that IFNβ significantly increased the mRNA expression of NKG2DLs in the three cell lines, compared to untreated controls (Fig. 5 F ) . NK/CIK lymphocytes exert antitumor activity against both bulk and OCT4 + cell fractions Since cGAS-STING activation increased NKG2DL expression in sarcoma cells upon trabectedin + olaparib treatment, we tested the antitumor activity of NK/CIK following drug treatment. Notably, NK-92 and CIK cells (immunophenotype detailed in Supplementary Figure S4 A-B) exhibited robust killing activity in three out of three cell lines, at the lowest effector:target ratio (1:12 for NK and 1:1 for CIK) after trabectedin and olaparib combination pretreatment compared to the other pretreatment conditions (Fig. 6 A-B). This potentiation was further validated using crystal violet staining. Both NK and CIK cells significantly decreased tumor cell growth after pretreatment with the combination compared to the other pretreatment conditions (Fig. 6 C). Next, we functionally validated the role of the cGAS-STING pathway in boosting the NK/CIK-killing activity. Blocking cGAS-STING activation by IRF3 silencing significantly reduced the antitumor activity of sequential treatment with trabectedin and olaparib combination followed by NK or CIK cells (Fig. 6 D-E). Since cGAS-STING pathway activation increases NKG2DLs and OCT4 expression, we investigated whether the OCT4 + population, enriched after drug treatment, might be killed by NK and CIK cells. We observed that both NK-92 (effector-target ratio 1:12) and CIK (effector-target ratio 1:1) significantly decreased the OCT4 + fraction in trabectedin and combination-pretreated cells (Fig. 6 F ) . DISCUSSION Innovative cancer treatments must focus on tackling persistent tumor cells, which are the primary drivers of disease relapse and metastatic reactivation. Previous studies have shown that while conventional treatments such as chemotherapy, radiation therapy, and targeted agents are effective against bulk tumor cells, they fail to eradicate persistent cells [ 37 – 39 ]. No universally accepted methods to identify persistent cells have emerged from these studies, highlighting the need to establish a consensus protocol for their characterization. The transcription factor OCT4 has a role in cancer maintenance and resistance to anticancer therapy, and its expression was studied for the characterization of tumor cells that persist after treatment [ 7 ]. Our group previously reported that the OCT4 + cell fraction increases after chemotherapy with doxorubicin and targeted therapy with sorafenib or pazopanib in sarcoma cells [ 8 ]. Moreover, we studied the antitumor activity of the alkylating agents trabectedin and olaparib, observing variable degrees of synergism against advanced sarcomas in preclinical and clinical settings [ 2 – 3 ]. However, half of the treated patients did not benefit from the combination, and progression or relapse occurred. In the present study, we assessed the phenotype of persistent cells spared by trabectedin and olaparib treatment, monitored the OCT4 marker, and elucidated the molecular mechanism that controls its expression. We showed that treatment with trabectedin, but not olaparib, significantly increased OCT4 + expression in surviving cells. Notably, PARP1 plays a central role in maintaining cell pluripotency and stemness-like features, and its inhibition reduces the tumorigenic potential of this treatment-resistant population [ 40 – 43 ]. In line with this, our results showed that olaparib counteracts trabectedin-induced OCT4 upregulation in sarcoma cell lines. To elucidate the molecular mechanism induced by drugs, we took advantage of transcriptomic analysis, which showed that genes involved in genotoxicity stress and cellular response to DNA damage were significantly modulated by the combination of trabectedin and olaparib. Previous studies have suggested the implications of trabectedin and its derivatives in genotoxicity stress, transcription-dependent replication stress, and genome instability [ 44 – 46 ]. Moreover, by directly blocking PARP1, olaparib impairs the DNA damage response [ 47 – 48 ] and, in combination with trabectedin, can synergistically perpetuate DNA damage [ 2 ]. The global modulation of 413 genes induced by the combination, but not by single agents, confirmed the effective drug synergism in these cell lines. Here, we showed that the perpetuation of DNA damage leads to the accumulation of cytosolic dsDNA, triggering the cGAS-STING pathway, activating the transcription factor IRF3, and eventually promoting IFNβ production in surviving sarcoma cells. Moreover, we observed that the OCT4 + subpopulation is sustained by IFNβ signaling, demonstrating for the first time that cGAS-STING activation promotes OCT4 expression, reinforcing previous evidence of Type I-IFN cancer-promoting effects [ 49 – 50 ]. We showed that IRF3 and IRF9 specifically bind to the OCT4 promoter region and directly stimulate OCT4 transcription in response to cGAS-STING activation in sarcoma cells. Notably, the direct inhibition of PARP1 by olaparib counteracts OCT4 upregulation caused by the activation of the cGAS-STING pathway, potentially controlling its transcription through alternative ways [ 51 ], which warrants further investigation. Moreover, the higher induction of the cGAS-STING pathway in sarcoma cells spared by trabectedin and olaparib combination led to increased expression of NK/CIK activating ligands. Using functional assays, we demonstrated that IFNβ treatment led to the upregulation of NKG2DLs and that IRF3 was implicated in this effect, adding further insights into the complex regulation of the crosstalk between innate immunity and cancer [ 52 – 55 ]. In this study, we elucidated the molecular mechanism that led to OCT4 and NKGD2L expression through cGAS-STING-IRF3-IFNβ pathway activation, as shown in Fig. 7 . Herein, we demonstrated, for the first time, the enhanced antitumor activity of NK and CIK cells against sarcoma cells pretreated with the trabectedin and olaparib combination, as well as the loss of this efficacy when the cGAS-STING-IRF3-IFNβ pathway is impaired. Notably, we showed that NK and CIK cells effectively targeted and killed both bulk tumor cells and persistent OCT4 + cell populations. Therefore, our findings endorse the use of NK/CIK-based immunotherapy to target sarcoma cells that survive drug treatments, consistent with prior evidence from other cancer types, including colorectal cancer, glioblastoma, and melanoma [ 56 – 58 ]. These promising results support further investigation of a therapeutic strategy with trabectedin and olaparib combination, followed by sequential treatment with NK/CIK lymphocytes. In conclusion, activation of the cGAS-STING pathway induced by trabectedin + olaparib treatment is a double-edged sword: it promotes the enrichment of OCT4 + persistent cells while simultaneously increasing the expression of NK/CIK ligands. Therefore, the efficient NK/CIK cell killing of both the bulk and OCT4 + populations supports the potential application of this innovative sequential chemo-targeted-immune therapeutic strategy against advanced sarcomas and deserves further investigation. Abbreviations HLA: human leukocyte antigen cGAS: Cyclic GMP–AMP synthase GMP: guanosine monophosphate AMP: adenosine monophosphate STING: Stimulator of Interferon Genes PARP1: Poly [ADP-ribose] polymerase 1 ADP: adenosine diphosphate NK: natural killer CIK: cytokine-induced killer NKG2DLs: natural killer group 2D ligands RT-qPCR: quantitative reverse transcription polymerase chain reaction IRF3: Interferon-regulatory factor 3 IFNβ: interferon-β OCT4: octamer-binding transcription factor 4 MICA/B: MHC class I chain-related proteins A and B MHC: Major histocompatibility complex ULBP: UL16 binding protein dsDNA: double-stranded deoxyribonucleic acid TBK1: TANK binding kinase-1 TANK: TRAF family member-associated nuclear factor-kappa-B activator TRAF: Tumor necrosis factor receptor-associated factor siCTRL: silencing control siIRF3: silencing IRF3 CD: cluster differentiation mRNA: messenger RNA ATCC: American Type Culture Collection EMEM: Eagle’s Minimum Essential Medium DMEM Dulbecco’s Modified Eagle Medium IL: Interleukine FBS: fetal bovine serum DMSO: dimethyl sulfoxide NOD/SCID: Nonobese diabetic/severe combined immunodeficient mice PBMCs: peripheral blood mononuclear cells RPMI: Roswell Park Memorial Institute Medium ChIP: chromatin immunoprecipitation IgG: Immunoglobulin G Declarations ETHICS APPROVAL AND CONSENT TO PARTICIPATE All participants provided written informed consent in accordance with a protocol that adheres to the Declaration of Helsinki, and the study was approved by the local Institutional Review Board and Ethics Committee (approval number 296-2020/14-OCT-2020). Animal study was approved by the Institutional Animal Welfare Organization (OPBA) of the Candiolo Cancer Institute according to Italian Legislative Decree no. 26 of 03.14.2014 and by the Italian Ministry of Health (Aut. Min. 178/2015-PR). CONSENT FOR PUBLICATION Not applicable. COMPETING INTERESTS AMe: PharmaMar, support for meeting participation and Institutional Grant LDA: PSI CRO Italy, GSK, AstraZeneca, Eisai, and Boehringer Ingelheim, Advisory Board; support for meeting participation from PharmaMar and AstraZeneca. GGr: fees for consulting and advisory board roles from PharmaMar, Lilly, Novartis, GSK, Bayer, and Eisai. FUNDINGS This work was supported by Ministero della Salute (Ricerca Finalizzata-Giovani Ricercatori GR-2016-02362726 to YP); AIRC (IG 23104 to GGr and IG 20259 to DS), Eta-Beta Linea 4" project - Bando UTT 2023-2025; Fondazione per la ricerca sui tumori dell'apparato muscoloscheletrico e rari ONLUS CRT