Endometrial cancer PDX-derived organoids (PDXOs) and PDXs with FGFR2c isoform expression are sensitive to FGFR inhibition.

Endometrial cancer PDX-derived organoids (PDXOs) and PDXs with FGFR2c isoform expression are sensitive to FGFR inhibition. | 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 Help Center Sign In Submit a Preprint Cite Share Download PDF Article Endometrial cancer PDX-derived organoids (PDXOs) and PDXs with FGFR2c isoform expression are sensitive to FGFR inhibition. Asmerom Sengal, Vanessa Bonazzi, Deborah Smith, Cristian Moiola, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2512859/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 12 You are reading this latest preprint version Abstract Endometrial cancer (EC) patients with metastatic/recurrent disease have limited treatment options and poor survival outcomes. Recently, we discovered the FGFR2c isoform is associated with poor prognosis in EC patients. Here we report the establishment of 14 EC patient-derived xenografts (PDX)-derived organoids (PDXOs) with or without FGFR2c expression. Treatment of 5 EC PDXOs with BGJ398 showed significant cell death in 3 models with FGFR2c expression. PDXs with FGFR2c+ showed significant tumour growth inhibition (TGI) following 21-day treatment with FGFR inhibitors (BGJ398 or pemigatinib) and significantly prolonged survival in 4/5 models. Pemigatinib + cisplatin combination therapy (n=5) resulted in significant TGI and prolonged survival in one of two p53abn PDXs. All five models treated with cisplatin alone showed de novo resistance and no survival benefit. Seven-day treatment with BGJ398 revealed a significant reduction in angiogenesis and CD206+ M2 macrophages. This data collectively supports the evaluation of FGFR inhibitors in a clinical trial. Health sciences/Oncology/Cancer/Gynaecological cancer/Endometrial cancer Biological sciences/Cancer/Cancer models Health sciences/Biomarkers/Predictive markers Health sciences/Medical research/Translational research Biological sciences/Biological techniques/Immunological techniques/Immunohistochemistry Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Background Endometrial cancer (EC) is the single gynaecological cancer that constantly showed a notable annual increase in both incidence and mortality in developed countries 1 . EC includes several histologic and molecular subtypes that have diverse prognostic outcomes. Traditionally, EC was classified as type I (well to moderately differentiated, endometrioid in histology associated with good prognosis) and Type II (poorly differentiated, non-oestrogen dependent with poor prognosis). In 2013, the Cancer Genome Atlas (TCGA) consortium identified four molecular subtypes with distinct prognostic significance 2 . These subtypes were subsequently validated using surrogate immunohistochemistry (IHC) biomarkers with Polymerase Ɛ-enzyme ( POLE) hotspot mutation analyses. For example, the McAlpine laboratory established a Proactive Molecular Risk Classifier for Endometrial Cancer (ProMisE) which simplified the TCGA approach 3 . The PORTEC consortium studies concurred with the ProMisE classification except for minor variations in nomenclature 4 . All published studies concluded that patients with POLE exodomain mutant have excellent prognoses; mismatch repair deficient (MMRd) and p53 wildtype (p53wt)/no specific molecular profile (NSMP) have intermediate prognoses, and p53 abnormal (p53abn) have the worst prognoses 2 , 5 , 6 . The molecular classification is endorsed by World Health Organisation and several clinical trials are currently evaluating using molecular subtyping in tailoring adjuvant treatment of EC 7 . Women diagnosed with metastasis or recurrent disease have limited treatment options with < 20% 5-year expected survival. The PORTEC-3 phase III clinical trial tested the addition of chemotherapy to radiotherapy compared to radiotherapy alone in high-risk women with EC 8 . Molecular subtyping of PORTEC-3 cohort tumour samples showed the combination of chemotherapy and radiotherapy increased survival for EC patients with the p53abn subtype but did not show significant benefit for patients with MMRd and NSMP/p53wt molecular subtypes 9 . The MMRd and p53wt/NSMP subtypes encompasses 80% of diagnosed ECs contributing to 50% of EC deaths and therefore, need additional treatment optimisation. Recently, immune checkpoint inhibitors (ICIs) have shown efficacy in EC patients with MMRd. The combination of pembrolizumab (anti-PD-1 antibody) + lenvatinib (primarily a VGFR inhibitor with reduced activity to other receptor tyrosine kinases (RTK) including FGFRs) has been shown to increase survival by 6 months in 40% and 30% of MMRd and proficient MMR (pMMR) patients, respectively in a large phase III clinical trial 10 . Following this study, the combination regimen was granted accelerated approval in several countries including USA, Europe and Australia. However, the combination of pembrolizumab + lenvatinib is not biomarker-driven and 90% of patients developed significant adverse effects 10 . Fibroblast Growth Factor Receptor 2 (FGFR2) is a member of the FGFR RTK family and has two main isoforms, FGFR2b and FGFR2c which are expressed in normal epithelial and mesenchymal cells respectively 11 , 12 . FGFR2 can be dysregulated via mutation, amplification, or gene fusion in different solid cancers. We have identified FGFR2 mutations in about 10–15% of EC 13 and shown that mutations were associated with shorter progression-free survival (PFS) and disease-specific survival (DSS). Recently, we discovered FGFR2c splice isoform expression due to isoform switching in ~ 50% and ~ 30% of MMRd and p53wt molecular subtypes respectively 14 . We demonstrated FGFR2c was an independent prognostic biomarker and was associated with shorter PFS and DSS compared to FGFR2b expressing tumours 12 . FGFR2c contributes to drive epithelial to mesenchymal transition (EMT), enhance cell motility and invasiveness and inhibits tumour differentiation in several solid cancer cell lines 15 – 18 . Previously, our laboratory demonstrated EC cell lines harbouring FGFR2 mutations are oncogene addicted and are sensitive to FGFR inhibition (PD173074 and BGJ398) in vitro 19 , 20 and in vivo using cell line xenograft models 20 , 21 . Currently, the US Food and Drug Administration (FDA) has approved infigratinib (BGJ398) and pemigatinib in intrahepatic cholangiocarcinoma with FGFR2 fusions as well as erdafitinib in advanced urothelial cancer with FGFR2/3 fusions 22 , 23 . Although we have identified FGFR2c isoform switching as a new mechanism of receptor activation in EC that is associated with poor prognosis, there is no functional data showing ECs with FGFR2c isoform expression show oncogene dependence and the role of FGFR inhibitors in preclinical models with FGFR2c expression is unknown. Despite the advances in molecular profiling of EC, discovery of novel targeted therapies for endometrial cancer is lagging, partly due to a lack of robust preclinical models that reflect the spectrum of molecular subtypes. Patient-Derived Xenografts (PDXs) and Patient-Derived Organoids (PDOs) are robust models for preclinical drug testing that better mimic patient tumour heterogeneity and molecular profiles. The objectives of this investigation were: 1) to establish 3D PDX derived organoids (PDXOs) from multiple EC PDX models representing advanced ECs with and without FGFR2c expression; 2) to assess the FGFR2c / FGFR2b status in the established PDXs and PDXOs and identify appropriate models with FGFR2c expression that can be targeted with FGFR inhibitors; 3) to target PDXOs expressing FGFR2c oncogenic splice isoform with FGFR inhibition 4) to assess the in vivo efficacy of FGFR inhibitors (infigratinib/BGJ398 and pemigatinib) as well as cisplatin, either alone or in combination in FGFR2c expressing EC PDX models representing various histologic and molecular subtypes. Results Characterization of EC PDXs-and PDXOs We have previously published detailed genomic profiling for 11 EC PDXs alongside the establishment of another 7 PDXs 24 . The current cohort contains total 21 PDXs including 3 newly established PDXs. The clinicopathologic characteristic and follow–up with clinical outcomes of patients from which these PDXs were derived is provided in supplementary Table 3. This panel of PDX represents the various histologic subtypes of EC including endometrioid endometrial carcinoma (n = 14), serous endometrial carcinoma (n = 2), clear cell carcinoma (n = 1) and uterine carcinosarcoma (n = 4). This cohort of PDX also included the four ProMiSE molecular subtypes including p53abn (n = 10), MMRd (n = 8), p53wt (n = 2), and POLE mut (n = 1) (Fig. 1 a). From the 15 independent PDXs tested for organoid culture 14/15 (93%) were successfully established and expanded. Nearly all the established PDXOs recapitulated the morphological pattern of the corresponding patients’ primary tumour and PDXs (Fig. 1 b). FGFR2 isoform status was determined for each primary tumour and matched PDX and PDXO via optimised and validated novel BaseScope RNA ISH assay which detects the FGFR2b and FGFR2c splice isoforms 14 . The expression of the FGFR2c splice isoform was highly consistent between the primary patient tumour and the matched PDXs and PDXOs (Fig. 1 c, Fig. 2 ). We observed four patterns of FGFR2c expression high (n = 5), moderate (n = 6), low (n = 5) and negative (n = 7) (Fig. 1 A) and representative images of patient primary tumours and matched PDXs and PDXOs are provided in Fig. 2 . There was no significant difference in morphology and proliferation across multiple passages of the PDXOs (P3-P10) ( supplementary Fig. 2 ). For the tested PDXOs multiple rounds of freeze thawing had no effect on organoid growth pattern, viability, and morphology ( supplementary Fig. 2 ). For 8 PDX models, PDXOs were generated from 3 or more independent mice carrying PDX tumours at multiple passages (F3-5) to ensure some in vitro functional experiments could be performed in biological triplicate. Differential Ligand Dependence Of Ec Pdxos Epithelial stem cell media contains a variety of growth factors to facilitate organoid proliferation including EGF, FGF2 as well as WNT3A, R-Spondin and Noggin (WRN). Based on our hypothesis that the splicing switch to FGFR2c establishes an autocrine loop in EC cells, we performed growth factor withdrawal experiments in PDXO59 and PDXO67 with FGFR2c expression and PDXO56 without FGFR2c expression (Fig. 3 ). Withdrawal of growth factors 24 hours following seeding showed that EC PDXOs with high FGFR2c expression (PDXO59 and PDXO67) did not require exogenous FGF2, EGF or WRN however, they were susceptible to treatment with 25 µg/ml anti-FGF2 and/or 100 nM BGJ398 and significant growth reduction and morphologic changes was evident. All PDXOs with FGFR2c expression were subsequently cultured without addition of exogeneous GF including FGF2, EGF and WRN and growth pattern was not altered. In contrast the PDXOs without FGFR2c expression showed a reduction in proliferation following removal of EGF and WRN and treatment with anti-FGF2 antibody and/or BGJ398 had no effect. This data suggested that initial growth of EC PDXOs with FGFR2c expression was dependent on endogenous FGF2 via establishment of an autocrine loop (Fig. 3 a-b). Indeed, IHC analyses of FGF2 on several primary patient tumour and corresponding PDXs and PDXOs revealed high FGF2 expression (Fig. 3 c, d). To confirm the specificity of FGF2 antibody and rule out the cross binding with other closely related FGF ligands, the FGF2 antibody used was validated via western blot analyses in BaF3 cell lines stably transduced with a subset of individual FGF ligands ( supplementary Fig. 3 ). For PDX67, initial culturing of the PDXO in 2D culture without Matrigel led to the development of an attached EC primary cell line (ASPX67-406) and named with first author initials and the PDX number. To assess whether FGFR2c was activated via endogenous FGF2 in EC, the ASPX67-406 EC primary cell line was incubated for 30 min with supplementation of 5 µg Heparin Sulphate (HS) with and without 10 ng/ml FGF2 in the presence of DMSO (control) or 100 nM BGJ398 in standard media (10% FBS in DMEM/F12) or following overnight (16 hr) serum starvation (0.5% FBS in DMEM/F12 media) (Fig. 4 ). High PLA signals was observed in cells stimulated with HS + FGF2 in 10% FBS full growth media however, a similar signal of PLA was noted without the addition of exogenous FGF2 (Fig. 3 b, c) and no PLA signals were seen when BGJ398 was added, confirming specificity of the PLA for detection of pFGFR2c. Following serum starvation for 16 hours, high PLA signals were seen with stimulation of exogenous FGF2 but not in the absence of FGF2 suggesting that serum starvation reduced FGF2 secretion. A reduction in endogenous FGF2 expression following serum starvation was then confirmed by IF in this cell line model (Fig. 4 d). We then performed PLA to detect phosphorylated FGFR2 to confirm autocrine activation of FGFR2c by endogenous FGF2 was driving EC organoid growth. Activation of FGFR2c was confirmed by PLA signals when PDXO67 was cultured in stem cell advanced media with and without exogenous FGF2 and these PLA signals were reduced in the presence of 100 nM BGJ398 (Fig. 2 e-f). To investigate further the downstream signalling activation, we have also determined expression pERK1/2 and pSTAT3 in BGJ398 treated ASPX67-406 primary cell line and PDXOs and phosphorylation was tracked using an in situ IF stain. Notably, BGJ398 treated ASPX67-406 cells and PDXOs demonstrated a significant reduction in pERK1/2 and pSTAT3 expression ( supplementary Fig. 4 ). While data in supplementary Fig. 3 showed initial culturing of organoids was sensitive to 100nM BGJ398, this does not reflect treatment of well-established cancers in patients. Therefore, we tested increasing concentrations of BGJ398 on well-established PDXO67 (FGFR2c high) and PDX56 (FGFR2c negative) organoids grown for 10 days at different time points treatment (24 hr, 48 hr and 72 hr). In this context 100nM BGJ398 had limited effect on organoid morphology, proliferative capacity, and viability at different time points of treatment whereas 300nM BGJ398 for 72 hr revealed marked reduction in proliferation, alongside marked cell death/necrosis and a cystic morphology in PDXO67 but not evident in PDX56 ( supplemental Fig. 5a, d ). Finally, five independent PDXO models were treated with 300nM BGJ398 or 0.01% DMSO vehicle for 72 hours. The three EC PDXOs with high FGFR2c isoform expression were established from PDX59 and PDX67 (MMRd) and PDX23 (p53abn) and the two PDXOs with very low or negative FGFR2c isoform expression (RNA ISH signals < 10/tumour cells) were established from PDX53 (EEC MMRd) and PDX56 (UCS p53abn). To ensure the most robust results, three independent organoid cultures were derived from three different mice each carrying the individual PDXs to represent true biological replicates. Using the LIVE/DEAD® assay followed by imaging, significant cell death ( P < 0.0001) was observed in PDXO models expressing high FGFR2c isoform (PDX23, PDX67, PDX59) but not in PDXOs with FGFR2c negative/low expression (PDX56 and PDX53) or vehicle treated organoids (Fig. 5 ). In vivo targeting of EC PDXs with Infigratinib/BGJ398 and Pemigatinib FGFR inhibitors Both infigratinib (BGJ398) and pemigatinib are orally bioavailable FGFR1-3 specific inhibitors that are approved as second line treatment for patients with advanced intrahepatic cholangiocarcinoma with FGFR2 dysregulation. Initially, five independent EC PDXs were treated with either 30 mg/kg BGJ398 (PDX52 and PDX59, MMRd FGFR2c high and PDX68 MMRd, FGFR2 mutant) or with 1 mg/kg Pemigatinib (PDX58 MMRd, PDX60 p53wt both FGFR2c moderate expression) daily for 21 days. Four of the five PDX models treated with FGFRi showed significant tumour growth inhibition (TGI) and had significantly longer survival compared with the vehicle treated arms (Fig. 6 a). PDX58 showed de novo resistance to FGFRi and currently we are investigating the mechanism of resistance in this model. Cisplatin is one of the first line standard of care chemotherapeutic agents for EC patients with metastatic disease or at high risk of recurrence. To evaluate if the addition of pemigatinib sensitises EC cells to cisplatin, 5 independent PDX models were treated with 1 mg/Kg pemigatinib alone, pemigatinib plus cisplatin or cisplatin alone (Fig. 6 b-c). All five models