Abstract Patient-derived xenografts (PDXs) are essential preclinical tools for modeling prostate cancer (PCa) biology and assessing therapeutic responses. Over the past years, we have established and comprehensively characterized a panel of 15 prostate cancer PDX models that recapitulate the histological and molecular heterogeneity of human disease. While PARP inhibitors such as olaparib provide clinical benefit to patients harboring BRCA1/2 mutations or homologous recombination deficiency (HRD), resistance inevitably emerges and remains a major clinical challenge. Here, we present insights that pave the way for characterizing mechanisms of PARP inhibitor resistance and identifying strategies to circumvent PARPi escape using our panel of PCa PDX models. Therapeutic responses to olaparib were evaluated across the biobank, alongside assessment of homologous recombination repair (HRR) status. Most PDX models displayed intrinsic resistance, whereas three showed marked sensitivity. Notably, a neuroendocrine prostate cancer (NEPC) PDX harboring DNA damage-repair alterations exhibited pronounced olaparib sensitivity, providing the first preclinical evidence supporting PARP inhibitor activity in a neuroendocrine context. These three sensitive models were then exposed to chronic treatment until tumor recurrence, generating acquired olaparib-resistant derivatives. Resistant PDXs were compared with their isogenic sensitive counterparts through histopathological analysis and transcriptomic and genomic profiling, with a focus on DNA repair and compensatory pathways. Multi-omic analyses indicate involvement of mechanisms such as partial restoration of homologous recombination and activation of alternative DNA repair pathways. Overall, this unique PCa PDX panel, together with the newly generated olaparib-resistant derivatives, constitutes a valuable preclinical resource for deciphering PARP inhibitor resistance mechanisms and informing the development of next-generation therapeutic combinations for prostate cancer. Citation Format: Nadège BIDAN, Claire BERAUD, Emilie INDERSIE, Marie TAVERNIER, Clementine KRUCKER, Eric POTIRON, Philippe LLUEL, Olivier DÉAS. Modeling olaparib resistance in prostate cancer PDXs to elucidate PARP inhibitor escape mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2154.

Fibroblast growth factor receptor 3 (FGFR3) is one of the most frequently altered genes in bladder cancer, primarily through activating mutations that drive oncogenesis and are enriched in luminal tumors. However, the underlying gene regulatory network (GRN) remains poorly characterized. Here, we constructed an FGFR3-mutated GRN using a bottom-up bioinformatics approach, integrating transcriptomic data from bladder cancer cell lines, FGFR3-mutated tumors, and FGFR3 perturbation experiments in human and mouse models. Using publicly available CRISPR/Cas9 screening data, we identified transcription factors from this GRN that regulate the viability of FGFR3-mutated cells, with a focus on p63 (TP63). We showed that FGFR3 activation upregulates p63 in patient-derived xenografts and cell lines, while single-cell RNA sequencing revealed heterogeneous p63 activation associated with basal differentiation. Functional studies, including TP63 knockdown in FGFR3-dependent in vitro and in vivo models and RNA-seq along with p63 ChIP-seq, demonstrated that p63 directly promotes cell proliferation and migration and uncovered a positive feedback loop between FGFR3 and p63. Together, these findings support p63 as a protumorigenic regulator in FGFR3-mutated tumors despite their luminal differentiation and provide a detailed FGFR3-driven GRN, offering insights into FGFR3-induced oncogenic dependency and potential strategies to circumvent resistance to FGFR inhibitors.