RF = 2016 -0917 to GGr; Alleanza contro il cancro-working group Sarcomi, Ricerca Corrente-Reti 2021 RCR 2021 WP8 to YP; FPRC 5x1000 Ministero della Salute 2015 ImGen, FPRC 5xmille MIUR 2014 to GGr and DS; FC was supported by AIRC fellowship “Volontari Comitato Abruzzo-Molise” Rif. 21173; AMe research activity was supported by PON 2014-2020 DM 1062/2021 PNR 2021-2027 and PharmaMar (Institutional Grant) AUTHORS’ CONTRIBUTIONS Conceptualization, GGi, YP, DS, FC; Methodology, GGi, SM, GF, DS, GM, FC, EL; Investigation, GGi, FC, SM, SB, CT, SCo, AMa., SB, and GF, SM; Resources, VL, GM, FG; Writing – Original Draft, GGi., FC, GF, AMe, and YP.; Writing –Review and Editing, GGi, YP, GGr, DS, LDA, MDB, SC, and CR; Visualization, GGi, GF, YP, EL, and SM; Funding Acquisition, YP, GGr, DS; Supervision, YP. All authors have read and approved the final version of the manuscript and declare that they have adhered to the ARRIVE guidelines. ACKNOWLEDGMENTS The authors would like to thank Dr. Daniela Cantarella, Dr. Barbara Martinoglio, and Dr. Paola Bernabei for RNA-Seq data, RT-qPCR, and assistance in cytofluorimetric analysis, respectively; the grant office and the Scientific Direction for continuous support; and PharmaMar for providing trabectedin. AVAILABILITY OF DATA AND MATERIALS The main data supporting the findings of this study are available in the article and its supplementary information. The raw RNA-Seq data are available on GEO ID: GSE239639 (token for Reviewer qbwbawqgjburxqf), all other raw data generated during the study are available from the corresponding authors upon reasonable request. References Blay JY, Honoré C, Stoeckle E, Meeus P, Jafari M, Gouin F, et al (2019) Surgery in reference centers improves survival of sarcoma patients: a nationwide study. 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J Immunol 182:3530–9. Pietra G, Manzini C, Vitale M, Balsamo M, Ognio E, Boitano M, et al (2009) Natural killer cells kill human melanoma cells with characteristics of cancer stem cells. Int Immunol 21:793–801. Supplementary Figure 4 Supplementary Figure 4 is not available with this version. Additional Declarations Competing interest reported. AMe: PharmaMar, support for meeting participation and Institutional Grant LDA: PSI CRO Italy, GSK, AstraZeneca, Eisai, and Boehringer Ingelheim, Advisory Board; support for meeting participation from PharmaMar and AstraZeneca. GGr: fees for consulting and advisory board roles from PharmaMar, Lilly, Novartis, GSK, Bayer, and Eisai. Supplementary Files SupplFig1.tif Supplementary Figure S1. Primer design and consensus motif of IRF3 and IRF9 binding to OCT4 protomer region. (A) OCT4 promoter region including the predicted IRF3 and IRF9 consensus sequence (highlighted in yellow) amplified by forward (in green) and reverse primers (in pink) employed for ChIP assay. (B) sLOGO motif of IRF3 and IRF9 binding site predicted on the amplified sequence by MEME tool, p<0.0001) (C) Position frequency matrices of IRF3 (matrix ID: MA1418.1) and IRF9 (matrix ID: MA0653.1) available on JASPAR 2024 database. SupplFigS2.tif Supplementary Figure S2. IFNβ pathway controls OCT4 expression. (A) Real-time PCR analyses of IFNβ receptor 1 and 2 (IFNAR1/2) mRNA expression (ΔCt) in DMR, HT-1080, S018 cells. (B) Representative western blot analysis and protein band intensity quantification of IFNAR1 and β-ACTIN in DMR, HT-1080, and S018 cell lines. (C-D) Western blot analysis and protein band intensity quantification of P-STAT1 (Y701) after 2’3’-cGAMP or IFNβ treatment, as single agents or in combination with itacitinib. (E) Real-time PCR analysis of OCT4 mRNA expression (fold change) in 2’3’-cGAMP or IFNβ as single agents or in combination with itacitinib. (F) Flow cytometry analysis of OCT4 + cells treated with 2’3’-cGAMP or IFNβ as single agents or in combination with itacitinib. (G) Real-time PCR analysis of IRF3 mRNA expression level (fold change vs. siCTRL NT) in siCTRL and siIRF3-transfected cells, after 24-hour drug treatment. (H) Real-time PCR analysis of IRF9 mRNA expression level in siCTRL and siIRF9-transfected cells. siCTRL=control siRNA-transfected cells, siIRF3=IRF3-silenced cells, siIRF9=IRF9-silenced cells NT=untreated, TR=trabectedin, OL=olaparib, CB=combination. ITA= itacitinib. Data are shown as mean ± standard deviation (n=3; *p <0.05, ** p <0.01, *** p <0.001, **** p<0.0001 vs. untreated; # p<0.05, ## p<0.01, ### p<0.001 vs. trabectedin; † p<0.05, †† p<0.01, ††† p<0.001, †††† p<0.0001 vs. olaparib; ◆ p<0.05, ◆◆ p<0.01, ◆◆◆ p<0.001, ◆◆◆◆ p<0.0001 vs . siCRTL; ▲ p<0.05, ▲▲ p<0.01, ▲▲▲ p<0.001, ▲▲▲▲ p<0.0001 vs. effector:target ratio=0:1). (*p<0.05, ** p <0.01, *** p <0.001, **** p<0.0001 vs. untreated; # p<0.05, ## p<0.01, ### p<0.001 vs. itacitinib; † p<0.05, †† p<0.01, ††† p<0.001, †††† p<0.0001 vs. itacitinib+2’3’-cGAMP; $ p<0.05, $$ p<0.01, $$$ p<0.001 vs itacitinib+IFNβ; ◆p<0.05, ◆◆p<0.01,◆◆◆p<0.001, ◆◆◆◆p<0.0001 vs . siCRTL.). SupplementaryFigureS3.tif Supplementary Figure S3. Effects of IRF3 silencing on IFNβ production and NKG2DL protein expression. (A) ELISA quantification of IFNβ production in siCTRL and siIRF3-transfected cells, after 24-hour drug treatment. (B) Representative western blot analysis of IRF3, NKG2DLs, and vinculin in siCTRL and siIRF3-transfected HT-1080 cells, after 24-hour drug treatment. (C) Quantification of protein band intensity normalized to vinculin housekeeping. siCTRL=control siRNA-transfected cells, siIRF3=IRF3-silenced cells. Data are shown as mean ± standard deviation (n=3; ◆ p<0.05, ◆◆ p<0.01, ◆◆◆ p<0.001, ◆◆◆◆ p<0.0001 vs . siCRTL). 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Torino","correspondingAuthor":false,"prefix":"","firstName":"Chiara","middleName":"","lastName":"Riganti","suffix":""},{"id":447169122,"identity":"804357a9-9c19-4df2-8eb2-69335ac7a901","order_by":14,"name":"Santina Cutrupi","email":"","orcid":"","institution":"University of Torino","correspondingAuthor":false,"prefix":"","firstName":"Santina","middleName":"","lastName":"Cutrupi","suffix":""},{"id":447169123,"identity":"53264bce-fa37-43bf-9eda-9c7c82ff400d","order_by":15,"name":"Michele Bortoli","email":"","orcid":"","institution":"University of Torino","correspondingAuthor":false,"prefix":"","firstName":"Michele","middleName":"","lastName":"Bortoli","suffix":""},{"id":447169124,"identity":"e2e7b718-77ae-4965-9ae2-434069fbc802","order_by":16,"name":"Lorenzo D’Ambrosio","email":"","orcid":"","institution":"University of Torino","correspondingAuthor":false,"prefix":"","firstName":"Lorenzo","middleName":"","lastName":"D’Ambrosio","suffix":""},{"id":447169125,"identity":"4e4137f7-f8ce-4a82-bdd1-f1be457d7585","order_by":17,"name":"Dario Sangiolo","email":"","orcid":"","institution":"FPO-IRCCS","correspondingAuthor":false,"prefix":"","firstName":"Dario","middleName":"","lastName":"Sangiolo","suffix":""},{"id":447169126,"identity":"ab8a1524-8ab5-4e68-83cf-8df59ebd9505","order_by":18,"name":"Giovanni Grignani","email":"","orcid":"","institution":"FPO-IRCCS","correspondingAuthor":false,"prefix":"","firstName":"Giovanni","middleName":"","lastName":"Grignani","suffix":""},{"id":447169127,"identity":"87166e03-b7cb-4790-913a-a6177e71bba6","order_by":19,"name":"Ymera Pignochino","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/klEQVRIiWNgGAWjYBADHhDBzGDAwMAPZByAiRCnRbIBqAWoh4egHogWIDA4QMAag2uHj334wXBHRj66+eDngoI7csY3cg8e/lDBIGOPS8vttOSZPQzPeAzvHEuWnmHwzNjsRl7CgQNncDvM4HaOMdAJh3kMZ+SYMfMYHE7cdiPH4MDBNnxa8j8z/kHWsnkGQS05zMwgW+QloFo2SBDQInk7zZhZxuAZj4FEWrI0UIuxxJk3BgfOnJHg4TmAXQvf7eTHjG8q7tjLz0g++Jnnz2E5/vYc4w8VFTb27A3YtSiAjQLGhQGamRI4nMXAIA8x6gCMMQpGwSgYBaMAEwAAoGpZ5zPqSygAAAAASUVORK5CYII=","orcid":"","institution":"FPO-IRCCS","correspondingAuthor":true,"prefix":"","firstName":"Ymera","middleName":"","lastName":"Pignochino","suffix":""}],"badges":[],"createdAt":"2025-04-16 13:23:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6463944/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6463944/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00262-025-04141-w","type":"published","date":"2025-09-23T15:57:18+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":92430711,"identity":"61467073-1447-4fdf-b982-1620d09f35ca","added_by":"auto","created_at":"2025-09-29 16:07:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1297570,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6463944/v1/a8078cc5-0bb0-45ab-adc7-b5c62fe15b64.pdf"},{"id":81813735,"identity":"29d24a12-2eda-4251-b731-c348ba069ad7","added_by":"auto","created_at":"2025-05-02 09:27:12","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":60111096,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure S1. Primer design and consensus motif of IRF3 and IRF9 binding to OCT4 protomer region. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) OCT4 promoter region including the predicted IRF3 and IRF9 consensus sequence (highlighted in yellow) amplified by forward (in green) and reverse primers (in pink) employed for ChIP assay. (\u003cstrong\u003eB\u003c/strong\u003e) sLOGO motif of IRF3 and IRF9 binding site predicted on the amplified sequence by MEME tool, p\u0026lt;0.0001) (\u003cstrong\u003eC\u003c/strong\u003e) Position frequency matrices of IRF3 (matrix ID: MA1418.1) and IRF9 (matrix ID: MA0653.1) available on JASPAR 2024 database.\u003c/p\u003e","description":"","filename":"SupplFig1.tif","url":"https://assets-eu.researchsquare.com/files/rs-6463944/v1/c684ae1c1bdaaf21a464bdbc.tif"},{"id":81813736,"identity":"f43d0466-f3f4-40d0-ac52-3d98aceea1a2","added_by":"auto","created_at":"2025-05-02 09:27:13","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":81193824,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure S2. IFNβ pathway controls OCT4 expression.