treated with cisplatin alone showed de novo resistance to cisplatin and no survival benefit. The addition of cisplatin to pemigatinib had also no significant benefit to increase survival in PDXs with MMRd molecular subtype (PDX52, PDX59 and PDX67) (Fig. 6 b). However, variable response was found in PDXs representing the p53abn subtype of EC (Fig. 6 c). We noted the combination of pemigatinib with cisplatin contributed to reduce tumour growth and prolonged survival in PDX23. In contrast FGFRi alone did show a significant improvement in survival for PDX61 with no additional benefit seen following combination with cisplatin (Fig. 6 c). Treatment related toxicity or weight reduction were not observed in mice treated with FGFRi implicating clinical safety in patients however, 95% of the mice treated with both FGFRi and cisplatin often presented with ruffled fur potentially due cisplatin toxicity. We have assessed and compared the PDX tumour morphology and tumour growth characteristics between vehicle treated (tumour collected when the target volume of 900mm 3 was reached) and the residual tumour regrown after completion of treatment (collected at last follow up day when tumour reached the target volume ~ 900mm 3 ). Consistent with the PDXO findings, treated PDX tumours demonstrated significant central necrosis, cystic formation and tumour differentiation that were not evident in mice from the vehicle treated control arm (supplementary Fig. 6) . We noted 21 days treated PDX tumours had small residual tumour with small area of viable cells that may not be sufficient to compare with vehicle treated tumours. Therefore, we treated 4 independent PDX models with 30mg/Kg BGJ398 for 7days to compare the tumour proliferation capacity and other tumour microenvironment markers (TME) between control and treated PDXs. As expected, a significant reduction in expression of Ki67 was observed in BGJ398 treated compared to vehicle treated PDX tumour (Fig. 7 a-b), consistent with our in vitro findings. Previous studies also demonstrated FGF2/FGFR signalling contributes to in vivo angiogenesis and immune modulation and treatment with FGFRi reduces angiogenesis and tumour growth 25 . In this study, we assessed the tumour microvessel density (MVD) via IHC staining of CD31 in four independent EC PDXs tumours (three with FGFR2c expression and one with FGFR2 mutation) treated with either BGJ398 or vehicle for 7 days. A significant reduction of MVD (CD31 reactive vessels) was found in BGJ398 treated PDXs compared to control (P < 0.0001) (Fig. 8 a-b). Several studies reported that FGF/FGFR signalling pathway modulates the tumour microenvironment (TME) and contributes to polarization of tissue associated macrophages (TAM) M2 macrophages 25 . We evaluated the effect of BGJ398 on EC PDXs TME and demonstrated a significant reduction in CD206 expressing macrophages (M2) following 7 day BGJ398 treatment (Fig. 9 a-b). Discussion Women with advanced EC have limited treatment options and current available treatment modalities are suboptimal. Identifying effective targeted therapies for metastatic and/or recurrent ECs and robust predictive biomarkers are a major unmet clinical need. Development of effective personalised targeted therapies requires authentic preclinical models that represent the morphological and molecular profiles of the patient tumours with high fidelity. In this study, we presented the establishment, expansion, and characterisation of EC PDXOs and demonstrated that EC PDXOs with expression of the oncogenic FGFR2c splice isoform were highly sensitive to FGFR inhibition. In vivo validation using corresponding PDX models confirmed that treatment with clinically approved FGFR1-3 specific inhibitors markedly reduced tumour growth and increased survival. We also showed the impact of FGFR inhibitors on reduction of EC cell proliferation and TME, evident by reduced angiogenesis (CD31 + microvessel density) as well as reduced TAMs (CD206 + M2 polarised macrophages). Establishment of PDOs from normal endometrium, benign diseases, and well-differentiated early-stage ECs have been previously reported 26 , 27 . The success rate of PDXOs establishment and expansion in this study was very high (93%) compared to previous published report (22%) from primary patient tumours 28 . The high success in our study likely reflects the selection of more aggressive tumours in those that initially established PDXs or an increased cancer stem cell population during expansion of the PDXs. Our established PDXOs predominantly represent G3 ECs with different histological types as well as the four molecular subtypes of EC that are clinically relevant for drug discovery and testing. The PDXOs are very stable and retain the morphological and molecular characteristics of original parental donor tumours after recovery from cryopreservation or several series of passaging (P15). This ensures the potential utility of our PDXOs for future drug or drugs combination screening to optimise precision therapy of EC patients. Initial screening of the primary patient tumours, PDXs and matched PDXOs for FGFR2 isoform status revealed four patterns of FGFR2c splice isoform expression (high, moderate, low and negative) consistent with the previous finding in large clinical cohort of EC patients 14 . Utilising three MMRd PDXO models, we noted the growth factor requirements were dependent on which pathways were activated to maintain stemness, regeneration and proliferation, with two PDXO models expressing FGFR2c not reliant on EGF, WNT5A, Noggin or R-Spondin. This finding suggests heterogeneity of EC PDXOs growth factors requirement. Previous studies reported different growth factor requirements for self-renewal for PDOs depending on activation of different driven oncogenic pathways 29 , 30 . Otte and colleagues reported colon cancer PDOs with KRAS mutation were dependent on FGF2 and EGF for regeneration and stemness 30 . We demonstrated PDXOs with FGFR2c expression were dependent on endogenous produced FGF2 for growth/proliferation and self-renewal and blocking of FGF2 with FGF2 specific antibody showed reduction in PDXOs number and size when incubated after initial 24 hr seeding period. We also showed that a lower concentration of BGJ398 was required to inhibit growth and regeneration of PDXOs if treatment was initiated 24 hrs following organoid seeding, but 300 nM of BGJ398 were required when treatment was initiated in mature and well established PDXOs (10 days after seeding), consistent with our previous finding with EC cell lines with FGFR2 mutations 20 . Morphological analyses of the well-established PDXOs revealed 72 hr of 300 nM BGJ398 results in degeneration of the organoids and inhibition of proliferation. The requirement for a higher dose of BGJ398 for killing PDXOs once they are well-established may reflect reduced drug diffusion/penetration in mature well established PDXOs or may reflect an increased dependency of FGF/FGFR signalling during initial organoid establishment, consistent with its role in stem cell maintenance. To better understand activation of the FGFR2c signalling pathway, receptor phosphorylation was assessed via a specific in situ PLA assay in a primary cell line derived from G3-EC PDX67 as well as in PDXO67 organoids with and without exogenous FGF2 stimulation and FGFR inhibition (BGJ398). This revealed high FGFR2c phosphorylation due to high endogenous FGF2 expression in 10% FBS with reduced endogenous FGF2 expression in serum starved conditions. In organoid culture, high FGFR2c phosphorylation was seen with or without addition of FGF2 indicating autocrine stimulation. IHC assessment of PDXs and PDXOs revealed high expression of FGF2 in the 8 models that were examined. However, there is redundancy in FGF/FGFR signalling and FGFR2c can be potentially activated by additional ligands including FGF1, FGF9 and FGF18. Our data showed the constitutive FGFR2c phosphorylation through an FGF2/FGFR2c autocrine loop was a principal pathway of FGFR2c isoform activation in contrast to the paracrine activation of FGFR2b in normal epithelial cells 12 . After optimisation of drug concentration, we assessed the effectiveness of FGFRi (BGJ398) in EC PDXOs with and without FGFR2c splice isoform expression. Treatment of EC PDXOs expressing FGFR2c led to cell death at 72 h only in the three PDXOs expressing FGFR2c and not in the two EC PDXOs with low/no expression. FGF2 is an empirical growth factor component of stem cell media and indicated to be a principal master regulator of cancer stem cells 31 , pluripotent adult stem cells, and embryonic stem cells 32 , 33 . Otte and colleagues reported FGF2/FGFR signalling is critical for growth, self-renewal of colon cancer PDOs and treatment with FGFR inhibitor supressed PDOs growth and regeneration 30 . Enhanced expression of FGFR2c has also been reported to play an essential role in pancreatic cancer cell proliferation and this also supports our current finding 34 . Studies have reported FGF2/FGFR downstream signalling through the ERK1/2-MAPK 32 and JAK/STAT3 35 pathway contribute to stemness. We demonstrated both pERK1/2 and pSTA3 are expressed in vehicle treated PDXOs and primary cell line but markedly reduced in BGJ398 treated counterparts. However, additional functional mechanistic investigations are needed to understand how these signalling pathways contributes to stemness maintenance in EC and whether additional FGF ligands are also involved in EC. Flix et al. reported FGF2 expression is associated with aggressive clinicopathologic markers and poor survival outcomes in EC 36 , consistent with our finding that FGFR2c isoform expression is associated with prognosis and survival in EC 12 . To validate our findings in vivo , multiple PDX models were treated with clinically relevant doses of 30 mg/Kg BGJ398 or 1 mg/Kg of Pemigatinib. Both BGJ398 and Pemigatinib demonstrated significant tumour growth inhibition consistent with our in vitro findings in 4/5 models expressing FGFR2c. This initial 21 day treatment also led to a significant doubling of survival for these four models sensitive to FGFR inhibition. Notably, morphologic assessment of tumours when they reached ~ 900mm 3 after withdrawal of treatment showed significant central necrosis and cystic formation with a small residual rim of tumour was evident. This indicates gross tumour volume did not correspond to tumour burden following FGFRi treatment. This observation has clinical relevance regarding the type of imaging used to assess tumour shrinkage and treatment response in patients in the clinic and suggests that CT morphological criteria or other functional imaging would be superior to RECIST size based criteria to more accurately reflect viable tumour burden. Molecular profiling of tumours collected in the PORTEC-3 clinical trial showed that chemotherapy had significant clinical benefit only in patients with p53abn tumours with no improvement seen in patients with MMRd tumours. FGFR inhibition has been shown to sensitise cell line and PDX xenografts to cisplatin in other cancer types 37 . For this reason, we wanted to assess if the combination might be effective in MMRd tumours where FGFR2c expression is most common. We report here that although cisplatin had some effect on TGI during the initial 21d treatment, neither cisplatin alone nor the addition of cisplatin to pemigatinib had any benefit on survival in three independent MMRd PDXs with FGFR2c expression. In contrast PDX61 representing a p53abn EC with moderate FGFR2c expression showed intrinsic in vivo resistance to cisplatin however, in vivo data showed significant TGI and improved survival with pemigatinib alone with no further benefit with the combination. PDX23 has wildtype p53 by sequencing but represents serous EC with a copy number high genomic profile 24 . For this model significant TGI was observed with all three treatments however only the combination of cisplatin and pemigatinib resulted in a significant improvement in survival. We have also demonstrated EC PDXs with FGFR2c expression or an FGFR2 C383R mutation treated with BGJ398 for 7 days had marked reduction in cancer cell proliferation, angiogenesis and pro-tumourigenic CD206 + M2 polarised macrophages revealing that FGFR inhibition plays a significant role in modulation the TME in addition to its anti-tumour effect. FGF/FGFR signalling has previously been shown to promotes tumour growth and metastasis through enhancing angiogenesis and treatment with FGFRi reduces CD31 + microvessel density in other solid cancers including breast and lung cancer 25 consistent with our finding. Microvessel density has also been associated with poor prognosis and shorter survival in EC 38 , 39 . Tumour associated Macrophages (M2 macrophages) expression is reported to be associated with EC progression and poor clinicopathologic biomarkers 40 , 41 . In other cancer types FGFR dysregulation plays a significant role in tumour immune evasion and importantly FGFRis have been shown to synergises to increase the efficacy of immune check inhibitors (ICI) 42 – 44 . We have also noted 7 days of BGJ398 treatment results in an increase in the differentiation of EC, with clear evidence of glandular morphology formation after treatment compared with an undifferentiated solid morphology in vehicle treated mice. This is consistent with the role of FGFR2c to promote dedifferentiation of cancer cells and suggests it may be another mechanism by which FGFR inhibition exerts its anti-tumour activity in ECs with FGFR2c expression. In conclusion, we have developed and characterised EC PDXOs that highly biomimic the morphological and molecular profile of advanced ECs and their corresponding PDX models. We have identified authentic preclinical models with different pattern of FGFR2c oncogenic splice isoform expression and showed PDXOs with FGFR2c expression are dependent on endogenous FGF2 for its autocrine activation. Finally, the study revealed PDXOs and matched PDXs with FGFR2c expression are highly sensitive to FGFRis (BGJ398 and pemigatinib). The combination of pembrolizumab and Lenvatinib has recently been approved for all EC subtypes however a very high proportion of patients (~ 90%) developed toxicity and complete responses are low. Our data suggest a combination of a FGFRi with ICI could potentially be more effective with less toxicity in those EC patients with dysregulation of FGFR2 either by mutation (15%) or isoform switching (30–40%) 12–14 . A phase II clinical trials are ongoing in EC patients with FGFR2 mutations/fusions and more EC patients may benefit with inclusion of Patients with FGFR2c expression. A study from urothelial carcinoma showed FGFR2/3 dysregulation contributes to ICI resistance 45 but more research into the role of the FGFR2c in immunotherapy resistance is warranted. We have also demonstrated that pemigatinib treatment could improve survival in a subset of p53abn EC models with intrinsic cisplatin resistance suggesting FGFRi + ICI may have some benefit in the second line cisplatin resistant setting. Collectively the data presented confirm the expression of the FGFR2c splice isoform as a precision predictive biomarker in EC. Due to the role of FGFRs in tumour immunosuppression, we propose a clinical trial of an FGFRi plus ICI in advanced EC with a MMRd arm (where FGFRi may overcome some mechanisms of ICI resistance) and a pMMR arm. Inclusion of the latter arm is supported by the activity of Lenvatinib and pembrolizumab in non-endometrioid ECs and our data showing sensitivity to FGFRi in 1/2 P53abn EC models and a p53wt model. As there is a range of FGFR2c expression in ECs, retrospective analysis could then be performed to determine the cut-off at which FGFR2c ISH functions as a predictive biomarker to improve survival. Materials And Methods PDXs tumour expansion and characterization We have previously established 18 EC PDXs with different histological and molecular subtypes 24 . Tumours were collected from EC patients following Human Research Ethics Committee (HREC) approval (HREC/15/MHS/127), QUT HREC (#1500000169, 1500000323). All participants provided written informed consent to take part in the study. Ethical clearance for laboratory animal use was granted from UQ Animal Ethics Committee (AEC) (UQ/TRI/021/19) and QUT (1900000701). We have also re-established three PDXs that were initially developed by the laboratory of Eva Colas and Antonio Gil-Moreno at the Vall Hebron Institute of Research (VHIR) in