Abstract Background: Inter-patient heterogeneity in bladder cancer (BC) remains a major driver of treatment failure, highlighting the need for more personalized therapeutic approaches. Org anoids represent valuable preclinical models capturing this diversity. We established a BC organoid biobank comprising 20 models that have been extensively characterized at phenotypic, pharmacological and molecular levels. The aim of this study was to demonstrate the relevance of our biobank in terms of specifications and disease representation. In addition, we illustrated the utility of these models for evaluating targeted therapies, using Enfortumab-vedotin (EV) as a proof of concept. Methods: Urothelial progenitors were isolated from tumor samples, seeded in Matrigel® and culture medium was added. Molecular characterization was performed by whole exome and transcriptome sequencing. Morphology of organoids was determined by optical microscopy. To evaluate targeted-therapy, organoids were treated with EV (from 0.3 to 10 µg/mL) for 5 days. Cell viability was measured with CellTiter-Glo3D® assay. Results: All organoid models were developed following a strict quality framework: cultures were expanded beyond passage 6, cryopreserved, and demonstrated a 100% post-thaw recovery rate. The biobank captured the full pathological spectrum, from non-invasive pTa to metastatic pT4 stages. It reflected the anatomical diversity of urothelial carcinoma, with models derived from both bladder and ureteral tumors. Omics analyses revealed the presence of key molecular alterations commonly observed in BC patients, including FGFR3 and TP53 mutations. Organoid morphology, categorized as solid, hollow, or mixed, correlated with tumor stage and metastatic models exhibited features of epithelial-mesenchymal transition. For EV evaluation, we assessed Nectin-4 expression across the biobank and observed heterogeneous expression levels. Interestingly, organoid responses to EV were not strictly correlated with Nectin-4 expression with low response for models with high Nectin-4 expression. Conclusion: Overall, our BC organoid biobank constitutes a high-quality collection of clinically annotated and comprehensively characterizes models ensuring robustness, stability, and reproducibility across experiments. It faithfully captured the pathological diversity and inter-patient heterogeneity observed in BC. These organoid models represent powerful tools for assessing novel targeted therapies, and the availability of a broad biobank enables the extrapolation of patient-specific responses, paving the way toward personalized medicine. Citation Format: Emilie Decaup, Céline Rouget, Amandine Prioux-Quartier, Claire Béraud, Nadège Bidan, Xavier Gamé, Philippe Lluel. Bladder cancer organoids as a translational models to model disease biology and assess new therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4879.

Abstract Background: Resistance to Enfortumab vedotin (EV) remains a major clinical challenge in bladder cancer (BC). The resistance can emerge through diverse mechanisms such as alterations in Nectin-4 expression, defects in ADC internalization and trafficking, or changes in downstream processing and payload response. We previously developed EV-resistant BC PDX and organoid models (poster 50, AACR 2025), providing validated preclinical tools to investigate the molecular basis of EV resistance. These models were designed both to elucidate resistance mechanisms and to enable direct comparison with patterns of resistance observed in the clinic. Methods: F659 PDX model was selected based on its high Nectin-4 mRNA expression and its strong response to EV treatment. Acquired resistance was generated by repeated EV administrations until tumors ceased to regress. From the resulting EV-resistant PDX (F659rP2), a matched organoid model was subsequently established. Both resistant and parental models were profiled using whole-exome sequencing, RNA-sequencing, and immunohistochemistry (IHC), and functionally characterized to assess EV activity and identify resistance mechanisms. Validation of these mechanisms was performed using pharmacological inhibition assays. Results: Resistance to EV was confirmed in both organoid and PDX models, as evidenced by a marked loss of sensitivity compared with their parental counterparts. Then, Nectin-4 expression was evaluated in resistant and parental models to investigate the underlying mechanisms, Flow cytometry and IHC analyses revealed a decreased cell surface expression of Nectin-4 in resistant models. Transcriptomic profiling identified significant changes in the expression of drug efflux transporters, FGFR3 and PPARγ pathways. Notably, the ABCB1 gene was markedly upregulated in resistant models, and corresponding P-glycoprotein (P-gp) overexpression was confirmed by flow cytometry and IHC.Given that ABC transporters promote drug efflux, reducing intracellular drug accumulation and therapeutic efficacy, we assessed the impact of P-gp inhibition. Our results demonstrate that co-treatment with the P-gp inhibitor tariquidar (XR9576) partially restored EV sensitivity in both resistant organoid and PDX models. Conclusion: We successfully generated paired in vitro and in vivo EV resistant models. Our findings highlight the role of ABC transporter upregulation in EV resistance associated with a reduction of Nectin4 expression consistent with clinical observations. Other pathways seem to be involved and will need further investigations. Importantly, these EV-resistant preclinical models provide powerful tools for evaluating innovative therapeutic strategies against resistant tumors. Citation Format: Emilie Decaup, Claire Béraud, Isabelle Bernard-Pierrot, Guerric Gilbert, Clémentine Krucker, Xavier Gamé, Philippe Lluel, Nadège Bidan. P-glycoprotein (ABCB1) overexpression confers resistance to the antibody-drug conjugate Enfortumab vedotin in EV-resistant organoid and PDX models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2958.