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Real-time PCR analyses of IFNβ receptor 1 and 2 (IFNAR1/2) mRNA expression (ΔCt) in DMR, HT-1080, S018 cells. (\u003cstrong\u003eB\u003c/strong\u003e) Representative western blot analysis and protein band intensity quantification of IFNAR1 and β-ACTIN in DMR, HT-1080, and S018 cell lines. (\u003cstrong\u003eC-D\u003c/strong\u003e) Western blot analysis and protein band intensity quantification of P-STAT1 (Y701) after 2’3’-cGAMP or IFNβ treatment, as single agents or in combination with itacitinib. (\u003cstrong\u003eE\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eReal-time PCR analysis of OCT4 mRNA expression (fold change) in 2’3’-cGAMP or IFNβ as single agents or in combination with itacitinib. (\u003cstrong\u003eF\u003c/strong\u003e) Flow cytometry analysis of OCT4\u003csup\u003e+ \u003c/sup\u003ecells treated with 2’3’-cGAMP or IFNβ as single agents or in combination with itacitinib. (\u003cstrong\u003eG\u003c/strong\u003e) Real-time PCR analysis of IRF3 mRNA expression level (fold change vs. siCTRL NT) in siCTRL and siIRF3-transfected cells, after 24-hour drug treatment. (\u003cstrong\u003eH\u003c/strong\u003e) Real-time PCR analysis of IRF9 mRNA expression level in siCTRL and siIRF9-transfected cells. siCTRL=control siRNA-transfected cells, siIRF3=IRF3-silenced cells, siIRF9=IRF9-silenced cells NT=untreated, TR=trabectedin, OL=olaparib, CB=combination. ITA= itacitinib. Data are shown as mean ± standard deviation (n=3; *p \u0026lt;0.05, ** p \u0026lt;0.01, *** p \u0026lt;0.001, **** p\u0026lt;0.0001 \u003cem\u003evs.\u003c/em\u003e untreated; # p\u0026lt;0.05, ## p\u0026lt;0.01, ### p\u0026lt;0.001\u003cem\u003e vs.\u003c/em\u003e trabectedin; † p\u0026lt;0.05, †† p\u0026lt;0.01, ††† p\u0026lt;0.001, †††† p\u0026lt;0.0001 \u003cem\u003evs.\u003c/em\u003e olaparib; ◆ p\u0026lt;0.05, ◆◆ p\u0026lt;0.01, ◆◆◆ p\u0026lt;0.001, ◆◆◆◆ p\u0026lt;0.0001 \u003cem\u003evs\u003c/em\u003e. siCRTL; ▲ p\u0026lt;0.05, ▲▲ p\u0026lt;0.01, ▲▲▲ p\u0026lt;0.001, ▲▲▲▲ p\u0026lt;0.0001\u0026nbsp; vs. effector:target ratio=0:1). (*p\u0026lt;0.05, ** p \u0026lt;0.01, *** p \u0026lt;0.001, **** p\u0026lt;0.0001 \u003cem\u003evs.\u003c/em\u003e untreated; # p\u0026lt;0.05, ## p\u0026lt;0.01, ### p\u0026lt;0.001\u003cem\u003e vs.\u003c/em\u003e itacitinib; † p\u0026lt;0.05, †† p\u0026lt;0.01, ††† p\u0026lt;0.001, †††† p\u0026lt;0.0001 \u003cem\u003evs.\u003c/em\u003e itacitinib+2’3’-cGAMP; $ p\u0026lt;0.05, $$ p\u0026lt;0.01, $$$ p\u0026lt;0.001 \u003cem\u003evs \u003c/em\u003eitacitinib+IFNβ; ◆p\u0026lt;0.05, ◆◆p\u0026lt;0.01,◆◆◆p\u0026lt;0.001, ◆◆◆◆p\u0026lt;0.0001 \u003cem\u003evs\u003c/em\u003e. siCRTL.).\u003c/p\u003e","description":"","filename":"SupplFigS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-6463944/v1/60b940c8ad75058830ee5b58.tif"},{"id":81813729,"identity":"fcbdff1d-abce-47db-8394-d7a42243748f","added_by":"auto","created_at":"2025-05-02 09:27:10","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":33236060,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure S3. Effects of IRF3 silencing on IFNβ production and NKG2DL protein expression. \u003c/strong\u003e(A) ELISA quantification of IFNβ production in siCTRL and siIRF3-transfected cells, after 24-hour drug treatment. (B) Representative western blot analysis of IRF3, NKG2DLs, and vinculin in siCTRL and siIRF3-transfected HT-1080 cells, after 24-hour drug treatment. (C) Quantification of protein band intensity normalized to vinculin housekeeping. siCTRL=control siRNA-transfected cells, siIRF3=IRF3-silenced cells. Data are shown as mean ± standard deviation (n=3; ◆ p\u0026lt;0.05, ◆◆ p\u0026lt;0.01, ◆◆◆ p\u0026lt;0.001, ◆◆◆◆ p\u0026lt;0.0001 \u003cem\u003evs\u003c/em\u003e. siCRTL).\u003c/p\u003e","description":"","filename":"SupplementaryFigureS3.tif","url":"https://assets-eu.researchsquare.com/files/rs-6463944/v1/8707e0bc0e7e769c869d09ed.tif"},{"id":81813700,"identity":"60b6f4d3-96aa-4819-9d33-f5b6a08f4cb9","added_by":"auto","created_at":"2025-05-02 09:27:08","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":19332,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS1S5.docx","url":"https://assets-eu.researchsquare.com/files/rs-6463944/v1/8182dab8e4367b193d5399e9.docx"},{"id":81813701,"identity":"0881fafe-0dbd-4785-88a9-285f86c79db7","added_by":"auto","created_at":"2025-05-02 09:27:09","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":8317595,"visible":true,"origin":"","legend":"","description":"","filename":"SupplTableS6.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6463944/v1/e41a6c2f9d7d7e335517cb48.xlsx"},{"id":81813965,"identity":"713e2c93-164a-4cd0-bad4-fe3aafc1aa35","added_by":"auto","created_at":"2025-05-02 09:35:09","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":206807,"visible":true,"origin":"","legend":"","description":"","filename":"SupplTable7EnrichmentMetascape.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6463944/v1/c814d62fca1b6737378d120a.xlsx"}],"financialInterests":"Competing interest reported. AMe: PharmaMar, support for meeting participation and Institutional Grant\nLDA: PSI CRO Italy, GSK, AstraZeneca, Eisai, and Boehringer Ingelheim, Advisory Board; support for meeting participation from PharmaMar and AstraZeneca.\nGGr: fees for consulting and advisory board roles from PharmaMar, Lilly, Novartis, GSK, Bayer, and Eisai.","formattedTitle":"The cGAS-STING pathway is a master regulator of OCT4 expression in persistent sarcoma cells and enhances cellular immunotherapy with NK and CIK lymphocytes","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eSarcomas are rare, heterogeneous, and malignant tumors of mesenchymal origin. Limited effective therapeutic options are available for the management of advanced, unresectable, or metastatic disease, and their prognosis remains poor [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. We have previously demonstrated that the combination of the alkylating agent trabectedin and the poly (ADP-ribose) polymerase-1 (PARP1) inhibitor olaparib is active against a subset of sarcomas in both preclinical and clinical settings [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In these studies, varying degrees of drug synergism were observed, and half of the treated patients did not benefit from the combination. Resistance to cancer treatments is typically attributed to a subset of cells that survive after treatment [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The characterization of persistent cells is challenging, and no univocal methods have been established, adding further complexity to the field. Octamer-binding transcription factor 4 (OCT4) is a well-recognized hallmark of pluripotency in embryonic and adult quiescent cells and plays a central role in the maintenance of several cancers, including sarcomas [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Our group previously reported that sarcoma cells spared by conventional treatment are significantly enriched in their OCT4\u003csup\u003e+\u003c/sup\u003e fraction [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In this context, the immune system's activity against persistent cells remains far from fully understood. Adoptive immune cell therapy may represent a promising approach to defeating treatment-resistant cancer cells [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The clinical use of innate immune cells such as natural killer (NK) cells and other human leukocyte antigen (HLA)-independent effectors such as cytokine-induced killer (CIK) lymphocytes has demonstrated a favorable clinical safety profile and represents an encouraging therapeutic alternative [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. We previously investigated the activity of immunotherapy with NK and CIK in preclinical \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e models of sarcomas [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], demonstrating their efficacy against the OCT4\u003csup\u003e+\u003c/sup\u003e fraction [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. NK and CIK kill their target cells without prior immunization or major histocompatibility complex (MHC) restriction. Several tumor histotypes, including sarcomas, express NK/CIK activating ligands, including the NKG2DLs, i.e. the stress-inducible ligands MHC class I chain-related protein A and B (MICA/B) and the UL16 binding proteins (ULBP2/3/5/6) [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. DNA damaging agents, such as trabectedin, may upregulate the expression of NKG2DLs on cancer cells, potentially enhancing NK/CIK activity [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In addition, olaparib may activate innate cellular immunity by triggering cyclic 2\u0026rsquo;3\u0026rsquo;GMP-AMP (2\u0026rsquo;3\u0026rsquo;-cGAMP) synthetase (cGAS), stimulator of interferon gene (STING), tank-binding kinase I (TBK1), and interferon regulatory factor 3 (IRF3) ultimately leading to interferon (IFN)-β production [\u003cspan additionalcitationids=\"CR20 CR21\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In this work, we show that the trabectedin and olaparib combination has a double-edged effect in sarcoma cells: it increases the OCT4\u003csup\u003e+\u003c/sup\u003e fraction on sarcoma cells by triggering the DNA-sensing cGAS-STING pathway, and in the same way, it increases and the expression of NK/CIK activating ligands eventually enhancing NK/CIK antitumor activity. NK/CIK cells kill OCT4\u003csup\u003e+\u003c/sup\u003e persistent sarcoma cells that survived after trabectedin and olaparib treatment. Hence, trabectedin and olaparib show immunomodulatory activity in a preclinical experimental setting, paving the way for sequential immunotherapeutic strategies with NK/CIK-based adoptive cell therapy.