Spain with ethical approval (PR(AMI) 276/2018) under QUT HREC approval (#5194). Fresh surgical resected tumours either from primary or distant metastatic site were collected from patients who had surgery and fragment of tumours were implanted subcutaneously into immunocompromised Nod Scid Gamma (NSG) 8 week female mice as described previously 24 . Serial passaging of the PDXs was carried out as previously published. In brief, when the PDX tumour reached a target volume of 900mm 3 , mice were euthanized with CO 2 and each tumour was collected under sterile technique, with ~ 1–2 mm3 fragments transplanted fresh into 2–3 mice for subsequent passaging as well as frozen with 90% FBS and 10% DMSO for later transplantation (biobanking). Portions of the tumour were also frozen for DNA/RNA and protein extraction, fixed in formalin and embedded in paraffin (FFPE) to assess for morphology and FGFR2 expression via IHC/RNA ISH as well as placed in media for subsequent organoid culture (Supplementary Fig. 1). Genomic characterization (WES, WGS) was performed in 11 PDXs as published previously 24 . IHC to assess FGFR2, p53, Ki67 expression, MMR protein status and RNA ISH was performed in matched patient tumours, PDXs and PDXOs at different passages (P2-10) to confirm PDXOs recapitulated PDX models. Ec Pdx Derived Organoids (Pdxos) Establishment And Expansion PDXOs culture was performed according to previously reported protocols 28 with slight modification to customise the need of growth factors depending on tumour type. In brief, PDX tumours were collected following a sterile procedure, dissected and a portion of tumour was placed in RPMI medium containing antibiotics (Penicillin/streptomycin) and antimycotic and transported on ice to the tissue culture (TC) room. PDX tumours were minced utilising a surgical blade and dissociated into clusters of tumour cells by mechanical dissociation and enzymatic degradation of the extracellular matrix using the HumanTumor Dissociation Kit (Cat#130-095-929, Miltenyl Biotech, VIC, Australia) following the manufacturer's protocol. The cell suspension was centrifuged, and the pellet was resuspended in DMEM/F12 medium containing 2 mmol/L L-glutamine with antibiotics (100 µg/ml Promicin (cat# amp-1, Jomar life Research, VIC, Australia) or 100 U/mL penicillin and 100 mg/mL streptomycin). The final suspension of tumour cells was then filtered through a 40 or 70-µm cell strainer (Biostrategy, VIC, Australia) to remove large clusters and the cells that were not filtered out were resuspended in DMEM/F12 medium containing 2 mmol/L L-glutamine, antibiotics (100 µg/ml Promicin (cat# amp-1, Jomar life Research, VIC, Australia) and 10 mmol/L (Y-27632) ROCK inhibitor (Selleck Chemicals Inc) ± growth factors WRN (WNT3A, R-Spondin-3, NOGGIN), EGF and FGF2 as required and was plated into the centre of 48 well plate precoated with 50% V/V) Matrigel™ (growth factor reduced, GFR) (cat# 354230, Corning, VIC, Australia) and incubated at 37 ⁰C and 5% CO 2 . When Matrigel™ was gelled, 200µL of expansion media was added to cover Matrigel droplets and media was changed every 3 days. PDXOs growth was monitored using an inverted phase contrast microscope and images were captured to document growth using a Nikon phase contrast microscope with camera attached. Established PDXOs were further passaged and were subjected to multiple sequential cycles of freezing and thawing to ensure the renewal stability and fitness of the organoids. PDXOs were banked at multiple passages by cryopreservation in liquid nitrogen. For establishment of primary PDX-derived EC cell lines, dissociated cells were cultured in 2D in a 10cm petri dish in 10% FBS with DMEM/F12 Media and cell selection was performed with differential trypsinisation (0.25% trypsin EDTA) to remove fibroblasts. After 4 successive passages, 100% purity was confirmed with a cytokeratin 7 stain. Mycoplasma testing was performed routinely using a commercial kit (MycoSEQ and Thermo-Fisher Scientific, VIC, Australia). PDXOs Formalin-Fixed Paraffin-Embedded (FFPE) Blocks Preparation. The medium was removed and replaced with cold medium, and the plate was cooled on ice to liquify the Matrigel to make PDXO suspension. The PDXOs suspension were gently transferred into yellow screw cap, flat base 5ml Polypropylene tube (Sarstedt, Ref.# 60.9921.524) and kept at room temperature (RT) for 20 min and then centrifuged at 300 g at 4°C for 4 min and then supernatant was discarded and PDXO pellets were washed with PBS twice and fixed with 4% PFA in PBS for 1 hour at RT. Then PDXO pellets were centrifuged at 300 g for 4 min at RT and the fixative was discarded. A 2% Histogel/agarose gel (Cat. # HG-4000-012, Thermo Scientific, VIC, Australia) in PBS was prepared and melted by heating in microwave and then cooled to 60°C prior to adding to the PDXO pellets. A 200 µL melted agarose gel was added to PDXO pellets, allowed to solidify at RT for 30 min or 10 min at -20°C, then carefully transferred to tissue cassettes with sponge to avoid loss of small PDXOs, placed in 70% ethanol and processed using an automated 4 hours processing protocol in the TRI-Histology core facility and embedded with paraffin to make FFPE blocks. H/E and IHC staining and BaseScope RNA ISH assay was performed as described previously 14 , 46 . Fgfr2 Isoform Determination FGFR2b and FGFR2c splice isoform expression on primary EC patient and matched PDX and PDXOs tumours was determined following our previously optimised and validated BaseScope RNA ISH assay 14 . Molecular Subtyping Molecular subtyping of the EC patient, PDX and matched PDOs tumours were performed using the ProMisE algorithm combining genomic analyses (tumour mutation burden, somatic copy number alteration) or IHC surrogate markers as previously reported 3 . IHC was done for several markers including p53, MLH1, MSH2, MSH6 and PMS2. Ligand Stimulation Of Primary Cell Line And Pdxos Primary cell line (ASPX67-406) cells (P4) were cultured in 10% FBS DMEM/F12 media and after reaching 70% confluence, they were trypsinised and 1 x 10 5 cells were seeded in 6 well plates containing 3 coverslip slides/well (1.13 mm Ø) with DMEM/F12 media for 48 hours at 37°C and 5% (v/v CO 2 ). After cells reached 60–70% confluence, media was removed, cells were washed with warm DPBS and either incubated with fresh 10% FBS media or starved overnight (16 hr) with 0.5% FBS DMEM/F12 media. At zero time point (T0) media was removed and washed with warm DPBS twice and stimulated with media containing 0.5% FBS with 5 µg/ml HS with or without 10 ng/ml FGF2 and incubated for 30 min at 37°C and 5% CO 2 . For the treatment arm 100nM/ml BGJ398 was added during stimulation. Cells were then washed with DPBS twice and fixed with 4% Paraformaldehyde (PFA) in PBS for 30 min at room temperature (RT) and PLA was performed. For PDXOs, culturing was performed in advanced DMEM/F12 Media with 30% (V/V) Matrigel™ with and without supplementation of FGF2 as described above and at Day 10 media was removed and washed with DPBS and fresh advanced media with and without HS + FGF2 and incubated for 30 min. For the treatment group 100nM BGJ398 was added during ligand stimulation. After 30min, the media was removed and PDXOs were fixed with PFA for 1 hour and resuspended and stained with Immunofluorescence (IF) or Proximity Ligation Assay (PLA) assay as described below. Immunofluorescence (If) And Proximity Ligation Assay (Pla) Staining Immunofluorescence and Proximity Ligation Assay (PLA) staining was performed manually using optimised and validated protocols. In brief, culture media was removed and cells and/or organoids were washed with PBS and fixed with 4% paraformaldehyde (PFA) with incubation for 30 min. PFA was removed and cells/organoids were washed three times with PBS/TPBS and permeabilised with 0.25% TritonX100 in PBS for 5 min at RT and washed with PBS/ TBS three times. Cells were blocked with 2% BSA in PBS for 1 hr at RT or overnight at 4°C. After that, cells were transferred into a humidity chamber and incubated with primary antibody diluted with 2% BSA in PBS (Suppl Table S1 for respective antibody source and dilution) and incubated for 90 min at room temperature or overnight at 4°C. Slides were then washed three times with TBS or PBS for 5 min each and incubated with secondary antibodies (Suppl Table S2 for dilutions and sources) for 45 min at RT. For PLA staining, primary antibodies (antiFGFR2 and pFGFR653) were incubated for 90 min after 3 times wash with TBS, cells/PDXOs were incubated for 90 min with PLA probes (oligonucleotide conjugated donkey anti-rabbit, PLUS (DUO82002) and donkey anti-mouse, MINUS (DUO82004)). After 3x washes with TBS a ligation and amplification step were followed according to the manufacture protocol (detail guideline suppl method A). To combine PLA with IF, the primary antibody was incubated for 60 min at 37 o C after the amplification step and then washed 3 times with TPS for 2 min each. Then, secondary antibody conjugated with FITC was incubated for 30 min at 37 o C and washed with TPS twice for 2 min each. Finally, cells/organoids were washed two times with PBS or TBS for five min each and final wash was performed with milliQ H 2 O to remove any precipitated salt and mounted with ProLong Gold Antifade with DAPI mounting medium (Thermo-Fisher, VIC, Australia). In vitro treatment of PDXOs with BGJ398 To test the effect of FGFR inhibitors in PDXOs regeneration and growth inhibition, PDXOs were initially treated at early time point following seeding (after 24 hr) with 100 nM BGJ398 or at a later timepoint when the organoids were well established (after 10 days) with different concentrations of BGJ398 (100 nM, 200 nM and 300 nM) at different time point (24 hr, 48 hr and 72 hr). To assess the effect of endogenous FGF2 on growth and regeneration, PDXOs were cultured with and without of anti-FGF2 antibody. After optimising the dose and duration of treatment, three independent PDXOs with high FGFR2c expression and two with negative or low FGFR2c expression) were grown for 10 days. After PDXOs reached 70–80% confluency, PDXOs were treated with either 300 nM infigratinib/BGJ398 (Selleck Chemicals Inc, VIC, Australia) or vehicle (equal volume of DMSO) and incubated for 72 hours at 37⁰C and 5% CO 2 . The viability of the PDXOs was then assessed using a Live/Dead assay kit (Cat# R3760, Thermo Fisher Scientific, VIC, Australia) following the manufacture’s protocol. This experiment was performed in 3 different PDXOs models derived from each PDX as well as in technical triplicate. Images were captured XYZ stack using an inverted rotatory confocal fluorescent laser microscope (FV1200) at 10x objective magnification view airy. Image analyses to quantify the live and dead cells were performed using Fiji ImageJ2 (National Institute of Health, Bethesda, Maryland, USA) as described previously 47 . In brief, the captured images were converted into TIF format and imported for Fiji ImageJ and image of PDXOs were imported to Fiji ImageJ2 platform. For each channel (Green, red, and blue) and Otsu’s threshold was used to create binary images (white and black) and level of pixel intensity was assigned between 1 (white) and 0 (black), object was identified using region of interest (ROI) and analysis set in parallel sequential. Total number of cells for each channel (live = green) and (red = dead) were calculated as follows; number of green positive cells divided by the number of nuclei (DAPI positive), likewise number of red positive cells divided by the total number of nuclei. The results were exported to SPSS for statistical analysis. Western Blot For Fgf2 Antibody Validation BaF3 cells (primarily sourced from ATCC, Manassas, VA) were previously transduced with FGF1, FGF2, FGF3, 7–10, 17, 18, 20 and 22 ligands tagged with Myc-DDK™ by our laboratory 48 . Protein extraction and western blot analysis were performed as previously published 20 . Twenty micrograms of protein lysates were loaded from BaF3 cell lysates expressing targeted ligands and probed with anti-FGF1, anti-FGF2, anti-FGF3 and anti-FGF7 antibodies (see supplementary Table S1 and 2 for source and cat#) to assess potential immune-cross reactivity. Cell lysates were probed with an anti-Myc antibody to demonstrate that each cell line was expressing the transduced FGF ligands. In Vivo treatment of PDXs The experiment was conducted according to the approved protocols AEC (#021/19, 399/20, 456/20) and the Australian National Health Medical Research Council (NHMRC) guidelines. EC tumour fragments were implanted into 6-8-week female NSG mice subcutaneously under sterile technique for each model. When the tumour reached 150-200mm 3 mice were randomized into vehicle, 30 mg/kg infigratinib/BGJ398 (3 models), 1 mg/Kg pemigatinib (Incyte) alone (7 models), pemigatinib + cisplatin (5 models), or cisplatin alone (5 models). Two PDX models (PDX52 and PDX59) were treated with both infigratinib and pemigatinib. Mice were treated for 21 days with infigratinib and pemigatinib via oral gavage and 5 mg/Kg cisplatin intravenous (IV) injection weekly. Tumours were measured using calliper 3 times/week until reaching 900mm 3 . Tumour volume (TV) was calculated as follows: Tumour Volume ( TV) = Length[width x width] 2 /2 . Follow up data regarding tumour size was collected 3x/w until tumours reached 900mm 3 . For tumour biomarkers assessment and mechanistic studies, short period (7 day) treatment was aimed to compare apparently equal tumour volume. For this aim, a separate cohort of 4 independent PDX models were treated with BGJ398 or vehicle (4 mice per arm) for 7 days. Mice were euthanised after 6 hr of the last treatment dose and their tumours were collected, cryopreserved or fixed with 4% paraformaldehyde for H/E morphologic examination and IHC biomarker evaluation. Immunohistochemistry And Quantification IHC staining of p53 and MMR proteins was performed via Ventana automated stainer (Ventana Medical Systems, Tucson, AZ, USA) using the diagnostic antibodies at Mater Pathology, Brisbane Australia. Detail information regarding these antibodies’ sources and cat# are provided in supplementary Table S1 and S2. The remaining IHC staining was performed manually following optimised protocols. In brief, whole FFPE tumour tissue sections were cut (4 uM thickness) and sections were dewaxed, hydrated and washed, and endogenous H2O2 was quenched with 3% H 2 O 2 for 10 min. Antigen retrieval was performed in 10 mM citrate buffer (PH = 6.0) with Decloaking Chamber™ (BIOCARE MEDICAL, Brisbane, Australia). Respective Biotin (Jackson Immunology Labs) or polymer conjugated secondary antibodies for all primary antibodies raised in rabbit or mouse (K406311-2, DAKO, Envision kit, Agilent Technologies, VIC, Australia) were used. Detailed information for all primary and secondary antibodies source, Cat#, period of incubation are provided in supplementary Tables 1 and 2. Chromogen development was performed using DAB (K3468, DAKO Envision) by incubating for 2–5 min and slides then counter stained with Mayer Haematoxylin (Sigma Aldrich). P53 and MMR proteins (MLH1, MSH2, MSH6 and PMS2) IHC scoring was performed as previously reported 3 . Quantification of other IHC/IF markers was performed using either automated Fiji ImageJ2 or QuPath bioimage analyses software following the guidelines as published previously 47 , 49 . In brief, whole section stained slides were scanned using whole slides scanner (3DHISTECH) at 40X magnification and images in the form of TIF files were imported to either ImageJ2 or QuPath software as indicated to create separate projects for each biomarker of interest or H/E stain. Quantification was performed after training and setting the required annotation, deconvolution and DAB vector intensity for each biomarker based on the algorithms and workflow of Fiji Image J2 or QuPath platform ( https://github.com/qupath/qupath ) 50 . Results were exported into SPSS or GraphPad Prism as required for data analyses. Statistical analysis Statistical analysis and data visualization was performed using SPSS version 23 and GraphPad Prism ver9.2 as required. Figures annotation. formatting and visualization were performed using Adobe illustrator (AI). Pearson’s (Chi X 2 ) test was used to evaluate correlations between categorical variables. Student T-Test (two-sided) was used to assess continuous variables. Two-way ANOVA with mixed model was used to assess tumour growth inhibition. Survival outcome differences between or among groups was computed using Kaplan Meier Curve with Mantel-Cox (Log-Rank) test of probability. Multiple comparison was corrected using either Bonferroni for categorical variables or Tukey test for continuous variables. P value < 0.05 (two sided) was considered statistically significant. Declarations Acknowledgement The authors would like to thank to all women for their generous support of offering their tumour samples for generation of PDXS. We would like to thank the TRI animal facility group for their enormous technical support for the mouse work. We are also grateful for the TRI histology and microscopy core facility as well as QUT histology core facility members at Kelvin Grove for whole slide scanning and other technical support. The authors also appreciate the generous support from John Hooper laboratory. We would like also to acknowledge the study was partially funded by Incyte INC. though investigator initiated study (IIS) scheme (Grant number # IPAP2020/1611). Funders have no role in study conception, design, data collection, analysis, interpretation, or writing. The Translational Research Institute (TRI) Australia is supported by a grant (APP108382) from the Australian Government. Authors contribution Conceptualisation and design: ATS and PMP Data curation: ATS and PMP Formal analysis: ATS Funding acquisition: ATS and PMP Investigation: ATS and PMP Methodology: ATS and PMP Project administration: ATS and PMP Resources: ATS, BV, DS, CPM, RL, RR, EC AGMSF, NC, LP, PMP Software: ATS Supervision: PMP Validation: ATS Visualization: ATS and PMP Writing—original draft: ATS Writing—review and editing: ATS, BV, DS, CPM, RL, RR, EC AGMSF, NC, LP, PMP. All authors approved and agreed manuscript publication References Sung, H. et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA: A Cancer Journal for Clinicians 71 , 209–249 (2021). The Cancer Genome Atlas Integrated genomic characterization of endometrial carcinoma. Nature 497 , 67–73 (2013). 