Significant therapeutic progress have been made for advanced bladder cancer (BC) particularly with the combination of Enfortumab Vedotin (EV) and the pembrolizumab (anti-PD1). This combination has shown a manageable safety profile and superior outcomes compared to chemotherapy, leading to FDA approval for patients with advanced BC. In clinical settings, decisions regarding PD1 immunotherapy are guided by PDL1 expression levels on tumor cells. We selected two organoid models (F659 and BLOU_191) out of 18 from our biobank based on their PDL1 and nectin-4 expression. An additional organoid model (F659R) with acquired-resistance to EV was also included. The objectives of this study were to highlight the advantages of a co-culture system, which combines organoids and immune cells, to evaluate the efficacy of EV and pembrolizumab combination. Furthermore, leveraging recent developments, effectiveness of this therapy was assessed in a context of EV resistance. Transcriptome sequencing of our biobank enabled the identification of mRNA levels expression for PDL-1 and nectin-4. Peripheral blood mononuclear cells (PBMCs) from healthy donors were introduced into organoid cultures at varying effector-to-target (E:T) ratios. Co-cultures were treated with pembrolizumab (200 nM) and EV (from 0.3 to 3 µg/mL), both individually and in combination. Organoid viability was assessed using the CellTiter-Glo® assay at intervals ranging from 48 to 96 hours post-treatment. The efficacy of the combination therapy was then compared to the effects of each compound alone. We have assessed the responses of the three models to pembrolizumab and EV, and correlated treatment efficacy with PDL1 and nectin-4 expression levels, respectively. Results demonstrated that pembrolizumab efficacy was related to PDL1 expression in organoid models, while EV efficacy did not show a strict correlation with nectin-4 expression. Indeed, despite its high Nectin-4 expression, BLOU_191 presented only a low response to EV. In co-culture systems using the EV-responsive organoid model (F659), the combination of EV and pembrolizumab exhibited significant 20% enhanced efficacy compared to the individual treatments whereas it did not in the low-responder model (BLOU_191). In the resistant model, neither EV nor pembrolizumab exhibited significant efficacy when used alone, and their combination did not enhance these effects. The co-culture system involving organoids and PBMCs demonstrated its utility in evaluating the combination of pembrolizumab and EV. Notably, this study highlighted a critical finding: while the combination has shown superiority over current treatment options, it does not overcome resistance to EV. Overall, this co-culture system is highly adaptable to other immunotherapeutic agents and presents a valuable tool for developing novel therapies, particularly in addressing resistance to EV. Anne-Sophie Bajeot, Céline Rouget, Nadège Bidan, Claire Béraud, Sarah Péricart, Xavier Gamé, Mathieu Roumiguié, Philippe Lluel, Emilie Decaup. Organoids and immune cells co-culture for evaluating the efficacy of enfortumab vedotin and pembrolizumab combination therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3996.

Nectin-4 has emerged as a promising target for antibody-drug conjugates (ADCs) due to its high expression in bladder cancer (BC) cells. Enfortumab vedotin (EV), an anti-Nectin-4 ADC conjugated with MMAE toxin, has demonstrated potent therapeutic activity, particularly in combination with pembrolizumab. However, resistance to EV presents a significant clinical challenge. This study aims to develop EV resistant models among Urosphere's biobank of BC patient-derived xenografts (PDX) and organoids for screening anti-Nectin-4 compounds and investigating mechanisms of resistance to EV. RNA sequencing/Affymetrix and immunohistochemistry (IHC) were performed to quantify Nectin-4 expression. Sixteen PDX models were treated with EV (4 mg/kg, twice) to evaluate antitumor activity. Two PDX models (coded as B521 and F659) highly responsive to EV were selected to generate acquired-resistant models. They were treated with repeated EV treatments until tumors no longer regressed. Subsequently, tumor fragments from both PDX models were implanted into new NMRI mice and treated with EV to validate the resistance acquisition. Starting from PDX tumors no longer responding to EV, organoid model was established and treated to EV from 0.003 up to 10 µg/mL for five days, followed by cell viability assessment using the CellTiter-Glo® 3D assay to confirm their resistance. Urosphere’s PDX biobank encompasses diverse Nectin-4 expression profiles. EV efficacy in vivo was not strictly correlated with Nectin-4 expression. B521 did not acquire any resistance whereas F659 PDX model developed resistance after ∼200 days of repeated administration. We established the corresponding organoids and we showed that IC50 and Emax were significantly different between sensitive and resistant models. Nectin-4 expression remained stable in resistant models (mRNA and protein levels), indicating that resistance was unrelated to target loss. Transcriptomic analyses highlighted several potential mechanisms, including the upregulation of efflux transporters such as MDR1 and MRP3, which may contribute to resistance. Urosphere’s PDX and organoid biobank serves as a powerful platform for advancing research on Nectin-4-targeted therapies. From the F659 PDX model, we successfully developed paired in vitro and in vivo models resistant to EV. These novel models provide valuable tools for assessing innovative therapeutic approaches for resistant tumors. Additionally, the EV-resistant models underscore the potential role of ABC transporter upregulation in resistance mechanisms, offering critical insights for the development of alternative treatment strategies. Claire Béraud, Emilie Decaup, Anne-Sophie Bajeot, Céline Rouget, Karine Rollet, Mathieu Roumiguié, Xavier Gamé, Philippe Lluel, Nadège Bidan. Development of paired enfortumab vedotin-resistant urothelial cancer models using organoid and PDX [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 50.