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e \u003cb\u003eCell lines\u003c/b\u003e, \u003cb\u003eex vivo\u003c/b\u003e \u003cb\u003eexpansion of CIK cells, and DMR xenografts\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBone and soft tissue sarcoma primary and commercially available cell lines (chondrosarcoma HT-1080 [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], leiomyosarcoma DMR [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], and undifferentiated pleomorphic sarcoma S018 [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]) were obtained from surgical resection or provided and cultured as previously described [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The NK-92 cell line was purchased from ATCC and cultured in Alpha Minimum Essential Medium with 12.5% horse serum (Gibco) and 12.5% FBS, supplemented with 0.2 mM inositol, 2-0.1 mM 2-mercaptoethanol, 0.02 mM folic acid, 100\u0026ndash;200 IU/ml recombinant interleukin (IL)-2 (Chiron Corporation). All the cell lines were maintained at 37\u0026deg;C in a humidified atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e. CIK cells were obtained from the expansion of peripheral blood mononuclear cells (PBMCs) collected as previously described from patients affected by histologically confirmed bone and soft tissue sarcomas [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. All participants provided written informed consent in accordance with a protocol that adheres to the Declaration of Helsinki, and the study was approved by the local Institutional Review Board and Ethics Committee (approval number 296\u0026ndash;2020/14-OCT-2020). DMR xenograft models were obtained by orthotopic injection of DMR cells into the uterine wall of non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice (Charles River), as previously described [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. This protocol was approved by the Institutional Animal Welfare Organization (OPBA) of the Candiolo Cancer Institute according to Italian Legislative Decree no. 26 of 03.14.2014 and by the Italian Ministry of Health (Aut. Min. 178/2015-PR).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePharmacological treatments\u003c/h2\u003e \u003cp\u003eTrabectedin (PharmaMar) and olaparib (Carbosynth) were dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich), stored at -80\u0026deg;C, and diluted in fresh media immediately before use at a final concentration of 0.5 nM trabectedin and 5 \u0026micro;M olaparib for 24 or 72-hour treatment. IFNβ (PHC4244, Thermo Fisher Scientific) was stored at -80\u0026deg;C and diluted to a final concentration of 10-100-1000 IU/mL in the culture medium for 24-hour treatments. Itacitinib (INCB39110, Selleck Chemicals) was stored at -80\u0026deg;C and diluted in fresh media immediately before use at a final concentration of 1 \u0026micro;M for 24-hour treatments. 2\u0026rsquo;3\u0026rsquo;-cGAMP (InvivoGen) was diluted to a final concentration of 10 \u0026micro;M and transfected into cells for 6 hours using Lipofectamine\u0026reg;RNAiMAX (Thermo Fisher Scientific) according to the manufacturer\u0026rsquo;s protocol. DMR xenografts were treated with trabectedin and olaparib as previously described [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCo-culture with NK-92 or CIK cells, and cell viability assays\u003c/h3\u003e\n\u003cp\u003eAfter 72 hours of drug treatment, tumor cells were co-cultured for additional 72 hours with NK or CIK cells at different effector:target ratios. Cell viability was measured by the CellTiter-Glo\u0026reg; Luminescent Cell Viability Assay (Promega) using a GloMax\u0026reg; Discover Microplate Reader (Promega). The killing activity was calculated as the mortality of each effector:target ratio normalized to the mortality in the absence of NK/CIK (effecor:target\u0026thinsp;=\u0026thinsp;0:1). In dedicated experiments, viable and total cell counts were performed by 0.1% Trypan blue dye exclusion (Thermo Fisher Scientific) and OCT4\u003csup\u003e+\u003c/sup\u003e cells were evaluated by flow cytometry, as described below. Cells were subjected to IRF3 silencing (siIRF3) for 48 hours, incubated for 24 hours with trabectedin and olaparib as single agents and in combination, and co-cultured for additional 24 hours with NK-92 (1:1) or CIK (10:1) cells to test cell viability in comparison with silencer negative control (siCTRL)-treated cells.\u003c/p\u003e\n\u003ch3\u003eRNA Silencing\u003c/h3\u003e\n\u003cp\u003eTumor cells were grown to 60% confluence in complete medium and then incubated with 10 nM siRNAs (listed in \u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e) and Lipofectamine\u0026reg;RNAiMAX (Thermo Fisher Scientific) diluted in Opti-MEM\u0026reg; (Gibco) without antibiotics, as recommended by the manufacturer. RT-qPCR and western blotting were performed to check silencing efficacy after 24 and 48 hours (methods described below).\u003c/p\u003e\n\u003ch3\u003eRNA extraction and RT-qPCR\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted from sarcoma cell lines and xenografts using the Maxwell\u0026reg; RSC miRNA Tissue Kit and the RSC Maxwell Instrument (Promega), following the manufacturer\u0026rsquo;s instructions. RNA quality and concentration were checked using a DS 11\u0026thinsp;+\u0026thinsp;spectrophotometer (Denovix Inc). Starting with 500 ng of total RNA, Superscript VILO IV Master Mix (Thermo Fisher Scientific) was used to obtain cDNA. RT-qPCR was performed using TaqMan Fast Advanced Mastermix (Thermo Fisher Scientific) and TaqMan probes (\u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e) using the ABI PRISM 7900HT System (Thermo Fisher Scientific). RT-qPCR for IFNAR1/2 was performed using SYBR Green Master mix (Thermo Fisher Scientific) and an appropriate set of primers (\u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e\u003c/b\u003e). The expression data were normalized to housekeeping genes, and the fold change for each group of treatments in comparison with untreated controls was calculated using the 2\u003csup\u003e\u0026minus;ΔΔct\u003c/sup\u003e formula.\u003c/p\u003e\n\u003ch3\u003eSpheroid-formation Assay\u003c/h3\u003e\n\u003cp\u003eHT-1080 cells were seeded in ultra-low-attachment round-bottom 96-well plates (Corning) at a density of 1000 viable cells/well in DMEM-F12 (Sigma Aldrich), B27 (Gibco), 10 ng/mL h-EGF, 10 ng/mL h-FGF, 4 \u0026micro;g/mL insulin (Thermo Fisher Scientific) and penicillin/streptomycin, with or without 2\u0026rsquo;3\u0026rsquo;-cGAMP (10 \u0026micro;M), centrifuged at 1000 rpm for 10 minutes, and cultured for 14 days, replacing half of the medium every 72 hours. Spheroid-forming ability was monitored by taking photographs at different time points (1, 3, 6, 10, and 14 days) using an inverted phase-contrast microscope at 10\u0026times; magnification (Leica). Digital images of the cultured spheroids were analyzed using ImageJ software [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] to calculate their area (\u0026micro;m\u003csup\u003e2\u003c/sup\u003e). The number of viable cells was counted by trypan blue dye exclusion, and spheroid growth was quantified at the end of the experiment (day 14) using the Cell Titer-Glo\u0026reg; assay.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eChromatin immunoprecipitation (ChIP)\u003c/h2\u003e \u003cp\u003eChIP was conducted using the Zymo-Spin ChIP kit (D5210, Zymo Research), following the manufacturer\u0026rsquo;s instructions. Briefly, 10\u003csup\u003e6\u003c/sup\u003e cells were used per 1 ml of ChIP reaction, and sonication was conducted using Bioruptor Pico (Diagenode) for five cycles, with 30 seconds ON and 30 seconds OFF at 40% amplitude. For each ChIP reaction, 2.5 \u0026micro;g of primary antibody (listed in \u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e\u003c/b\u003e) was added. qPCR analysis was performed on immunoprecipitated samples or input controls in each reaction mixed with SYBR Green Master mix (Thermo Fisher Scientific) and an appropriate set of primers designed for the promoter region of POUF5F1 (\u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e\u003c/b\u003e). Briefly, the genomic region from \u0026minus;\u0026thinsp;2000 to +\u0026thinsp;400 bp of the OCT4 gene (\u003cem\u003ePOU5F1\u003c/em\u003e) Transcription Start Site (TSS) was explored for candidate IRF3 and IRF9 binding motifs using the Eukaryotic Promoter Database (EPD), Expasy [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and Multiple Em for Motif Elicitation (MEME) tool [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] considering the positional weight matrices annotated in JASPAR CORE 2024 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Specifically, IRF3 and IRF9 binding motifs annotated in this database were analyzed and only those associated with a p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant. IRF3 and IRF9 consensus sequences in the \u003cem\u003ePOU5F1\u003c/em\u003e promoter region were identified at -1598 bp (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and \u0026minus;\u0026thinsp;1596 bp (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) from the TSS, respectively (\u003cb\u003eSupplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA-C\u003c/b\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eWestern blot\u003c/h3\u003e\n\u003cp\u003eCells were lysed in Lysis Buffer 6 (R\u0026amp;D Systems) to obtain protein extracts, as previously described [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Protein concentrations were determined using the BCA Protein Assay (Thermo Fisher Scientific) and Glomax Discover System (Promega). After gel electrophoresis and blotting, nonspecific binding sites were blocked with 10% bovine serum albumin for 1 hour. Primary antibodies (listed in \u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e\u003c/b\u003e) were incubated overnight at 4\u0026deg;C, followed by 1-hour incubation of with HRP-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories). Imaging and quantification were performed using the Bio-Rad Chemidoc\u0026trade; Touch Imaging System (Bio-Rad Laboratories), and intensity was quantified using QuantityOne (Bio-Rad Laboratories), using Vinculin or ACTB as a normalizer.