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Additional Declarations There is a conflict of interest A Sengal and P Pollock received IIS grant from Incyte Biopharma. Supplementary Files ASengaletalSupplementaryFiguresandlegendsV3.pdf Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: revise 27 Feb, 2023 Review # 3 received at journal 22 Feb, 2023 Review # 2 received at journal 17 Feb, 2023 Review # 1 received at journal 14 Feb, 2023 Reviewer # 3 agreed at journal 13 Feb, 2023 Reviewer # 2 agreed at journal 10 Feb, 2023 Reviewer # 1 agreed at journal 10 Feb, 2023 Reviewers invited by journal 10 Feb, 2023 Editor assigned by journal 09 Feb, 2023 Submission checks completed at journal 07 Feb, 2023 First submitted to journal 06 Feb, 2023 Unknown event 27 Jan, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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(VHIR)","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Antonio","middleName":"","lastName":"Gil-Moreno","suffix":""},{"id":174893036,"identity":"8dd4ca9a-5eb1-4e0a-8727-272e27a8aa2f","order_by":8,"name":"Sophia Frentzas","email":"","orcid":"","institution":"Monash Health","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sophia","middleName":"","lastName":"Frentzas","suffix":""},{"id":174893037,"identity":"d4f86822-3b13-426f-9846-b56c2ea1fe14","order_by":9,"name":"Naven Chetty","email":"","orcid":"","institution":"Mater Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Naven","middleName":"","lastName":"Chetty","suffix":""},{"id":174893038,"identity":"66617f9d-ed10-4bd1-b435-55e157fe11fb","order_by":10,"name":"Lewis Perrin","email":"","orcid":"","institution":"Mater Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lewis","middleName":"","lastName":"Perrin","suffix":""},{"id":174893039,"identity":"485cc38d-edb4-46a2-9bda-66a86754d207","order_by":11,"name":"Pamela Pollock","email":"","orcid":"","institution":"Queensland University of Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Pamela","middleName":"","lastName":"Pollock","suffix":""}],"badges":[],"createdAt":"2023-01-25 07:55:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2512859/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2512859/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":32933590,"identity":"06e7cbbd-b53d-4c5a-9f14-93f7dc5761b5","added_by":"auto","created_at":"2023-02-14 18:38:39","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":928794,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of patients’ primary endometrial cancer, PDXs and PDX-derived organoids (PDXOs).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea) Histologic and molecular characterization of 21 endometrial cancer PDXs b) Representative images from different histologic and molecular subtypes of endometrial cancer showing 7 primary patients’ tumours with different histologic and molecular types (upper panel) recapitulated their morphology in corresponding PDXOs (phase contrast micrography of individual PDXOs, middle panel) and (H/E stained morphology of PDXOs, lower panel). c) Representative images for PDX23 demonstrating histologic morphology (H/E), FGFR2 protein expression detected via pan-FGFR2 antibody IHC and FGFR2b and FGFR2c mRNA expression in patients’ primary tumour (upper panel), PDX 23 tumour passage 3 (F3) (middle panel) and corresponding histologic morphology of PDXO23 at passage 3 (P3) (lower panel); Scale bar indicates 50µm. CC, Clear cell carcinoma; CNV, Copy number variation; EEC, Endometrial endometrioid carcinoma; H/E, haematoxylin/eosin; FGFR2c, Fibroblast growth factor receptor 2c splice isoform; FIGO, International Federation Gynaecological Oncology; IHC, Immunohistochemistry; LVSI, Lymphovascular space invasion, Mol, Molecular; Myo, Myometrial; PDX, patient-derived xenograft; PDXO, patient-derived xenograft organoids; EEC; Serous endometrial carcinoma; TMB, Tumour mutation burden, UCS, Uterine carcinosarcoma\u003c/p\u003e","description":"","filename":"ATSengalFigure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2512859/v1/5d003d8f77c77d254b3f02ef.jpg"},{"id":32933045,"identity":"6d1fde28-829f-4587-9280-a5118f8706d6","added_by":"auto","created_at":"2023-02-14 18:30:39","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":747103,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePattern of FGFR2c splice isoform expression in primary patient tumour and matched EC PDXs and PDXOs that recapitulate corresponding primary patient tumours.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe micrograph images illustrate five EC patients and corresponding PDXs and PDXOs representing the different patterns of FGFR2c splice isoform expression. The first and second column represents models with high FGFR2c splice isoform expression with an RNA ISH signal score of 4 (cluster of signals without distinct dots and a score of 3 (\u0026gt;10 signals/cell with multiple clusters) respectively. The third column represents a model of moderate FGFR2c splice isoform expression with an RNA ISH score of 2 (4-10 signals/cell without clustered dots) the fourth column represents a model with a low FGFR2c splice isoform RNA ISH score of 1 (2-3 signals/cell) and the fifth column is a model with negative FGFR2c splice isoform with RNA ISH score of 0 (\u0026lt;1 signal/10 tumour cells).\u003c/p\u003e","description":"","filename":"ATSengalFigure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2512859/v1/a6ff241a307d00794264be1a.jpg"},{"id":32933030,"identity":"34edd96b-a58f-4b53-8acb-d30a3ee20603","added_by":"auto","created_at":"2023-02-14 18:30:39","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":662475,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of growth factor withdrawal and FGFR inhibition on EC PDXOs growth and regeneration. \u003c/strong\u003ea) Representative images of EC PDXO59 (upper panel) and PDXO67 (lower panel) cultured for 14 days with the indicated withdrawal of growth factors and treatment with neutralising anti-FGF2 antibody alone or in combination with FGFR inhibitor (BGJ398). b) Bar graph showing the relative growth reduction in the number of PDXOs cultured with the withdrawal of target growth factors or treatment with FGFR inhibitor (BGJ398) with and without anti-FGF2 neutralising antibody of three independent PDXO models. This experiment was done in biological triplicate using organoids established from three different mice carrying respective PDX models and technical duplicates. Organoid counting was performed by capturing 3 independent fields at a 10X microscopic objective view of each experiment. The error bar indicates the standard error of the mean (SEM) of the biological triplicates. c) Representative IHC images showing expression of FGF2 in primary tumour and corresponding PDX and PDXO. d) FGF2 IHC H-Score of primary patient tumour and matched PDXs and PDXOs. Standard media is advanced DMED/F12 media supplemented with growth factors including FGF2, EGF, WRN (Wnt3A, R-Spondin 3, Noggin) and L-glutamine, Rho-associated kinase (ROCK) inhibitor (Y27632). All experiments were performed in advanced media with Growth Factor reduced (GFR) Matrigel. The scale bar indicates 200µm for the PDXOs and 50 µm for the IHC. Ab, antibody; PDX, Patient-Derived Xenograft\u003c/p\u003e","description":"","filename":"ATSengalFigure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2512859/v1/85d89473f8a3ae32d12f01cf.jpg"},{"id":32934387,"identity":"9ae71465-aa16-4a9e-a39b-8c85a2becd92","added_by":"auto","created_at":"2023-02-14 18:46:39","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":487730,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIn situ\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e detection of autocrine loop activated (phosphorylated) pFGFR2c using PLA assay in endometrial cancer cell line and PDXO\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea) Schematic diagram illustrating the principle of PLA assay and autocrine loop mode of activation in carcinoma cells with FGFR2c splice isoform expression (I); \u003cem\u003eIn situ\u003c/em\u003eindirect PLA assay design, with one primary antibody omitted (technical negative control) without signal formation (II) and positive signal formation with incubation of both primary antibodies (III). b) Representative images of PLA assay in ASPX67-406 primary EC cell line grown in full growth media (10% FBS) or 16 hr of serum starvation followed by stimulation with and without 25 ng/ml FGF2 + 0.01% DMSO for 30 min (\u003cstrong\u003etop panels\u003c/strong\u003e) alongside 300nM BGJ398 (\u003cstrong\u003ebottom panels\u003c/strong\u003e). c) Representative images demonstrating co-expression of FGF2 and pFGFR2c in vehicle and BGJ398 treated ASPX67-406 primary EC cell line. FGF2 is markedly reduced when cells were serum starved (\u003cstrong\u003eleft panels\u003c/strong\u003e). d) Graph showing pFGFR2c signal analyses in ASPX67-406 primary endometrial cancer cell line. e) Representative images of PLA assay in EC PDXO67 cultured with standard media with or without supplantation of FGF2 in vehicle-treated (\u003cstrong\u003eleft panels\u003c/strong\u003e) and BGJ398-treated (right panels). f) Analyses of pFGFR2c signal in EC PDXO67 cultured in stem cell media with or without FGF2 in vehicle and BGJ38 treated organoids. All PLA signals data analyses were performed using automated Fiji ImageJ2. Error bars indicate standard error of the mean (SEM). The experiment was performed in biological triplicate in this single organoid line. Small red dots indicate PLA signals of pFGFR2c and blue indicates nuclei stained with DAPI. Green IF in the PDXOs shows Histone-3 nuclear stain. EC endometrial cancer; FGF2, Fibroblast Growth Factor; PLA, proximity ligation assay; pFGFR2c, phosphorylated Fibroblast Growth Factor Receptor 2c splice isoform\u003c/p\u003e","description":"","filename":"ATSengalFigure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2512859/v1/91dc80e1e12815a7f053ed71.jpg"},{"id":32933055,"identity":"ea99a4b9-d734-4ecf-822a-4ce4e82f7fce","added_by":"auto","created_at":"2023-02-14 18:30:39","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":593121,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSignificant cell death was observed in established PDXO models expressing FGFR2c following 72 hr treatment with 300nM BGJ398\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003ea) Representative images from three PDXOs expressing FGFR2c (\u003cstrong\u003etop three panels\u003c/strong\u003e) and PDXOs without FGFR2c expression (\u003cstrong\u003ebottom two panels\u003c/strong\u003e) treated with vehicle (DMSO) or 300nM BGJ398 for 72 hours. Images were captured in XYZ stack at 10x microscopic objective view airy using an Inverted Laser rotatory Confocal Fluorescent Olympus Microscope with (FV1200 software). Green fluorescence shows esterase activity of live cells (Calcein AM), red fluorescence is generated upon binding of Ethidium homodimer-1 to DNA in damaged cells and nuclei were stained with DAPI. b) Bar graph showing the proportion of live (green) and dead (red) cells in 5 independent PDO models treated with DMSO vehicle and 300nM BGJ398. The experiment was performed in biological triplicatesusing organoids cultured from different mice (n=3) carrying each PDX model and technical triplicates. Live and dead data was performed using automated Fiji ImageJ2 analyses. **** two-sided student’s T-test P \u0026lt;0.0001; Error bars indicate standard error of the mean (SEM), Scale bar 50µm. DMSO, Dimethyl-sulphoxide; DAPI; Diamidino-2-phenylindole, FR2c+, Fibroblast Growth Factor Receptor 2c positive; FR2c-, Fibroblast Growth Factor Receptor 2c negative; PDOs Patient-Derived Organoids; PDX, Patient Derived Xenograft. NS, not statistically significant\u003c/p\u003e","description":"","filename":"ATSengalFigure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2512859/v1/ed55f8a24a3e3682dff8c535.jpg"},{"id":32934388,"identity":"3534277c-369e-4921-ac89-2768e49a844b","added_by":"auto","created_at":"2023-02-14 18:46:39","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":686834,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIn vivo data showing significant tumour growth inhibition (TGI) and longer survival in EC PDXs with FGFR2 activation treated with FGFRi alone or in combination with Cisplatin\u003c/strong\u003e a) Five independent EC PDX models treated with either BGJ398/infigratinib PDX52, PDX59 and PDX68) or pemigatinib (PDX60 and PDX58). The four PDXs demonstrated significant tumour growth inhibition (upper panel) and significantly increased survival outcome (lower panel) but PDX58 showed de novo resistance. b) Three independent EC PDX models with MMRd molecular subtype with FGFR2c expression treated with cisplatin alone, pemigatinib alone or pemigatinib Plus cisplatin (tumour growth inhibition, upper panel) and (prolonged survival outcome, lower panel). c) Two independent EC PDX models with p53abn molecular subtype with FGFR2c expression treated with cisplatin alone, pemigatinib alone or pemigatinib + cisplatin (tumour growth inhibition, upper panel) and (survival outcome, lower panel). Significance for TGI was assessed with a two-way ANOVA and significance for survival curves was assessed with a log-rank test * P\u0026lt;0.05, ** P\u0026lt;0.001 *** P\u0026lt;0.0001, **** P\u0026lt;0.00001. ANOVA, Analysis of variance; cis, cisplatin; EC, endometrial cancer, PDX, patient-derived xenograft\u003c/p\u003e","description":"","filename":"ATSengalFigure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2512859/v1/9a27cea60634137633237926.jpg"},{"id":32933592,"identity":"d776068e-e4ed-4f78-a77b-5be210a6903c","added_by":"auto","created_at":"2023-02-14 18:38:39","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":769581,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKi67 expression in EC PDX models treated with either vehicle or BGJ398 for 7 days\u003c/strong\u003e. a) Representative micrography images of Ki67 expression on four indicated PDX tumours treated for 7 days with either vehicle (left panel) or BGJ398 (right panel) b) Bar graph showing a significant reduction in Ki67 expression on BGJ398 treated versus vehicle on indicated PDX tumours. Ki67 was quantified using automated digital image analyses (Qu Path) and score was reported as average/0.1mm\u003csup\u003e2\u003c/sup\u003e area. (**** two-sided T-test, P\u0026lt;0.0001); Error Bars indicate standard error of the mean (SEM) and scale bar 50µm\u003c/p\u003e","description":"","filename":"ATSengalFigure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2512859/v1/5038559cd91bb9b7c3a9bcfb.jpg"},{"id":32933056,"identity":"ffcf3d2b-2c12-474e-a44b-2365c85f9386","added_by":"auto","created_at":"2023-02-14 18:30:40","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":757963,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFGFR inhibitor significantly reduced tumour microvessel density (MVD) in EC PDXs with FGFR2 dysregulation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea) Representative microphotography images of IHC stain of CD31 in four indicated independent PDX tumours treated with either vehicle (left panel) or 30 mg/KgBGJ398 (right panel). b) Bar illustrating a significant reduction of microvessel density (MVD) in PDXs with FGFR2c expressing models (PDX52, PDX59 and PDX67) and PDX68 FGFR2\u003csup\u003eC383R\u003c/sup\u003e mutant treated with 30 mg/Kg BGJ398 versus vehicle. MVD was counted using the automated image analyses and reported an average count of MVD/0.5mm\u003csup\u003e2\u003c/sup\u003e. **** two-sided student’s T-test P \u0026lt;0.0001; Error bars indicate standard error of the mean (SEM), Scale bar 50µm\u003c/p\u003e","description":"","filename":"ATSengalFigure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2512859/v1/9a13b8e6942b3ece079bf17f.jpg"},{"id":32933591,"identity":"218e56c3-576d-4e6b-8dbd-54168614f4d0","added_by":"auto","created_at":"2023-02-14 18:38:39","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":775790,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCD206 expression in EC PDX models treated with vehicle or BGJ398 for 7 days. \u003c/strong\u003ea) Representative images of CD206+ M2 macrophage from 4 EC PDX models treated with Vehicle (\u003cstrong\u003etop panels\u003c/strong\u003e) and 30 mg/Kg BGJ398 (\u003cstrong\u003ebottom panels\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eb) Bar graph showing a significant reduction in M2 macrophages on BGJ398 treated EC PDX models compared vehicle-treated ((**** two-sided T-test, P\u0026lt;0.0001); Error Bars indicate Standard error of the mean (SEM) and scale bar 50µm. EC, Endometrial cancer; FGFR2c, Fibroblast Growth Factor Receptor 2c splice isoform; PDX, Patient-Derived Xenografts; MMRd, Mismatch Repair deficient; p53wt, p53 wildtype\u003c/p\u003e","description":"","filename":"ATSengalFigure9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2512859/v1/a38e3791e4cbed4c5174ccf7.jpg"},{"id":32934390,"identity":"6caae6c0-1eb4-40ee-9c0c-2ea676c73f08","added_by":"auto","created_at":"2023-02-14 18:46:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2245635,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2512859/v1/6ec5c93b-978e-4e9b-8ac6-fb7580db4cd0.pdf"},{"id":32933057,"identity":"3330ae0a-3a83-4aa0-8c69-7ef9a34cc112","added_by":"auto","created_at":"2023-02-14 18:30:40","extension":"pdf","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":2173851,"visible":true,"origin":"","legend":"","description":"","filename":"ASengaletalSupplementaryFiguresandlegendsV3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2512859/v1/8d800352857ceab1720579b7.pdf"}],"financialInterests":"There is a conflict of interest\nA Sengal and P Pollock received IIS grant from Incyte Biopharma.","formattedTitle":"Endometrial cancer PDX-derived organoids (PDXOs) and PDXs with FGFR2c isoform expression are sensitive to FGFR inhibition.","fulltext":[{"header":"Background","content":"\u003cp\u003eEndometrial cancer (EC) is the single gynaecological cancer that constantly showed a notable annual increase in both incidence and mortality in developed countries\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. EC includes several histologic and molecular subtypes that have diverse prognostic outcomes. Traditionally, EC was classified as type I (well to moderately differentiated, endometrioid in histology associated with good prognosis) and Type II (poorly differentiated, non-oestrogen dependent with poor prognosis). In 2013, the Cancer Genome Atlas (TCGA) consortium identified four molecular subtypes with distinct prognostic significance\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. These subtypes were subsequently validated using surrogate immunohistochemistry (IHC) biomarkers with Polymerase Ɛ-enzyme (\u003cem\u003ePOLE)\u003c/em\u003e hotspot mutation analyses. For example, the McAlpine laboratory established a Proactive Molecular Risk Classifier for Endometrial Cancer (ProMisE) which simplified the TCGA approach\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. The PORTEC consortium studies concurred with the ProMisE classification except for minor variations in nomenclature\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. All published studies concluded that patients with \u003cem\u003ePOLE\u003c/em\u003e exodomain mutant have excellent prognoses; mismatch repair deficient (MMRd) and p53 wildtype (p53wt)/no specific molecular profile (NSMP) have intermediate prognoses, and p53 abnormal (p53abn) have the worst prognoses\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. The molecular classification is endorsed by World Health Organisation and several clinical trials are currently evaluating using molecular subtyping in tailoring adjuvant treatment of EC\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWomen diagnosed with metastasis or recurrent disease have limited treatment options with \u0026lt;\u0026thinsp;20% 5-year expected survival. The PORTEC-3 phase III clinical trial tested the addition of chemotherapy to radiotherapy compared to radiotherapy alone in high-risk women with EC \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Molecular subtyping of PORTEC-3 cohort tumour samples showed the combination of chemotherapy and radiotherapy increased survival for EC patients with the p53abn subtype but did not show significant benefit for patients with MMRd and NSMP/p53wt molecular subtypes\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. The MMRd and p53wt/NSMP subtypes encompasses 80% of diagnosed ECs contributing to 50% of EC deaths and therefore, need additional treatment optimisation. Recently, immune checkpoint inhibitors (ICIs) have shown efficacy in EC patients with MMRd. The combination of pembrolizumab (anti-PD-1 antibody)\u0026thinsp;+\u0026thinsp;lenvatinib (primarily a VGFR inhibitor with reduced activity to other receptor tyrosine kinases (RTK) including FGFRs) has been shown to increase survival by 6 months in 40% and 30% of MMRd and proficient MMR (pMMR) patients, respectively in a large phase III clinical trial\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Following this study, the combination regimen was granted accelerated approval in several countries including USA, Europe and Australia. However, the combination of pembrolizumab\u0026thinsp;+\u0026thinsp;lenvatinib is not biomarker-driven and 90% of patients developed significant adverse effects\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFibroblast Growth Factor Receptor 2 (FGFR2) is a member of the FGFR RTK family and has two main isoforms, FGFR2b and FGFR2c which are expressed in normal epithelial and mesenchymal cells respectively \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. FGFR2 can be dysregulated via mutation, amplification, or gene fusion in different solid cancers. We have identified \u003cem\u003eFGFR2\u003c/em\u003e mutations in about 10\u0026ndash;15% of EC \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e and shown that mutations were associated with shorter progression-free survival (PFS) and disease-specific survival (DSS). Recently, we discovered \u003cem\u003eFGFR2c\u003c/em\u003e splice isoform expression due to isoform switching in ~\u0026thinsp;50% and ~\u0026thinsp;30% of MMRd and p53wt molecular subtypes respectively \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. We demonstrated \u003cem\u003eFGFR2c\u003c/em\u003e was an independent prognostic biomarker and was associated with shorter PFS and DSS compared to FGFR2b expressing tumours \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. FGFR2c contributes to drive epithelial to mesenchymal transition (EMT), enhance cell motility and invasiveness and inhibits tumour differentiation in several solid cancer cell lines\u003csup\u003e\u003cspan additionalcitationids=\"CR16 CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePreviously, our laboratory demonstrated EC cell lines harbouring FGFR2 mutations are oncogene addicted and are sensitive to FGFR inhibition (PD173074 and BGJ398) \u003cem\u003ein vitro\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e and \u003cem\u003ein vivo\u003c/em\u003e using cell line xenograft models\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Currently, the US Food and Drug Administration (FDA) has approved infigratinib (BGJ398) and pemigatinib in intrahepatic cholangiocarcinoma with FGFR2 fusions as well as erdafitinib in advanced urothelial cancer with FGFR2/3 fusions\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Although we have identified FGFR2c isoform switching as a new mechanism of receptor activation in EC that is associated with poor prognosis, there is no functional data showing ECs with FGFR2c isoform expression show oncogene dependence and the role of FGFR inhibitors in preclinical models with FGFR2c expression is unknown.\u003c/p\u003e \u003cp\u003eDespite the advances in molecular profiling of EC, discovery of novel targeted therapies for endometrial cancer is lagging, partly due to a lack of robust preclinical models that reflect the spectrum of molecular subtypes. Patient-Derived Xenografts (PDXs) and Patient-Derived Organoids (PDOs) are robust models for preclinical drug testing that better mimic patient tumour heterogeneity and molecular profiles.\u003c/p\u003e \u003cp\u003eThe objectives of this investigation were: 1) to establish 3D PDX derived organoids (PDXOs) from multiple EC PDX models representing advanced ECs with and without \u003cem\u003eFGFR2c\u003c/em\u003e expression; 2) to assess the \u003cem\u003eFGFR2c\u003c/em\u003e/\u003cem\u003eFGFR2b\u003c/em\u003e status in the established PDXs and PDXOs and identify appropriate models with \u003cem\u003eFGFR2c\u003c/em\u003e expression that can be targeted with FGFR inhibitors; \u003cem\u003e3)\u003c/em\u003e to target PDXOs expressing \u003cem\u003eFGFR2c\u003c/em\u003e oncogenic splice isoform with FGFR inhibition 4) to assess the \u003cem\u003ein vivo\u003c/em\u003e efficacy of FGFR inhibitors (infigratinib/BGJ398 and pemigatinib) as well as cisplatin, either alone or in combination in \u003cem\u003eFGFR2c\u003c/em\u003e expressing EC PDX models representing various histologic and molecular subtypes.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of EC PDXs-and PDXOs\u003c/h2\u003e \u003cp\u003eWe have previously published detailed genomic profiling for 11 EC PDXs alongside the establishment of another 7 PDXs\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. The current cohort contains total 21 PDXs including 3 newly established PDXs. The clinicopathologic characteristic and follow\u0026ndash;up with clinical outcomes of patients from which these PDXs were derived is provided in supplementary Table\u0026nbsp;3. This panel of PDX represents the various histologic subtypes of EC including endometrioid endometrial carcinoma (n\u0026thinsp;=\u0026thinsp;14), serous endometrial carcinoma (n\u0026thinsp;=\u0026thinsp;2), clear cell carcinoma (n\u0026thinsp;=\u0026thinsp;1) and uterine carcinosarcoma (n\u0026thinsp;=\u0026thinsp;4). This cohort of PDX also included the four ProMiSE molecular subtypes including p53abn (n\u0026thinsp;=\u0026thinsp;10), MMRd (n\u0026thinsp;=\u0026thinsp;8), p53wt (n\u0026thinsp;=\u0026thinsp;2), and \u003cem\u003ePOLE\u003c/em\u003e mut (n\u0026thinsp;=\u0026thinsp;1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). From the 15 independent PDXs tested for organoid culture 14/15 (93%) were successfully established and expanded. Nearly all the established PDXOs recapitulated the morphological pattern of the corresponding patients\u0026rsquo; primary tumour and PDXs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFGFR2 isoform status was determined for each primary tumour and matched PDX and PDXO via optimised and validated novel BaseScope RNA ISH assay which detects the FGFR2b and FGFR2c splice isoforms\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. The expression of the FGFR2c splice isoform was highly consistent between the primary patient tumour and the matched PDXs and PDXOs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). We observed four patterns of FGFR2c expression high (n\u0026thinsp;=\u0026thinsp;5), moderate (n\u0026thinsp;=\u0026thinsp;6), low (n\u0026thinsp;=\u0026thinsp;5) and negative (n\u0026thinsp;=\u0026thinsp;7) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) and representative images of patient primary tumours and matched PDXs and PDXOs are provided in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. There was no significant difference in morphology and proliferation across multiple passages of the PDXOs (P3-P10) (\u003cb\u003esupplementary Fig.\u0026nbsp;2\u003c/b\u003e). For the tested PDXOs multiple rounds of freeze thawing had no effect on organoid growth pattern, viability, and morphology (\u003cb\u003esupplementary Fig.\u0026nbsp;2\u003c/b\u003e). For 8 PDX models, PDXOs were generated from 3 or more independent mice carrying PDX tumours at multiple passages (F3-5) to ensure some \u003cem\u003ein vitro\u003c/em\u003e functional experiments could be performed in biological triplicate.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDifferential Ligand Dependence Of Ec Pdxos\u003c/h3\u003e\n\u003cp\u003eEpithelial stem cell media contains a variety of growth factors to facilitate organoid proliferation including EGF, FGF2 as well as WNT3A, R-Spondin and Noggin (WRN). Based on our hypothesis that the splicing switch to FGFR2c establishes an autocrine loop in EC cells, we performed growth factor withdrawal experiments in PDXO59 and PDXO67 with FGFR2c expression and PDXO56 without FGFR2c expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Withdrawal of growth factors 24 hours following seeding showed that EC PDXOs with high FGFR2c expression (PDXO59 and PDXO67) did not require exogenous FGF2, EGF or WRN however, they were susceptible to treatment with 25 \u0026micro;g/ml anti-FGF2 and/or 100 nM BGJ398 and significant growth reduction and morphologic changes was evident. All PDXOs with FGFR2c expression were subsequently cultured without addition of exogeneous GF including FGF2, EGF and WRN and growth pattern was not altered. In contrast the PDXOs without FGFR2c expression showed a reduction in proliferation following removal of EGF and WRN and treatment with anti-FGF2 antibody and/or BGJ398 had no effect. This data suggested that initial growth of EC PDXOs with FGFR2c expression was dependent on endogenous FGF2 via establishment of an autocrine loop (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-b). Indeed, IHC analyses of FGF2 on several primary patient tumour and corresponding PDXs and PDXOs revealed high FGF2 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, d). To confirm the specificity of FGF2 antibody and rule out the cross binding with other closely related FGF ligands, the FGF2 antibody used was validated via western blot analyses in BaF3 cell lines stably transduced with a subset of individual FGF ligands (\u003cb\u003esupplementary Fig.\u0026nbsp;3\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor PDX67, initial culturing of the PDXO in 2D culture without Matrigel led to the development of an attached EC primary cell line (ASPX67-406) and named with first author initials and the PDX number. To assess whether FGFR2c was activated via endogenous FGF2 in EC, the ASPX67-406 EC primary cell line was incubated for 30 min with supplementation of 5 \u0026micro;g Heparin Sulphate (HS) with and without 10 ng/ml FGF2 in the presence of DMSO (control) or 100 nM BGJ398 in standard media (10% FBS in DMEM/F12) or following overnight (16 hr) serum starvation (0.5% FBS in DMEM/F12 media) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). High PLA signals was observed in cells stimulated with HS\u0026thinsp;+\u0026thinsp;FGF2 in 10% FBS full growth media however, a similar signal of PLA was noted without the addition of exogenous FGF2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, c) and no PLA signals were seen when BGJ398 was added, confirming specificity of the PLA for detection of pFGFR2c. Following serum starvation for 16 hours, high PLA signals were seen with stimulation of exogenous FGF2 but not in the absence of FGF2 suggesting that serum starvation reduced FGF2 secretion. A reduction in endogenous FGF2 expression following serum starvation was then confirmed by IF in this cell line model (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). We then performed PLA to detect phosphorylated FGFR2 to confirm autocrine activation of FGFR2c by endogenous FGF2 was driving EC organoid growth. Activation of FGFR2c was confirmed by PLA signals when PDXO67 was cultured in stem cell advanced media with and without exogenous FGF2 and these PLA signals were reduced in the presence of 100 nM BGJ398 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee-f).