\u003c/p\u003e\n\u003ch3\u003eFlow cytometry\u003c/h3\u003e\n\u003cp\u003eTumor cells were incubated for one hour at +\u0026thinsp;4\u0026deg;C in the dark with fluorochrome-conjugated primary antibodies against NKG2DLs (\u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e\u003c/b\u003e). The fluorescent signal was detected by ADP Cyan (Beckman Coulter Srl) and analyzed using Summit v4.3 software (Beckman Coulter Srl). In dedicated experiments, cells were permeabilized with a fix-and-perm kit (Thermo Fisher) and incubated with OCT4 antibody (\u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e\u003c/b\u003e). NK and CIK cell phenotypes were evaluated using fluorochrome-conjugated monoclonal antibodies against CD3, CD8, CD56, CD134, and CD226 (\u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e\u003c/b\u003e).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eRNA sequencing and Bioinformatic analysis\u003c/h2\u003e \u003cp\u003eAfter RNA extraction, RNA concentration was determined using the Qubit RNA BR (broad range) assay kit (Thermo Fisher Scientific) and a Qubit\u0026reg; 3.0 Fluorometer (Thermo Fisher Scientific). RNA fragmentation was assessed using the 2100 Bioanalyzer High-Sensitivity RNA Assay Kit (Agilent Technologies). The total RNA was processed for RNA-sequencing analysis with TruSeq Stranded Total RNA Gold (Illumina) following the manufacturer\u0026rsquo;s instructions and sequenced on a NovaSeq6000 (Illumina). Sequencing reads were preprocessed and filtered using fastp software v0.23.1 [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Filtered reads were aligned to transcript annotations using Salmon v1.4.0 in default settings with seqBias and gcBias options to correct for hexamer priming and GC-content biases, respectively [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Gencode v36 was used as a reference transcriptome. Read alignments were preprocessed with the tximport v1.18.0 R package [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] and expression levels were normalized to transcripts per million (TPM). DESeq2 v1.30.1 [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] was used to perform the differential expression analysis. A gene was considered differentially expressed if it was associated with an adjusted p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05, and median TPM\u0026thinsp;\u0026gt;\u0026thinsp;1 in all samples. Metascape v3.5 [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] was used in the default settings for functional enrichment analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence\u003c/h2\u003e \u003cp\u003eCytosolic dsDNA was visualized in drug-treated cells and formalin-fixed paraffin-embedded (FFPE) DMR xenografts. Cells were seeded in an 8-well chamber (Ibidi), treated with trabectedin, olaparib, or their combination for 24 h, and fixed with cold 4% paraformaldehyde (#sc-281692; Santa Cruz Biotechnology) for 10 min at room temperature. FFPE-DMR xenografts were 2-\u0026micro;m sectioned using an RM 2125 microtome (Leica Biosystems), mounted on polarized glass slides (Leica Biosystem), and dried at 40\u0026deg;C for 30 min. Sections were deparaffinized, subjected to antigen retrieval with sodium citrate buffer pH 6 (#C9999, Sigma Aldrich) at 96\u0026deg;C for 30 min, and permeabilized in 0.01% Triton-X100, 0.1% Tween-20, and phosphate-buffer saline (PBST) for 10 min. Nonspecific binding sites were blocked for 30 min in blocking buffer (1% BSA, 2.25% Glycine, PBST). Finally, the sections were incubated overnight at 4\u0026deg;C with dsDNA antibody (\u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e\u003c/b\u003e), followed by a 1-hour incubation with Alexa Fluor 488 secondary antibody (Thermo Fisher Scientific), DAPI (Thermo Fisher Scientific), and phalloidin (PHN-Alexa 647, Sigma Aldrich). Imaging was performed using an SPE II confocal microscope (Leica) with an oil immersion objective (63x). The number of intracellular dsDNA spots was quantified using ImageJ software as the total occupied area within the cell.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCrystal violet growth assay\u003c/h2\u003e \u003cp\u003eCells were seeded in 12-well plates in complete medium. After 24 h, cells were treated with trabectedin, olaparib, or their combination for 72 h. The medium was then replaced, and surviving cells were cultured for another 72 h in the presence or absence of NK-92 (effector:target ratio\u0026thinsp;=\u0026thinsp;1:12) or CIK (effector:target ratio\u0026thinsp;=\u0026thinsp;1:1) cells. The cells were treated with 2\u0026rsquo;3\u0026rsquo;-cGAMP for 72 h. At the end of the experiment, the cells were stained with 0.1% crystal violet (Sigma-Aldrich), and images were captured using the high-throughput imaging platform LIPSI.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eELISA assay\u003c/h2\u003e \u003cp\u003eCell supernatants were collected and analyzed using the Human IFNβ Beta ELISA Kit (DIFNB0, R\u0026amp;D Systems) according to the manufacturer\u0026rsquo;s instructions. Absorbance was measured at 450 nM, with wavelength correction at 570 nM, using the Glomax Discover System (Promega).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStudy design and Statistical analysis\u003c/h2\u003e \u003cp\u003eNo prior sample-size calculations were performed. In vitro experiments were performed with three cell lines and validation of specific molecular mechanisms was conducted with one cell line, as specified in the results section. No inclusion or exclusion criteria were applied. No randomization, blinding, or confounding controls were applied. Differences between treatment groups (including untreated controls) were analyzed with two-way ANOVA (Tukey\u0026rsquo;s test with Bonferroni\u0026rsquo;s correction for multiple comparisons) using Prism 8 GraphPad Software. The results are reported as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation of at least three replicates. No replicate exclusions were made. Results with a p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were deemed to be statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003ePersistent sarcoma cells spared by trabectedin and olaparib combination express OCT4\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBased on the previous observation of the increase in OCT4\u003csup\u003e+\u003c/sup\u003e cell population fraction upon targeted and conventional chemotherapeutic treatments [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], we investigated its expression in persistent sarcoma cells treated with trabectedin and olaparib. In three out of three sarcoma cell lines (DMR, HT-1080, and S018), OCT4 mRNA expression levels were significantly higher in persistent cells surviving trabectedin compared to untreated controls, as well as in the olaparib and combination treatments. Moreover, the combination treatment significantly increased OCT4 mRNA expression compared to that in the untreated control (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In \u003cem\u003evivo\u003c/em\u003e, DMR xenografts that survived trabectedin treatment exhibited higher levels of OCT4 mRNA compared to untreated controls, as well as olaparib and combination treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Furthermore, we confirmed the modulation of OCT4 expression at the protein level by flow cytometry, showing that trabectedin as a single agent significantly increased the percentage of OCT4\u003csup\u003e+\u003c/sup\u003e cells in three out of three cell lines compared to untreated controls. Olaparib, a single agent, did not modify OCT4\u003csup\u003e+\u003c/sup\u003e population fraction. Furthermore, in cells treated with the combination, the percentage of OCT4\u003csup\u003e+\u003c/sup\u003e cells was significantly higher than that in untreated cells, but lower than that in trabecular-treated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe combination of trabectedin and olaparib upregulates the genotoxicity pathway and inhibits the cellular response to DNA damage\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe transcriptomic profiles of persistent sarcoma cells treated with trabectedin and olaparib were analyzed in cell lines and xenograft models. Total RNA sequencing revealed that 437 genes were differentially expressed between treated \u003cem\u003eversus\u003c/em\u003e untreated cells (adj. p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, median TPM\u0026thinsp;\u0026gt;\u0026thinsp;1) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Specifically, trabectedin, olaparib, and their combination significantly modulated 23, 1, and 434 genes, respectively. Remarkably, 21 genes were modulated by both trabectedin and its combination, whereas 413 genes were significantly modulated only by the combination \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Notably, S018 cells showed the most relevant gene expression variation between untreated and combination-treated samples, accounting for 347 differentially expressed genes (adj. p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e\u003c/b\u003e). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, the fold-change in expression was consistent among DMR, HT-1080, and S018, with a prevalence of gene downregulation. These expression patterns were also observed \u003cem\u003ein vivo\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Functional enrichment analysis of both cell lines and xenografts showed that the \u003cem\u003eGenotoxicity pathway (WP4286)\u003c/em\u003e was the most enriched term for upregulated genes, and the \u003cem\u003eCellular response to DNA damage stimulus (GO:0006974)\u003c/em\u003e was the most downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC \u003cb\u003eand Supplementary Table S7\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eTrabectedin and olaparib combination increases cytosolic dsDNA and activates the cGAS-STING-IRF3-IFNβ pathway\u003c/h2\u003e \u003cp\u003eThe activation of genotoxic stress and inhibition of the DNA damage response may induce dsDNA leakage into the cytosol. Indeed, we observed by confocal microscopy the accumulation of cytosolic dsDNA after 24 h of treatment, and the effect of the combination was significantly higher than that of both single agents and controls \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-B\u003cb\u003e)\u003c/b\u003e, which was further confirmed \u003cem\u003ein vivo.\u003c/em\u003e Consistently, DMR xenografts treated with trabectedin and olaparib, and their combination, showed a significant increase in cytosolic dsDNA content compared to untreated controls. Notably, the effect induced by trabectedin\u0026thinsp;+\u0026thinsp;olaparib was significantly higher than that induced by either agent (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe increment of cytosolic dsDNA triggers the cGAS-STING pathway [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. First, we measured the expression of cGAS-STING pathway transducers (STING, TBK1, IRF3) in cell lines and DMR xenografts, showing that trabectedin and olaparib combination induced a significant increase of their mRNA expression if compared to untreated controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Moreover, to evaluate the cGAS-STING pathway activation, we performed western blot analyses of the phosphorylated TBK1 (S172) and IRF3 (S396) and the corresponding total protein in all cell lines and observed a significant increase in both phosphorylation in trabectedin- and combination-treated cells, but not in olaparib-treated cells, as compared to controls without differences between trabectedin as a single agent or in combination (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-E). Moreover, the final product of cGAS-pathway activation, IFNβ, was significantly increased in all cell lines after combination treatment compared to untreated controls \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eThe cGAS-STING-IRF3-IFNβ pathway controls OCT4 gene expression\u003c/h2\u003e \u003cp\u003eTo understand whether cGAS-STING pathway activation plays a role in persistent cells spared by drug treatment, we investigated its effect on OCT4 expression. Treatment with 2'3'-cGAMP induced a significant upregulation of both IFNβ and OCT4 mRNA expression levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The functional role of this upregulation was investigated using sphere-forming and growth assays in the presence or absence of 2\u0026rsquo;3\u0026rsquo;-cGAMP. DMR and S018 cells were unable to grow in sphere-forming conditions (data not shown), otherwise, HT-1080 cells grown as spheroids and the addition of 2\u0026rsquo;3\u0026rsquo;-cGAMP significantly increased this growth, if compared to untreated controls \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-D\u003cb\u003e).\u003c/b\u003e To further confirm this evidence, we tested the colony growth ability of HT-1080 cells treated with 2\u0026rsquo;3\u0026rsquo;-cGAMP and found that the number of colonies was significantly higher than that in the untreated control \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition, after verifying the expression of IFNβ receptors (IFNAR1/2) in sarcoma cells at both the mRNA and protein levels (\u003cb\u003eSupplementary Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA-B\u003c/b\u003e), we directly treated sarcoma cells with IFNβ, showing a dose-dependent OCT4 upregulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF-G). To further validate the role of IFNβ in the control of OCT4 expression, we inhibited its production by silencing IFNB1 expression in three cell lines. HT-1080 and S018 cells died after IFNβ silencing (data not shown), but not after scramble treatment (siCTRL), suggesting a potential role of IFNβ in maintaining cell viability. In contrast, DMR cells survived IFNβ silencing, and a significant decrease in OCT4 mRNA expression was observed in IFNβ-silenced cells compared to siCTRL cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). In addition, we investigated the role of the IFNβ downstream pathway through JAK/STAT signaling in the control of OCT4 expression. Treatment with the JAK1 inhibitor itacitinib blocked 2\u0026rsquo;3\u0026rsquo;-cGAMP and IFNβ-induced STAT1 phosphorylation (Y701) (\u003cb\u003eSupplementary Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eC-D)\u003c/b\u003e and OCT4 upregulation (\u003cb\u003eSupplementary Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eE-F).\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo further elucidate the regulation of OCT4 expression, we explored whether OCT4 gene transcription could be directly activated by the transcription factors IRF3 and IRF9 as key upstream and downstream components of the IFNβ signaling pathway, respectively. ChIP assay showed that both IRF3 and IRF9 bind to the OCT4 promoter region. Moreover, treatment with 2'3'-cGAMP significantly increased this binding compared with that in the untreated controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI\u003cb\u003e).\u003c/b\u003e To validate their role in OCT4 expression modulation after drug treatment, we silenced IRF3 and IRF9 in the three cell lines. siIRF3 significantly reduced IRF3 expression under all treatment conditions, whereas siIRF9 did not properly silence IRF9 (\u003cb\u003eSupplementary S2G-H)\u003c/b\u003e. Notably, OCT4 mRNA expression in IRF3-silenced cells was significantly reduced compared with that in siCTRL mock-treated cells. Moreover, silencing of IRF3 also reduced OCT4 expression in drug-treated cells (trabectedin single-agent, olaparib single-agent, and trabectedin\u0026thinsp;+\u0026thinsp;olaparib combination) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ).\u003c/p\u003e \u003cp\u003eOCT4 upregulation in combination-treated cells was significantly lower than that in trabecular-treated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), even though cGAS-STING was activated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). To explain this observation, we assessed OCT4 expression in the presence of 2\u0026rsquo;3\u0026rsquo;-cGAMP and olaparib. Notably, PARP1 inhibition counteracted 2\u0026rsquo;3\u0026rsquo;-cGAMP-induced OCT4 mRNA upregulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK), evoking the effects observed in trabectedin-olaparib combination-treated cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eThe cGAS-STING-IRF3-IFNβ pathway activation increases the NKG2DLs\u003c/h2\u003e \u003cp\u003eBased on previous observations regarding the induction of NK/CIK-activating ligand expression through the cGAS-STING pathway [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], we hypothesized the potential application of adoptive cell immunotherapy to target persistent tumor cells spared by drug treatment.\u003c/p\u003e \u003cp\u003eWe tested whether the combination of trabectedin and olaparib boosts NK/CIK antitumor activity against sarcoma cells by upregulating NK/CIK activating ligands. The expression of these ligands was measured at both the mRNA and protein levels, and it was observed that the combination upregulated the expression of MICA/B and ULBPs in three out of three sarcoma cell lines, compared to untreated controls and single agents (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B). Similar results were obtained \u003cem\u003ein vivo\u003c/em\u003e in combination-treated DMR xenografts (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo evaluate the involvement of the cGAS-STING pathway in drug-induced NKG2DL upregulation, we blocked its activation by silencing the crucial component, IRF3. In IRF3-silenced cells, IFNβ production (\u003cb\u003eSupplementary Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA\u003c/b\u003e) and drug-induced NKG2DL upregulation were impaired in three of the three cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-E). These results were confirmed at the protein level by western blot analysis. As shown in \u003cb\u003eSupplementary Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eB-C\u003c/b\u003e, IRF3 silencing significantly impeded the upregulation of all NKG2DLs induced by trabectedin and combination treatment. To further validate the direct role of cGAS-STING-IRF3-IFNβ in the upregulation of NKG2DLs, we treated sarcoma cells with increasing concentrations of IFNβ, showing that IFNβ significantly increased the mRNA expression of NKG2DLs in the three cell lines, compared to untreated controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eNK/CIK lymphocytes exert antitumor activity against both bulk and OCT4\u003csup\u003e+\u003c/sup\u003e cell fractions\u003c/h2\u003e \u003cp\u003eSince cGAS-STING activation