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo investigate further the downstream signalling activation, we have also determined expression pERK1/2 and pSTAT3 in BGJ398 treated ASPX67-406 primary cell line and PDXOs and phosphorylation was tracked using an \u003cem\u003ein situ\u003c/em\u003e IF stain. Notably, BGJ398 treated ASPX67-406 cells and PDXOs demonstrated a significant reduction in pERK1/2 and pSTAT3 expression (\u003cb\u003esupplementary Fig.\u0026nbsp;4\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eWhile data in \u003cb\u003esupplementary Fig.\u0026nbsp;3\u003c/b\u003e showed initial culturing of organoids was sensitive to 100nM BGJ398, this does not reflect treatment of well-established cancers in patients. Therefore, we tested increasing concentrations of BGJ398 on well-established PDXO67 (FGFR2c high) and PDX56 (FGFR2c negative) organoids grown for 10 days at different time points treatment (24 hr, 48 hr and 72 hr). In this context 100nM BGJ398 had limited effect on organoid morphology, proliferative capacity, and viability at different time points of treatment whereas 300nM BGJ398 for 72 hr revealed marked reduction in proliferation, alongside marked cell death/necrosis and a cystic morphology in PDXO67 but not evident in PDX56 (\u003cb\u003esupplemental Fig.\u0026nbsp;5a, d\u003c/b\u003e). Finally, five independent PDXO models were treated with 300nM BGJ398 or 0.01% DMSO vehicle for 72 hours. The three EC PDXOs with high FGFR2c isoform expression were established from PDX59 and PDX67 (MMRd) and PDX23 (p53abn) and the two PDXOs with very low or negative FGFR2c isoform expression (RNA ISH signals\u0026thinsp;\u0026lt;\u0026thinsp;10/tumour cells) were established from PDX53 (EEC MMRd) and PDX56 (UCS p53abn). To ensure the most robust results, three independent organoid cultures were derived from three different mice each carrying the individual PDXs to represent true biological replicates. Using the LIVE/DEAD\u0026reg; assay followed by imaging, significant cell death (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) was observed in PDXO models expressing high FGFR2c isoform (PDX23, PDX67, PDX59) but not in PDXOs with FGFR2c negative/low expression (PDX56 and PDX53) or vehicle treated organoids (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eIn vivo\u003c/span\u003e \u003cb\u003etargeting of EC PDXs with Infigratinib/BGJ398 and Pemigatinib FGFR inhibitors\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBoth infigratinib (BGJ398) and pemigatinib are orally bioavailable FGFR1-3 specific inhibitors that are approved as second line treatment for patients with advanced intrahepatic cholangiocarcinoma with FGFR2 dysregulation. Initially, five independent EC PDXs were treated with either 30 mg/kg BGJ398 (PDX52 and PDX59, MMRd FGFR2c high and PDX68 MMRd, FGFR2 mutant) or with 1 mg/kg Pemigatinib (PDX58 MMRd, PDX60 p53wt both FGFR2c moderate expression) daily for 21 days. Four of the five PDX models treated with FGFRi showed significant tumour growth inhibition (TGI) and had significantly longer survival compared with the vehicle treated arms (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). PDX58 showed de novo resistance to FGFRi and currently we are investigating the mechanism of resistance in this model.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCisplatin is one of the first line standard of care chemotherapeutic agents for EC patients with metastatic disease or at high risk of recurrence. To evaluate if the addition of pemigatinib sensitises EC cells to cisplatin, 5 independent PDX models were treated with 1 mg/Kg pemigatinib alone, pemigatinib plus cisplatin or cisplatin alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb-c). All five models treated with cisplatin alone showed de novo resistance to cisplatin and no survival benefit. The addition of cisplatin to pemigatinib had also no significant benefit to increase survival in PDXs with MMRd molecular subtype (PDX52, PDX59 and PDX67) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). However, variable response was found in PDXs representing the p53abn subtype of EC (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). We noted the combination of pemigatinib with cisplatin contributed to reduce tumour growth and prolonged survival in PDX23. In contrast FGFRi alone did show a significant improvement in survival for PDX61 with no additional benefit seen following combination with cisplatin (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). Treatment related toxicity or weight reduction were not observed in mice treated with FGFRi implicating clinical safety in patients however, 95% of the mice treated with both FGFRi and cisplatin often presented with ruffled fur potentially due cisplatin toxicity.\u003c/p\u003e \u003cp\u003eWe have assessed and compared the PDX tumour morphology and tumour growth characteristics between vehicle treated (tumour collected when the target volume of 900mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e was reached) and the residual tumour regrown after completion of treatment (collected at last follow up day when tumour reached the target volume\u0026thinsp;~\u0026thinsp;900mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e). Consistent with the PDXO findings, treated PDX tumours demonstrated significant central necrosis, cystic formation and tumour differentiation that were not evident in mice from the vehicle treated control arm \u003cb\u003e(supplementary Fig.\u0026nbsp;6)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eWe noted 21 days treated PDX tumours had small residual tumour with small area of viable cells that may not be sufficient to compare with vehicle treated tumours. Therefore, we treated 4 independent PDX models with 30mg/Kg BGJ398 for 7days to compare the tumour proliferation capacity and other tumour microenvironment markers (TME) between control and treated PDXs. As expected, a significant reduction in expression of Ki67 was observed in BGJ398 treated compared to vehicle treated PDX tumour (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea-b), consistent with our \u003cem\u003ein vitro\u003c/em\u003e findings.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePrevious studies also demonstrated FGF2/FGFR signalling contributes to in vivo angiogenesis and immune modulation and treatment with FGFRi reduces angiogenesis and tumour growth \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. In this study, we assessed the tumour microvessel density (MVD) via IHC staining of CD31 in four independent EC PDXs tumours (three with FGFR2c expression and one with FGFR2 mutation) treated with either BGJ398 or vehicle for 7 days. A significant reduction of MVD (CD31 reactive vessels) was found in BGJ398 treated PDXs compared to control (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea-b). Several studies reported that FGF/FGFR signalling pathway modulates the tumour microenvironment (TME) and contributes to polarization of tissue associated macrophages (TAM) M2 macrophages\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. We evaluated the effect of BGJ398 on EC PDXs TME and demonstrated a significant reduction in CD206 expressing macrophages (M2) following 7 day BGJ398 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea-b).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWomen with advanced EC have limited treatment options and current available treatment modalities are suboptimal. Identifying effective targeted therapies for metastatic and/or recurrent ECs and robust predictive biomarkers are a major unmet clinical need. Development of effective personalised targeted therapies requires authentic preclinical models that represent the morphological and molecular profiles of the patient tumours with high fidelity. In this study, we presented the establishment, expansion, and characterisation of EC PDXOs and demonstrated that EC PDXOs with expression of the oncogenic FGFR2c splice isoform were highly sensitive to FGFR inhibition. \u003cem\u003eIn vivo\u003c/em\u003e validation using corresponding PDX models confirmed that treatment with clinically approved FGFR1-3 specific inhibitors markedly reduced tumour growth and increased survival. We also showed the impact of FGFR inhibitors on reduction of EC cell proliferation and TME, evident by reduced angiogenesis (CD31\u0026thinsp;+\u0026thinsp;microvessel density) as well as reduced TAMs (CD206\u0026thinsp;+\u0026thinsp;M2 polarised macrophages).\u003c/p\u003e \u003cp\u003eEstablishment of PDOs from normal endometrium, benign diseases, and well-differentiated early-stage ECs have been previously reported\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. The success rate of PDXOs establishment and expansion in this study was very high (93%) compared to previous published report (22%) from primary patient tumours\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. The high success in our study likely reflects the selection of more aggressive tumours in those that initially established PDXs or an increased cancer stem cell population during expansion of the PDXs. Our established PDXOs predominantly represent G3 ECs with different histological types as well as the four molecular subtypes of EC that are clinically relevant for drug discovery and testing. The PDXOs are very stable and retain the morphological and molecular characteristics of original parental donor tumours after recovery from cryopreservation or several series of passaging (P15). This ensures the potential utility of our PDXOs for future drug or drugs combination screening to optimise precision therapy of EC patients.\u003c/p\u003e \u003cp\u003eInitial screening of the primary patient tumours, PDXs and matched PDXOs for FGFR2 isoform status revealed four patterns of FGFR2c splice isoform expression (high, moderate, low and negative) consistent with the previous finding in large clinical cohort of EC patients\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Utilising three MMRd PDXO models, we noted the growth factor requirements were dependent on which pathways were activated to maintain stemness, regeneration and proliferation, with two PDXO models expressing FGFR2c not reliant on EGF, WNT5A, Noggin or R-Spondin. This finding suggests heterogeneity of EC PDXOs growth factors requirement. Previous studies reported different growth factor requirements for self-renewal for PDOs depending on activation of different driven oncogenic pathways\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Otte and colleagues reported colon cancer PDOs with KRAS mutation were dependent on FGF2 and EGF for regeneration and stemness\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe demonstrated PDXOs with FGFR2c expression were dependent on endogenous produced FGF2 for growth/proliferation and self-renewal and blocking of FGF2 with FGF2 specific antibody showed reduction in PDXOs number and size when incubated after initial 24 hr seeding period. We also showed that a lower concentration of BGJ398 was required to inhibit growth and regeneration of PDXOs if treatment was initiated 24 hrs following organoid seeding, but 300 nM of BGJ398 were required when treatment was initiated in mature and well established PDXOs (10 days after seeding), consistent with our previous finding with EC cell lines with FGFR2 mutations\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Morphological analyses of the well-established PDXOs revealed 72 hr of 300 nM BGJ398 results in degeneration of the organoids and inhibition of proliferation. The requirement for a higher dose of BGJ398 for killing PDXOs once they are well-established may reflect reduced drug diffusion/penetration in mature well established PDXOs or may reflect an increased dependency of FGF/FGFR signalling during initial organoid establishment, consistent with its role in stem cell maintenance.\u003c/p\u003e \u003cp\u003eTo better understand activation of the FGFR2c signalling pathway, receptor phosphorylation was assessed via a specific \u003cem\u003ein situ\u003c/em\u003e PLA assay in a primary cell line derived from G3-EC PDX67 as well as in PDXO67 organoids with and without exogenous FGF2 stimulation and FGFR inhibition (BGJ398). This revealed high FGFR2c phosphorylation due to high endogenous FGF2 expression in 10% FBS with reduced endogenous FGF2 expression in serum starved conditions. In organoid culture, high FGFR2c phosphorylation was seen with or without addition of FGF2 indicating autocrine stimulation. IHC assessment of PDXs and PDXOs revealed high expression of FGF2 in the 8 models that were examined. However, there is redundancy in FGF/FGFR signalling and FGFR2c can be potentially activated by additional ligands including FGF1, FGF9 and FGF18. Our data showed the constitutive FGFR2c phosphorylation through an FGF2/FGFR2c autocrine loop was a principal pathway of FGFR2c isoform activation in contrast to the paracrine activation of FGFR2b in normal epithelial cells\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAfter optimisation of drug concentration, we assessed the effectiveness of FGFRi (BGJ398) in EC PDXOs with and without FGFR2c splice isoform expression. Treatment of EC PDXOs expressing FGFR2c led to cell death at 72 h only in the three PDXOs expressing FGFR2c and not in the two EC PDXOs with low/no expression. FGF2 is an empirical growth factor component of stem cell media and indicated to be a principal master regulator of cancer stem cells \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, pluripotent adult stem cells, and embryonic stem cells\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Otte and colleagues reported FGF2/FGFR signalling is critical for growth, self-renewal of colon cancer PDOs and treatment with FGFR inhibitor supressed PDOs growth and regeneration\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Enhanced expression of FGFR2c has also been reported to play an essential role in pancreatic cancer cell proliferation and this also supports our current finding\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Studies have reported FGF2/FGFR downstream signalling through the ERK1/2-MAPK\u003csup\u003e32\u003c/sup\u003e and JAK/STAT3 \u003csup\u003e35\u003c/sup\u003e pathway contribute to stemness. We demonstrated both pERK1/2 and pSTA3 are expressed in vehicle treated PDXOs and primary cell line but markedly reduced in BGJ398 treated counterparts. However, additional functional mechanistic investigations are needed to understand how these signalling pathways contributes to stemness maintenance in EC and whether additional FGF ligands are also involved in EC. Flix et al. reported FGF2 expression is associated with aggressive clinicopathologic markers and poor survival outcomes in EC \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, consistent with our finding that FGFR2c isoform expression is associated with prognosis and survival in EC \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo validate our findings \u003cem\u003ein vivo\u003c/em\u003e, multiple PDX models were treated with clinically relevant doses of 30 mg/Kg BGJ398 or 1 mg/Kg of Pemigatinib. Both BGJ398 and Pemigatinib demonstrated significant tumour growth inhibition consistent with our \u003cem\u003ein vitro\u003c/em\u003e findings in 4/5 models expressing FGFR2c. This initial 21 day treatment also led to a significant doubling of survival for these four models sensitive to FGFR inhibition. Notably, morphologic assessment of tumours when they reached\u0026thinsp;~\u0026thinsp;900mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e after withdrawal of treatment showed significant central necrosis and cystic formation with a small residual rim of tumour was evident. This indicates gross tumour volume did not correspond to tumour burden following FGFRi treatment. This observation has clinical relevance regarding the type of imaging used to assess tumour shrinkage and treatment response in patients in the clinic and suggests that CT morphological criteria or other functional imaging would be superior to RECIST size based criteria to more accurately reflect viable tumour burden.\u003c/p\u003e \u003cp\u003eMolecular profiling of tumours collected in the PORTEC-3 clinical trial showed that chemotherapy had significant clinical benefit only in patients with p53abn tumours with no improvement seen in patients with MMRd tumours. FGFR inhibition has been shown to sensitise cell line and PDX xenografts to cisplatin in other cancer types\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. For this reason, we wanted to assess if the combination might be effective in MMRd tumours where FGFR2c expression is most common. We report here that although cisplatin had some effect on TGI during the initial 21d treatment, neither cisplatin alone nor the addition of cisplatin to pemigatinib had any benefit on survival in three independent MMRd PDXs with FGFR2c expression. In contrast PDX61 representing a p53abn EC with moderate FGFR2c expression showed intrinsic \u003cem\u003ein vivo\u003c/em\u003e resistance to cisplatin however, \u003cem\u003ein vivo\u003c/em\u003e data showed significant TGI and improved survival with pemigatinib alone with no further benefit with the combination. PDX23 has wildtype p53 by sequencing but represents serous EC with a copy number high genomic profile \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. For this model significant TGI was observed with all three treatments however only the combination of cisplatin and pemigatinib resulted in a significant improvement in survival.\u003c/p\u003e \u003cp\u003eWe have also demonstrated EC PDXs with FGFR2c expression or an FGFR2\u003csup\u003eC383R\u003c/sup\u003e mutation treated with BGJ398 for 7 days had marked reduction in cancer cell proliferation, angiogenesis and pro-tumourigenic CD206\u0026thinsp;+\u0026thinsp;M2 polarised macrophages revealing that FGFR inhibition plays a significant role in modulation the TME in addition to its anti-tumour effect. FGF/FGFR signalling has previously been shown to promotes tumour growth and metastasis through enhancing angiogenesis and treatment with FGFRi reduces CD31\u0026thinsp;+\u0026thinsp;microvessel density in other solid cancers including breast and lung cancer \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e consistent with our finding. Microvessel density has also been associated with poor prognosis and shorter survival in EC\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Tumour associated Macrophages (M2 macrophages) expression is reported to be associated with EC progression and poor clinicopathologic biomarkers\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. In other cancer types FGFR dysregulation plays a significant role in tumour immune evasion and importantly FGFRis have been shown to synergises to increase the efficacy of immune check inhibitors (ICI)\u003csup\u003e\u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. We have also noted 7 days of BGJ398 treatment results in an increase in the differentiation of EC, with clear evidence of glandular morphology formation after treatment compared with an undifferentiated solid morphology in vehicle treated mice. This is consistent with the role of FGFR2c to promote dedifferentiation of cancer cells and suggests it may be another mechanism by which FGFR inhibition exerts its anti-tumour activity in ECs with FGFR2c expression.\u003c/p\u003e \u003cp\u003eIn conclusion, we have developed and characterised EC PDXOs that highly biomimic the morphological and molecular profile of advanced ECs and their corresponding PDX models. We have identified authentic preclinical models with different pattern of \u003cem\u003eFGFR2c\u003c/em\u003e oncogenic splice isoform expression and showed PDXOs with FGFR2c expression are dependent on endogenous FGF2 for its autocrine activation. Finally, the study revealed PDXOs and matched PDXs with FGFR2c expression are highly sensitive to FGFRis (BGJ398 and pemigatinib). The combination of pembrolizumab and Lenvatinib has recently been approved for all EC subtypes however a very high proportion of patients (~\u0026thinsp;90%) developed toxicity and complete responses are low. Our data suggest a combination of a FGFRi with ICI could potentially be more effective with less toxicity in those EC patients with dysregulation of FGFR2 either by mutation (15%) or isoform switching (30\u0026ndash;40%)\u003csup\u003e12\u0026ndash;14\u003c/sup\u003e. A phase II clinical trials are ongoing in EC patients with FGFR2 mutations/fusions and more EC patients may benefit with inclusion of Patients with FGFR2c expression. A study from urothelial carcinoma showed FGFR2/3 dysregulation contributes to ICI resistance\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e but more research into the role of the FGFR2c in immunotherapy resistance is warranted. We have also demonstrated that pemigatinib treatment could improve survival in a subset of p53abn EC models with intrinsic cisplatin resistance suggesting FGFRi\u0026thinsp;+\u0026thinsp;ICI may have some benefit in the second line cisplatin resistant setting. Collectively the data presented confirm the expression of the FGFR2c splice isoform as a precision predictive biomarker in EC. Due to the role of FGFRs in tumour immunosuppression, we propose a clinical trial of an FGFRi plus ICI in advanced EC with a MMRd arm (where FGFRi may overcome some mechanisms of ICI resistance) and a pMMR arm. Inclusion of the latter arm is supported by the activity of Lenvatinib and pembrolizumab in non-endometrioid ECs and our data showing sensitivity to FGFRi in 1/2 P53abn EC models and a p53wt model. As there is a range of FGFR2c expression in ECs, retrospective analysis could then be performed to determine the cut-off at which FGFR2c ISH functions as a predictive biomarker to improve survival.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003ePDXs tumour expansion and characterization\u003c/h2\u003e \u003cp\u003eWe have previously established 18 EC PDXs with different histological and molecular subtypes \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Tumours were collected from EC patients following Human Research Ethics Committee (HREC) approval (HREC/15/MHS/127), QUT HREC (#1500000169, 1500000323). All participants provided written informed consent to take part in the study. Ethical clearance for laboratory animal use was granted from UQ Animal Ethics Committee (AEC) (UQ/TRI/021/19) and QUT (1900000701). We have also re-established three PDXs that were initially developed by the laboratory of Eva Colas and Antonio Gil-Moreno at the Vall Hebron Institute of Research (VHIR) in Spain with ethical approval (PR(AMI) 276/2018) under QUT HREC approval (#5194).\u003c/p\u003e \u003cp\u003eFresh surgical resected tumours either from primary or distant metastatic site were collected from patients who had surgery and fragment of tumours were implanted subcutaneously into immunocompromised Nod Scid Gamma (NSG) 8 week female mice as described previously \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Serial passaging of the PDXs was carried out as previously published. In brief, when the PDX tumour reached a target volume of 900mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, mice were euthanized with CO\u003csub\u003e2\u003c/sub\u003e and each tumour was collected under sterile technique, with ~\u0026thinsp;1\u0026ndash;2 mm3 fragments transplanted fresh into 2\u0026ndash;3 mice for subsequent passaging as well as frozen with 90% FBS and 10% DMSO for later transplantation (biobanking). Portions of the tumour were also frozen for DNA/RNA and protein extraction, fixed in formalin and embedded in paraffin (FFPE) to assess for morphology and FGFR2 expression via IHC/RNA ISH as well as placed in media for subsequent organoid culture (Supplementary Fig.\u0026nbsp;1). Genomic characterization (WES, WGS) was performed in 11 PDXs as published previously \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. IHC to assess FGFR2, p53, Ki67 expression, MMR protein status and RNA ISH was performed in matched patient tumours, PDXs and PDXOs at different passages (P2-10) to confirm PDXOs recapitulated PDX models.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEc Pdx Derived Organoids (Pdxos) Establishment And Expansion\u003c/h3\u003e\n\u003cp\u003ePDXOs culture was performed according to previously reported protocols\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e with slight modification to customise the need of growth factors depending on tumour type. In brief, PDX tumours were collected following a sterile procedure, dissected and a portion of tumour was placed in RPMI medium containing antibiotics (Penicillin/streptomycin) and antimycotic and transported on ice to the tissue culture (TC) room. PDX tumours were minced utilising a surgical blade and dissociated into clusters of tumour cells by mechanical dissociation and enzymatic degradation of the extracellular matrix using the HumanTumor Dissociation Kit (Cat#130-095-929, Miltenyl Biotech, VIC, Australia) following the manufacturer's protocol. The cell suspension was centrifuged, and the pellet was resuspended in DMEM/F12 medium containing 2 mmol/L L-glutamine with antibiotics (100 \u0026micro;g/ml Promicin (cat# amp-1, Jomar life Research, VIC, Australia) or 100 U/mL penicillin and 100 mg/mL streptomycin). The final suspension of tumour cells was then filtered through a 40 or 70-\u0026micro;m cell strainer (Biostrategy, VIC, Australia) to remove large clusters and the cells that were not filtered out were resuspended in DMEM/F12 medium containing 2 mmol/L L-glutamine, antibiotics (100 \u0026micro;g/ml Promicin (cat# amp-1, Jomar life Research, VIC, Australia) and 10 mmol/L (Y-27632) ROCK inhibitor (Selleck Chemicals Inc)\u0026thinsp;\u0026plusmn;\u0026thinsp;growth factors WRN (WNT3A, R-Spondin-3, NOGGIN), EGF and FGF2 as required and was plated into the centre of 48 well plate precoated with 50% V/V) Matrigel\u0026trade; (growth factor reduced, GFR) (cat# 354230, Corning, VIC, Australia) and incubated at 37 ⁰C and 5% CO\u003csub\u003e2\u003c/sub\u003e. When Matrigel\u0026trade; was gelled, 200\u0026micro;L of expansion media was added to cover Matrigel droplets and media was changed every 3 days. PDXOs growth was monitored using an inverted phase contrast microscope and images were captured to document growth using a Nikon phase contrast microscope with camera attached. Established PDXOs were further passaged and were subjected to multiple sequential cycles of freezing and thawing to ensure the renewal stability and fitness of the organoids. PDXOs were banked at multiple passages by cryopreservation in liquid nitrogen. For establishment of primary PDX-derived EC cell lines, dissociated cells were cultured in 2D in a 10cm petri dish in 10% FBS with DMEM/F12 Media and cell selection was performed with differential trypsinisation (0.25% trypsin EDTA) to remove fibroblasts. After 4 successive passages, 100% purity was confirmed with a cytokeratin 7 stain. Mycoplasma testing was performed routinely using a commercial kit (MycoSEQ and Thermo-Fisher Scientific, VIC, Australia).\u003c/p\u003e \u003cp\u003e \u003cb\u003ePDXOs Formalin-Fixed Paraffin-Embedded (FFPE) Blocks Preparation.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe medium was removed and replaced with cold medium, and the plate was cooled on ice to liquify the Matrigel to make PDXO suspension. The PDXOs suspension were gently transferred into yellow screw cap, flat base 5ml Polypropylene tube (Sarstedt, Ref.# 60.9921.524) and kept at room temperature (RT) for 20 min and then centrifuged at 300 g at 4\u0026deg;C for 4 min and then supernatant was discarded and PDXO pellets were washed with PBS twice and fixed with 4% PFA in PBS for 1 hour at RT. Then PDXO pellets were centrifuged at 300 g for 4 min at RT and the fixative was discarded. A 2% Histogel/agarose gel (Cat. # HG-4000-012, Thermo Scientific, VIC, Australia) in PBS was prepared and melted by heating in microwave and then cooled to 60\u0026deg;C prior to adding to the PDXO pellets. A 200 \u0026micro;L melted agarose gel was added to PDXO pellets, allowed to solidify at RT for 30 min or 10 min at -20\u0026deg;C, then carefully transferred to tissue cassettes with sponge to avoid loss of small PDXOs, placed in 70% ethanol and processed using an automated 4 hours processing protocol in the TRI-Histology core facility and embedded with paraffin to make FFPE blocks. H/E and IHC staining and BaseScope RNA ISH assay was performed as described previously \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eFgfr2 Isoform Determination\u003c/h3\u003e\n\u003cp\u003eFGFR2b and FGFR2c splice isoform expression on primary EC patient and matched PDX and PDXOs tumours was determined following our previously optimised and validated BaseScope RNA ISH assay\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eMolecular Subtyping\u003c/h3\u003e\n\u003cp\u003eMolecular subtyping of the EC patient, PDX and matched PDOs tumours were performed using the ProMisE algorithm combining genomic analyses (tumour mutation burden, somatic copy number alteration) or IHC surrogate markers as previously reported\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. IHC was done for several markers including p53, MLH1, MSH2, MSH6 and PMS2.\u003c/p\u003e\n\u003ch3\u003eLigand Stimulation Of Primary Cell Line And Pdxos\u003c/h3\u003e\n\u003cp\u003ePrimary cell line (ASPX67-406) cells (P4) were cultured in 10% FBS DMEM/F12 media and after reaching 70% confluence, they were trypsinised and 1 x 10\u003csup\u003e5\u003c/sup\u003e cells were seeded in 6 well plates containing 3 coverslip slides/well (1.13 mm \u0026Oslash;) with DMEM/F12 media for 48 hours at 37\u0026deg;C and 5% (v/v CO\u003csub\u003e2\u003c/sub\u003e). After cells reached 60\u0026ndash;70% confluence, media was removed, cells were washed with warm DPBS and either incubated with fresh 10% FBS media or starved overnight (16 hr) with 0.5% FBS DMEM/F12 media. At zero time point (T0) media was removed and washed with warm DPBS twice and stimulated with media containing 0.5% FBS with 5 \u0026micro;g/ml HS with or without 10 ng/ml FGF2 and incubated for 30 min at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e. For the treatment arm 100nM/ml BGJ398 was added during stimulation. Cells were then washed with DPBS twice and fixed with 4% Paraformaldehyde (PFA) in PBS for 30 min at room temperature (RT) and PLA was performed. For PDXOs, culturing was performed in advanced DMEM/F12 Media with 30% (V/V) Matrigel\u0026trade; with and without supplementation of FGF2 as described above and at Day 10 media was removed and washed with DPBS and fresh advanced media with and without HS\u0026thinsp;+\u0026thinsp;FGF2 and incubated for 30 min. For the treatment group 100nM BGJ398 was added during ligand stimulation. After 30min, the media was removed and PDXOs were fixed with PFA for 1 hour and resuspended and stained with Immunofluorescence (IF) or Proximity Ligation Assay (PLA) assay as described below.