increased NKG2DL expression in sarcoma cells upon trabectedin\u0026thinsp;+\u0026thinsp;olaparib treatment, we tested the antitumor activity of NK/CIK following drug treatment. Notably, NK-92 and CIK cells (immunophenotype detailed in \u003cb\u003eSupplementary Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eA-B)\u003c/b\u003e exhibited robust killing activity in three out of three cell lines, at the lowest effector:target ratio (1:12 for NK and 1:1 for CIK) after trabectedin and olaparib combination pretreatment compared to the other pretreatment conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-B). This potentiation was further validated using crystal violet staining. Both NK and CIK cells significantly decreased tumor cell growth after pretreatment with the combination compared to the other pretreatment conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Next, we functionally validated the role of the cGAS-STING pathway in boosting the NK/CIK-killing activity. Blocking cGAS-STING activation by IRF3 silencing significantly reduced the antitumor activity of sequential treatment with trabectedin and olaparib combination followed by NK or CIK cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD-E). Since cGAS-STING pathway activation increases NKG2DLs and OCT4 expression, we investigated whether the OCT4\u003csup\u003e+\u003c/sup\u003e population, enriched after drug treatment, might be killed by NK and CIK cells. We observed that both NK-92 (effector-target ratio 1:12) and CIK (effector-target ratio 1:1) significantly decreased the OCT4\u003csup\u003e+\u003c/sup\u003e fraction in trabectedin and combination-pretreated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eInnovative cancer treatments must focus on tackling persistent tumor cells, which are the primary drivers of disease relapse and metastatic reactivation. Previous studies have shown that while conventional treatments such as chemotherapy, radiation therapy, and targeted agents are effective against bulk tumor cells, they fail to eradicate persistent cells [\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. No universally accepted methods to identify persistent cells have emerged from these studies, highlighting the need to establish a consensus protocol for their characterization. The transcription factor OCT4 has a role in cancer maintenance and resistance to anticancer therapy, and its expression was studied for the characterization of tumor cells that persist after treatment [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Our group previously reported that the OCT4\u003csup\u003e+\u003c/sup\u003e cell fraction increases after chemotherapy with doxorubicin and targeted therapy with sorafenib or pazopanib in sarcoma cells [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Moreover, we studied the antitumor activity of the alkylating agents trabectedin and olaparib, observing variable degrees of synergism against advanced sarcomas in preclinical and clinical settings [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, half of the treated patients did not benefit from the combination, and progression or relapse occurred. In the present study, we assessed the phenotype of persistent cells spared by trabectedin and olaparib treatment, monitored the OCT4 marker, and elucidated the molecular mechanism that controls its expression. We showed that treatment with trabectedin, but not olaparib, significantly increased OCT4\u003csup\u003e+\u003c/sup\u003e expression in surviving cells. Notably, PARP1 plays a central role in maintaining cell pluripotency and stemness-like features, and its inhibition reduces the tumorigenic potential of this treatment-resistant population [\u003cspan additionalcitationids=\"CR41 CR42\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. In line with this, our results showed that olaparib counteracts trabectedin-induced OCT4 upregulation in sarcoma cell lines.\u003c/p\u003e \u003cp\u003eTo elucidate the molecular mechanism induced by drugs, we took advantage of transcriptomic analysis, which showed that genes involved in \u003cem\u003egenotoxicity stress\u003c/em\u003e and \u003cem\u003ecellular response to DNA damage\u003c/em\u003e were significantly modulated by the combination of trabectedin and olaparib. Previous studies have suggested the implications of trabectedin and its derivatives in genotoxicity stress, transcription-dependent replication stress, and genome instability [\u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Moreover, by directly blocking PARP1, olaparib impairs the DNA damage response [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] and, in combination with trabectedin, can synergistically perpetuate DNA damage [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The global modulation of 413 genes induced by the combination, but not by single agents, confirmed the effective drug synergism in these cell lines. Here, we showed that the perpetuation of DNA damage leads to the accumulation of cytosolic dsDNA, triggering the cGAS-STING pathway, activating the transcription factor IRF3, and eventually promoting IFNβ production in surviving sarcoma cells. Moreover, we observed that the OCT4\u003csup\u003e+\u003c/sup\u003e subpopulation is sustained by IFNβ signaling, demonstrating for the first time that cGAS-STING activation promotes OCT4 expression, reinforcing previous evidence of Type I-IFN cancer-promoting effects [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. We showed that IRF3 and IRF9 specifically bind to the OCT4 promoter region and directly stimulate OCT4 transcription in response to cGAS-STING activation in sarcoma cells. Notably, the direct inhibition of PARP1 by olaparib counteracts OCT4 upregulation caused by the activation of the cGAS-STING pathway, potentially controlling its transcription through alternative ways [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e], which warrants further investigation.\u003c/p\u003e \u003cp\u003eMoreover, the higher induction of the cGAS-STING pathway in sarcoma cells spared by trabectedin and olaparib combination led to increased expression of NK/CIK activating ligands. Using functional assays, we demonstrated that IFNβ treatment led to the upregulation of NKG2DLs and that IRF3 was implicated in this effect, adding further insights into the complex regulation of the crosstalk between innate immunity and cancer [\u003cspan additionalcitationids=\"CR53 CR54\" citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. In this study, we elucidated the molecular mechanism that led to OCT4 and NKGD2L expression through cGAS-STING-IRF3-IFNβ pathway activation, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHerein, we demonstrated, for the first time, the enhanced antitumor activity of NK and CIK cells against sarcoma cells pretreated with the trabectedin and olaparib combination, as well as the loss of this efficacy when the cGAS-STING-IRF3-IFNβ pathway is impaired. Notably, we showed that NK and CIK cells effectively targeted and killed both bulk tumor cells and persistent OCT4\u003csup\u003e+\u003c/sup\u003e cell populations. Therefore, our findings endorse the use of NK/CIK-based immunotherapy to target sarcoma cells that survive drug treatments, consistent with prior evidence from other cancer types, including colorectal cancer, glioblastoma, and melanoma [\u003cspan additionalcitationids=\"CR57\" citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. These promising results support further investigation of a therapeutic strategy with trabectedin and olaparib combination, followed by sequential treatment with NK/CIK lymphocytes.\u003c/p\u003e \u003cp\u003eIn conclusion, activation of the cGAS-STING pathway induced by trabectedin\u0026thinsp;+\u0026thinsp;olaparib treatment is a double-edged sword: it promotes the enrichment of OCT4\u003csup\u003e+\u003c/sup\u003e persistent cells while simultaneously increasing the expression of NK/CIK ligands. Therefore, the efficient NK/CIK cell killing of both the bulk and OCT4\u003csup\u003e+\u003c/sup\u003e populations supports the potential application of this innovative sequential chemo-targeted-immune therapeutic strategy against advanced sarcomas and deserves further investigation.