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence (If) And Proximity Ligation Assay (Pla) Staining\u003c/h3\u003e\n\u003cp\u003eImmunofluorescence and Proximity Ligation Assay (PLA) staining was performed manually using optimised and validated protocols. In brief, culture media was removed and cells and/or organoids were washed with PBS and fixed with 4% paraformaldehyde (PFA) with incubation for 30 min. PFA was removed and cells/organoids were washed three times with PBS/TPBS and permeabilised with 0.25% TritonX100 in PBS for 5 min at RT and washed with PBS/ TBS three times. Cells were blocked with 2% BSA in PBS for 1 hr at RT or overnight at 4\u0026deg;C. After that, cells were transferred into a humidity chamber and incubated with primary antibody diluted with 2% BSA in PBS (Suppl Table S1 for respective antibody source and dilution) and incubated for 90 min at room temperature or overnight at 4\u0026deg;C. Slides were then washed three times with TBS or PBS for 5 min each and incubated with secondary antibodies (Suppl Table S2 for dilutions and sources) for 45 min at RT. For PLA staining, primary antibodies (antiFGFR2 and pFGFR653) were incubated for 90 min after 3 times wash with TBS, cells/PDXOs were incubated for 90 min with PLA probes (oligonucleotide conjugated donkey anti-rabbit, PLUS (DUO82002) and donkey anti-mouse, MINUS (DUO82004)). After 3x washes with TBS a ligation and amplification step were followed according to the manufacture protocol (detail guideline suppl method A). To combine PLA with IF, the primary antibody was incubated for 60 min at 37\u003csup\u003eo\u003c/sup\u003eC after the amplification step and then washed 3 times with TPS for 2 min each. Then, secondary antibody conjugated with FITC was incubated for 30 min at 37\u003csup\u003eo\u003c/sup\u003eC and washed with TPS twice for 2 min each. Finally, cells/organoids were washed two times with PBS or TBS for five min each and final wash was performed with milliQ H\u003csub\u003e2\u003c/sub\u003eO to remove any precipitated salt and mounted with ProLong Gold Antifade with DAPI mounting medium (Thermo-Fisher, VIC, Australia).\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eIn vitro\u003c/span\u003e \u003cb\u003etreatment of PDXOs with BGJ398\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo test the effect of FGFR inhibitors in PDXOs regeneration and growth inhibition, PDXOs were initially treated at early time point following seeding (after 24 hr) with 100 nM BGJ398 or at a later timepoint when the organoids were well established (after 10 days) with different concentrations of BGJ398 (100 nM, 200 nM and 300 nM) at different time point (24 hr, 48 hr and 72 hr). To assess the effect of endogenous FGF2 on growth and regeneration, PDXOs were cultured with and without of anti-FGF2 antibody. After optimising the dose and duration of treatment, three independent PDXOs with high FGFR2c expression and two with negative or low FGFR2c expression) were grown for 10 days. After PDXOs reached 70\u0026ndash;80% confluency, PDXOs were treated with either 300 nM infigratinib/BGJ398 (Selleck Chemicals Inc, VIC, Australia) or vehicle (equal volume of DMSO) and incubated for 72 hours at 37⁰C and 5% CO\u003csub\u003e2\u003c/sub\u003e. The viability of the PDXOs was then assessed using a Live/Dead assay kit (Cat# R3760, Thermo Fisher Scientific, VIC, Australia) following the manufacture\u0026rsquo;s protocol. This experiment was performed in 3 different PDXOs models derived from each PDX as well as in technical triplicate. Images were captured XYZ stack using an inverted rotatory confocal fluorescent laser microscope (FV1200) at 10x objective magnification view airy. Image analyses to quantify the live and dead cells were performed using Fiji ImageJ2 (National Institute of Health, Bethesda, Maryland, USA) as described previously\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. In brief, the captured images were converted into TIF format and imported for Fiji ImageJ and image of PDXOs were imported to Fiji ImageJ2 platform. For each channel (Green, red, and blue) and Otsu\u0026rsquo;s threshold was used to create binary images (white and black) and level of pixel intensity was assigned between 1 (white) and 0 (black), object was identified using region of interest (ROI) and analysis set in parallel sequential. Total number of cells for each channel (live\u0026thinsp;=\u0026thinsp;green) and (red\u0026thinsp;=\u0026thinsp;dead) were calculated as follows; number of green positive cells divided by the number of nuclei (DAPI positive), likewise number of red positive cells divided by the total number of nuclei. The results were exported to SPSS for statistical analysis.\u003c/p\u003e\n\u003ch3\u003eWestern Blot For Fgf2 Antibody Validation\u003c/h3\u003e\n\u003cp\u003eBaF3 cells (primarily sourced from ATCC, Manassas, VA) were previously transduced with FGF1, FGF2, FGF3, 7\u0026ndash;10, 17, 18, 20 and 22 ligands tagged with Myc-DDK\u0026trade; by our laboratory\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Protein extraction and western blot analysis were performed as previously published\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Twenty micrograms of protein lysates were loaded from BaF3 cell lysates expressing targeted ligands and probed with anti-FGF1, anti-FGF2, anti-FGF3 and anti-FGF7 antibodies (see supplementary Table S1 and 2 for source and cat#) to assess potential immune-cross reactivity. Cell lysates were probed with an anti-Myc antibody to demonstrate that each cell line was expressing the transduced FGF ligands.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eIn Vivo\u003c/span\u003e \u003cb\u003etreatment of PDXs\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe experiment was conducted according to the approved protocols AEC (#021/19, 399/20, 456/20) and the Australian National Health Medical Research Council (NHMRC) guidelines. EC tumour fragments were implanted into 6-8-week female NSG mice subcutaneously under sterile technique for each model. When the tumour reached 150-200mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e mice were randomized into vehicle, 30 mg/kg infigratinib/BGJ398 (3 models), 1 mg/Kg pemigatinib (Incyte) alone (7 models), pemigatinib\u0026thinsp;+\u0026thinsp;cisplatin (5 models), or cisplatin alone (5 models). Two PDX models (PDX52 and PDX59) were treated with both infigratinib and pemigatinib. Mice were treated for 21 days with infigratinib and pemigatinib via oral gavage and 5 mg/Kg cisplatin intravenous (IV) injection weekly. Tumours were measured using calliper 3 times/week until reaching 900mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Tumour volume (TV) was calculated as follows: Tumour Volume (\u003cem\u003eTV)\u0026thinsp;=\u0026thinsp;Length[width\u003c/em\u003e x \u003cem\u003ewidth]\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/2\u003c/em\u003e. Follow up data regarding tumour size was collected 3x/w until tumours reached 900mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. For tumour biomarkers assessment and mechanistic studies, short period (7 day) treatment was aimed to compare apparently equal tumour volume. For this aim, a separate cohort of 4 independent PDX models were treated with BGJ398 or vehicle (4 mice per arm) for 7 days. Mice were euthanised after 6 hr of the last treatment dose and their tumours were collected, cryopreserved or fixed with 4% paraformaldehyde for H/E morphologic examination and IHC biomarker evaluation.\u003c/p\u003e\n\u003ch3\u003eImmunohistochemistry And Quantification\u003c/h3\u003e\n\u003cp\u003eIHC staining of p53 and MMR proteins was performed via Ventana automated stainer (Ventana Medical Systems, Tucson, AZ, USA) using the diagnostic antibodies at Mater Pathology, Brisbane Australia. Detail information regarding these antibodies\u0026rsquo; sources and cat# are provided in supplementary Table S1 and S2. The remaining IHC staining was performed manually following optimised protocols. In brief, whole FFPE tumour tissue sections were cut (4 uM thickness) and sections were dewaxed, hydrated and washed, and endogenous H2O2 was quenched with 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 10 min. Antigen retrieval was performed in 10 mM citrate buffer (PH\u0026thinsp;=\u0026thinsp;6.0) with Decloaking Chamber\u0026trade; (BIOCARE MEDICAL, Brisbane, Australia). Respective Biotin (Jackson Immunology Labs) or polymer conjugated secondary antibodies for all primary antibodies raised in rabbit or mouse (K406311-2, DAKO, Envision kit, Agilent Technologies, VIC, Australia) were used. Detailed information for all primary and secondary antibodies source, Cat#, period of incubation are provided in supplementary Tables\u0026nbsp;1 and 2. Chromogen development was performed using DAB (K3468, DAKO Envision) by incubating for 2\u0026ndash;5 min and slides then counter stained with Mayer Haematoxylin (Sigma Aldrich).\u003c/p\u003e \u003cp\u003eP53 and MMR proteins (MLH1, MSH2, MSH6 and PMS2) IHC scoring was performed as previously reported\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Quantification of other IHC/IF markers was performed using either automated Fiji ImageJ2 or QuPath bioimage analyses software following the guidelines as published previously\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. In brief, whole section stained slides were scanned using whole slides scanner (3DHISTECH) at 40X magnification and images in the form of TIF files were imported to either ImageJ2 or QuPath software as indicated to create separate projects for each biomarker of interest or H/E stain. Quantification was performed after training and setting the required annotation, deconvolution and DAB vector intensity for each biomarker based on the algorithms and workflow of Fiji Image J2 or QuPath platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/qupath/qupath\u003c/span\u003e\u003cspan address=\"https://github.com/qupath/qupath\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003csup\u003e50\u003c/sup\u003e. Results were exported into SPSS or GraphPad Prism as required for data analyses.\u003c/p\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis and data visualization was performed using SPSS version 23 and GraphPad Prism ver9.2 as required. Figures annotation. formatting and visualization were performed using Adobe illustrator (AI). Pearson\u0026rsquo;s (Chi X\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e) test was used to evaluate correlations between categorical variables. Student T-Test (two-sided) was used to assess continuous variables. Two-way ANOVA with mixed model was used to assess tumour growth inhibition. Survival outcome differences between or among groups was computed using Kaplan Meier Curve with Mantel-Cox (Log-Rank) test of probability. Multiple comparison was corrected using either Bonferroni for categorical variables or Tukey test for continuous variables. \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (two sided) was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank to all women for their generous support of offering their tumour samples for generation of PDXS. We would like to thank the TRI animal facility group for their enormous technical support for the mouse work. We are also grateful for the TRI histology and microscopy core facility as well as QUT histology core facility members at Kelvin Grove for whole slide scanning and other technical support. The authors also appreciate the generous support from John Hooper laboratory. We would like also to acknowledge the study was partially funded by Incyte INC. though investigator initiated study (IIS) scheme (Grant number #\u0026nbsp;IPAP2020/1611). Funders have no role in study conception, design, data collection, analysis, interpretation, or writing. \u0026nbsp;The Translational Research Institute (TRI) Australia is supported by a grant (APP108382) from the Australian Government.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualisation and design: ATS and PMP Data curation: ATS and PMP Formal analysis: ATS Funding acquisition: ATS and PMP Investigation: ATS and PMP Methodology: ATS and PMP Project administration: ATS and PMP Resources: ATS, BV, DS, CPM, RL, RR, EC AGMSF, NC, LP, PMP Software: ATS Supervision: PMP Validation: ATS Visualization: ATS and PMP Writing\u0026mdash;original draft: ATS Writing\u0026mdash;review and editing: ATS, BV, DS, CPM, RL, RR, EC AGMSF, NC, LP, PMP. All authors approved and agreed manuscript publication\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSung, H. \u003cem\u003eet al.\u003c/em\u003e Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA: A Cancer Journal for Clinicians \u003cb\u003e71\u003c/b\u003e, 209\u0026ndash;249 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThe Cancer Genome Atlas Integrated genomic characterization of endometrial carcinoma. Nature \u003cb\u003e497\u003c/b\u003e, 67\u0026ndash;73 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTalhouk, A., McConechy, M. \u0026amp; Leung, S.e.a. A clinically applicable molecular-based classification for endometrial cancers. 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Scientific Reports \u003cb\u003e7\u003c/b\u003e, 16878 (2017).\u003c/span\u003e\u003c/li\u003e\u003c/ol\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":"npj-precision-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjprecisiononcology","sideBox":"Learn more about [npj Precision Oncology](http://www.nature.com/npjprecisiononcology/)","snPcode":"41698","submissionUrl":"https://submission.springernature.com/new-submission/41698/3","title":"npj Precision Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-2512859/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2512859/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Endometrial cancer (EC) patients with metastatic/recurrent disease have limited treatment options and poor survival outcomes. Recently, we discovered the FGFR2c isoform is associated with poor prognosis in EC patients. Here we report the establishment of 14 EC patient-derived xenografts (PDX)-derived organoids (PDXOs) with or without FGFR2c expression. Treatment of 5 EC PDXOs with BGJ398 showed significant cell death in 3 models with FGFR2c expression. PDXs with FGFR2c+ showed significant tumour growth inhibition (TGI) following 21-day treatment with FGFR inhibitors (BGJ398 or pemigatinib) and significantly prolonged survival in 4/5 models. Pemigatinib + cisplatin combination therapy (n=5) resulted in significant TGI and prolonged survival in one of two p53abn PDXs. All five models treated with cisplatin alone showed de novo resistance and no survival benefit. Seven-day treatment with BGJ398 revealed a significant reduction in angiogenesis and CD206+ M2 macrophages. This data collectively supports the evaluation of FGFR inhibitors in a clinical trial.","manuscriptTitle":"Endometrial cancer PDX-derived organoids (PDXOs) and PDXs with FGFR2c isoform expression are sensitive to FGFR inhibition.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-02-14 18:30:34","doi":"10.21203/rs.3.rs-2512859/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2023-02-27T14:13:39+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2023-02-22T13:54:48+00:00","index":3,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2023-02-18T03:27:55+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2023-02-14T18:56:01+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2023-02-13T15:19:31+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2023-02-10T19:51:05+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2023-02-10T15:44:02+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2023-02-10T15:06:50+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-02-09T21:30:22+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-02-07T09:55:17+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Precision Oncology","date":"2023-02-07T01:48:24+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2023-01-27T09:35:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-precision-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjprecisiononcology","sideBox":"Learn more about [npj Precision Oncology](http://www.nature.com/npjprecisiononcology/)","snPcode":"41698","submissionUrl":"https://submission.springernature.com/new-submission/41698/3","title":"npj Precision Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"aca167d6-84e7-4fef-a24c-10ab64aeb7af","owner":[],"postedDate":"February 14th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":19100866,"name":"Health sciences/Oncology/Cancer/Gynaecological cancer/Endometrial cancer"},{"id":19100867,"name":"Biological sciences/Cancer/Cancer models"},{"id":19100868,"name":"Health sciences/Biomarkers/Predictive markers"},{"id":19100869,"name":"Health sciences/Medical research/Translational research"},{"id":19100870,"name":"Biological sciences/Biological techniques/Immunological techniques/Immunohistochemistry"}],"tags":[{"value":"featured","date":"2023-02-14 19:31:38"}],"updatedAt":"2023-11-14T21:00:47+00:00","versionOfRecord":[],"versionCreatedAt":"2023-02-14 18:30:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2512859","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2512859","identity":"rs-2512859","version":["v1"]},"buildId":"omnImTCwR2MFx8CMYfrG7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}