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eHLA: human leukocyte antigen\u003c/p\u003e\n\u003cp\u003ecGAS: Cyclic GMP\u0026ndash;AMP synthase\u003c/p\u003e\n\u003cp\u003eGMP: guanosine monophosphate\u003c/p\u003e\n\u003cp\u003eAMP: adenosine monophosphate\u003c/p\u003e\n\u003cp\u003eSTING: Stimulator of Interferon Genes\u003c/p\u003e\n\u003cp\u003ePARP1: Poly [ADP-ribose] polymerase 1\u003c/p\u003e\n\u003cp\u003eADP: adenosine diphosphate\u003c/p\u003e\n\u003cp\u003eNK: natural killer\u003c/p\u003e\n\u003cp\u003eCIK: cytokine-induced killer\u003c/p\u003e\n\u003cp\u003eNKG2DLs: natural killer group 2D ligands\u003c/p\u003e\n\u003cp\u003eRT-qPCR: quantitative reverse transcription polymerase chain reaction\u003c/p\u003e\n\u003cp\u003eIRF3: Interferon-regulatory factor\u0026nbsp;3\u003c/p\u003e\n\u003cp\u003eIFN\u0026beta;: interferon-\u0026beta;\u003c/p\u003e\n\u003cp\u003eOCT4: octamer-binding transcription factor 4\u003c/p\u003e\n\u003cp\u003eMICA/B: MHC class I chain-related proteins A and B\u003c/p\u003e\n\u003cp\u003eMHC: Major histocompatibility complex\u003c/p\u003e\n\u003cp\u003eULBP: UL16 binding protein\u003c/p\u003e\n\u003cp\u003edsDNA: double-stranded deoxyribonucleic acid \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTBK1: TANK binding kinase-1\u003c/p\u003e\n\u003cp\u003eTANK: TRAF family member-associated nuclear factor-kappa-B activator\u003c/p\u003e\n\u003cp\u003eTRAF: Tumor necrosis factor receptor-associated factor\u003c/p\u003e\n\u003cp\u003esiCTRL: silencing control\u0026nbsp;\u003c/p\u003e\n\u003cp\u003esiIRF3: silencing IRF3\u003c/p\u003e\n\u003cp\u003eCD: cluster differentiation\u0026nbsp;\u003c/p\u003e\n\u003cp\u003emRNA: messenger RNA\u003c/p\u003e\n\u003cp\u003eATCC: American Type Culture Collection\u003c/p\u003e\n\u003cp\u003eEMEM: Eagle\u0026rsquo;s Minimum Essential Medium\u003c/p\u003e\n\u003cp\u003eDMEM Dulbecco\u0026rsquo;s Modified Eagle Medium\u003c/p\u003e\n\u003cp\u003eIL: Interleukine\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFBS: fetal bovine serum\u003c/p\u003e\n\u003cp\u003eDMSO: dimethyl sulfoxide\u003c/p\u003e\n\u003cp\u003eNOD/SCID: Nonobese diabetic/severe combined immunodeficient mice\u003c/p\u003e\n\u003cp\u003ePBMCs: peripheral blood mononuclear cells\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRPMI: Roswell Park Memorial Institute Medium\u003c/p\u003e\n\u003cp\u003eChIP: chromatin immunoprecipitation\u003c/p\u003e\n\u003cp\u003eIgG: Immunoglobulin G\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eETHICS APPROVAL AND CONSENT TO PARTICIPATE\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll participants provided written informed consent in accordance with a protocol that adheres to the Declaration of Helsinki, and the study was approved by the local Institutional Review Board and Ethics Committee (approval number 296-2020/14-OCT-2020). Animal study was approved by the Institutional Animal Welfare Organization (OPBA) of the Candiolo Cancer Institute according to Italian Legislative Decree no. 26 of 03.14.2014 and by the Italian Ministry of Health (Aut. Min. 178/2015-PR).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONSENT FOR PUBLICATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCOMPETING INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAMe: PharmaMar, support for meeting participation and Institutional Grant\u003c/p\u003e\n\u003cp\u003eLDA: PSI CRO Italy, GSK, AstraZeneca, Eisai, and Boehringer Ingelheim, Advisory Board; support for meeting participation from PharmaMar and AstraZeneca.\u003c/p\u003e\n\u003cp\u003eGGr: fees for consulting and advisory board roles from PharmaMar, Lilly, Novartis, GSK, Bayer, and Eisai.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDINGS\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Ministero della Salute (Ricerca Finalizzata-Giovani Ricercatori GR-2016-02362726 to YP); AIRC (IG 23104 to GGr and IG 20259 to DS), Eta-Beta Linea 4\" project - Bando UTT 2023-2025; Fondazione per la ricerca sui tumori dell'apparato muscoloscheletrico e rari ONLUS CRT RF = 2016 -0917 to GGr; Alleanza contro il cancro-working group Sarcomi, Ricerca Corrente-Reti 2021 RCR 2021 WP8 to YP; FPRC 5x1000 Ministero della Salute 2015 ImGen, FPRC 5xmille MIUR 2014 to GGr and DS; FC was supported by AIRC fellowship “Volontari Comitato\u0026nbsp;Abruzzo-Molise” Rif. 21173; AMe research activity was supported by PON 2014-2020 DM 1062/2021 PNR 2021-2027 and PharmaMar (Institutional Grant)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHORS’ CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, GGi, YP, DS, FC; Methodology, GGi, SM, GF, DS, GM, FC, EL; Investigation, GGi, FC, SM, SB, CT, SCo, AMa., SB, and GF, SM; Resources, VL, GM, FG; Writing – Original Draft, GGi., FC, GF, AMe, and YP.; Writing –Review and Editing, GGi, YP, GGr, DS, LDA, MDB, SC, and CR; Visualization, GGi, GF, YP, EL, and SM; Funding Acquisition, YP, GGr, DS; Supervision, YP. All authors have read and approved the final version of the manuscript and declare that they have adhered to the ARRIVE guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Dr. Daniela Cantarella, Dr. Barbara Martinoglio, and Dr. Paola Bernabei for RNA-Seq data, RT-qPCR, and assistance in cytofluorimetric analysis, respectively; the grant office and the Scientific Direction for continuous support; and PharmaMar for providing trabectedin.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAVAILABILITY OF DATA AND MATERIALS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe main data supporting the findings of this study are available in the article and its supplementary information. The raw RNA-Seq data are available on GEO ID: GSE239639 (token for Reviewer qbwbawqgjburxqf), all other raw data generated during the study are available from the corresponding authors upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBlay JY, Honor\u0026eacute; C, Stoeckle E, Meeus P, Jafari M, Gouin F, et al (2019) Surgery in reference centers improves survival of sarcoma patients: a nationwide study. Ann Oncol 30:1143\u0026ndash;53.\u003c/li\u003e\n\u003cli\u003ePignochino Y, Capozzi F, D\u0026rsquo;Ambrosio L, Dell\u0026rsquo;Aglio C, Basiric\u0026ograve; M, Canta M, et al (2017) PARP1 expression drives the synergistic antitumor activity of trabectedin and PARP1 inhibitors in sarcoma preclinical models. 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Int Immunol 21:793\u0026ndash;801.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Supplementary Figure 4","content":"\u003cp\u003eSupplementary Figure 4 is not available with this version.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cancer-immunology-immunotherapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ciim","sideBox":"Learn more about [Cancer Immunology, Immunotherapy](http://link.springer.com/journal/262)","snPcode":"262","submissionUrl":"https://submission.nature.com/new-submission/262/3","title":"Cancer Immunology, Immunotherapy","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"NK/CIK cellular immunotherapy, cGAS-STING-IRF3-IFNβ pathway, trabectedin, olaparib, OCT4, persistent sarcoma cells","lastPublishedDoi":"10.21203/rs.3.rs-6463944/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6463944/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAdvanced sarcomas have poor prognosis and limited therapeutic options. Disease recurrence is caused by persistent cells that survive drug treatments. The alkylating agent trabectedin, when combined with the poly(ADP-ribose) polymerase 1 (PARP1) inhibitor olaparib, exhibits variable antitumor effects in advanced sarcomas. In this study, we demonstrated that the expression of the transcription factor OCT4 is upregulated in persistent cells surviving trabectedin and olaparib treatment through the cGAS-STING-IRF3-IFNβ pathway. This route also leads to the upregulation of natural killer (NK) and cytokine-induced killer (CIK) lymphocyte activating ligands. This upregulation promoted the antitumor effect of immunotherapy with NK and CIK cells against both bulk and persistent cells that survive drug treatment. In conclusion, activation of the cGAS-STING pathway due to trabectedin\u0026thinsp;+\u0026thinsp;olaparib treatment has a double-edged sword effect, enriching the OCT4\u003csup\u003e+\u003c/sup\u003e persistent cell population while increasing the expression of NK/CIK ligands. Therefore, sequential treatment with trabectedin and olaparib followed by NK/CIK immunotherapy is a promising strategy against advanced sarcomas and deserves further investigation.\u003c/p\u003e","manuscriptTitle":"The cGAS-STING pathway is a master regulator of OCT4 expression in persistent sarcoma cells and enhances cellular immunotherapy with NK and CIK lymphocytes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-02 09:27:03","doi":"10.21203/rs.3.rs-6463944/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-29T07:08:17+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-29T05:49:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"338512913647270981040367446603450387778","date":"2025-04-24T01:24:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"69148391139136326346219440575149480053","date":"2025-04-23T15:22:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"223357396933962628099541890133593798482","date":"2025-04-21T16:03:40+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-18T21:14:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"135102203589281955383152327610317549371","date":"2025-04-18T19:57:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"54904494958087998325534117572965453306","date":"2025-04-18T15:04:36+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-18T07:14:51+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-17T10:08:47+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-17T10:07:32+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cancer Immunology, Immunotherapy","date":"2025-04-16T13:15:24+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cancer-immunology-immunotherapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ciim","sideBox":"Learn more about [Cancer Immunology, Immunotherapy](http://link.springer.com/journal/262)","snPcode":"262","submissionUrl":"https://submission.nature.com/new-submission/262/3","title":"Cancer Immunology, Immunotherapy","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"149a3f31-a441-491e-9411-a1011ab337da","owner":[],"postedDate":"May 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-09-29T16:05:52+00:00","versionOfRecord":{"articleIdentity":"rs-6463944","link":"https://doi.org/10.1007/s00262-025-04141-w","journal":{"identity":"cancer-immunology-immunotherapy","isVorOnly":false,"title":"Cancer Immunology, Immunotherapy"},"publishedOn":"2025-09-23 15:57:18","publishedOnDateReadable":"September 23rd, 2025"},"versionCreatedAt":"2025-05-02 09:27:03","video":"","vorDoi":"10.1007/s00262-025-04141-w","vorDoiUrl":"https://doi.org/10.1007/s00262-025-04141-w","workflowStages":[]},"version":"v1","identity":"rs-6463944","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6463944","identity":"rs-6463944","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}