Abstract In patients with bladder cancer (BC) immune therapies have recently demonstrated an improvement for overall survival rates compared to chemotherapy alone. However, several challenges persist and it remains an unmet need to develop novel therapeutic strategies. Organoids have emerged as a promising animal-free tool revealing patients’ heterogeneity and allowing large preclinical therapeutic screening. Urosphere developed a BC organoids biobank with phenotypic, pharmacological and molecular characterization. The aim of this study was to demonstrate the relevance of our organoids models and their use for validation of therapeutic approaches such as targeted or CAR-T cell therapies using EGFR as target. Molecular characterization of the organoids was performed by exome and whole transcriptome sequencing. Cell architecture and differentiation were assessed by labeling cytokeratins (CK) 5, 17 and 20, and uroplakin 3 (UPK3). Proliferation and tumor status were validated with Ki67 and GATA3, respectively. To evaluate targeted-therapy, organoids were treated with erlotinib for 5 days and cell viability was measured with CellTiter-Glo3D® (CTG) assay. To validate CAR-T cell approach, organoids were co-cultured with anti-EGFR or control anti-CD19 CAR-T cells at 4 effector:target ratios. Cell viability was measured with CTG and Caspase-Glo® 3/7 3D assays. Pro-inflammatory and granzyme B release were analyzed by Luminex® assay. Phenotypically, organoids showed proliferating structures whose tumor status was confirmed by GATA3 expression. Cellular heterogeneity present in original tumors was also found, with differential expression of CKs showing the presence of basal (CK5/CD17) and/or differentiated superficial (CK20/UPK3) cells. Organoids from 2 models with high transcriptomic EGFR expression were selected and protein expression was confirmed by immunofluorescence. For targeted therapy, erlotinib presented significant efficacy in both models with a better activity in the model presenting the highest EGFR expression. After 48h of co-culture with anti-EGFR CAR-T cells, a decrease of cell viability associated with an increase of apoptosis was observed. After 72h of co-culture, an increase in the secretion of pro-inflammatory cytokines (IFN-γ and TNF-α) and granzyme B by anti-EGFR CAR-T cells was also observed. In co-cultures with organoids higher expressing EGFR, CAR-T cells activation appeared higher compared to those co-cultured with organoids lower expressing EGFR. Organoids reproduce characteristics of the tumor from which they are derived, making them a predictive preclinical model. In an EGFR targeting approach, the use of organoids to evaluate both targeted- and CAR-T cell therapies was validated with concordant results. This robust translational model is fully transposable to other therapeutic approaches, whether in a traditional culture model or in a co-culture system. Citation Format: Emilie Decaup, Claire Béraud, Anne Sophie Bajeot, Xavier Gamé, Nicolas Monjotin, Pascal Rischmann, Philippe Lluel, Mathieu Roumiguié. A biobank of bladder cancer organoids as a platform for screening new therapeutic approaches [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4231.

BACKGROUND AND PURPOSE:Castration-resistant prostate cancer (CRPC) is a common male malignancy that requires new therapeutic strategies due to acquired resistance to its first-line treatment, docetaxel. The benefits of vitamin D on prostate cancer (PCa) progression have been previously reported. This study aimed to investigate the effects of vitamin D on chemoresistance in CRPC. EXPERIMENTAL APPROACH:Structure function relationships of potent vitamin D analogues were determined. The combination of the most potent analogue and docetaxel was explored in chemoresistant primary PCa spheroids and in a xenograft mouse model derived from a patient with a chemoresistant CRPC. KEY RESULTS:Here, we show that Xe4MeCF3 is more potent than the natural ligand to induce vitamin D receptor (VDR) transcriptional activities and that it has a larger therapeutic window. Moreover, we demonstrate that VDR agonists restore docetaxel sensitivity in PCa spheroids. Importantly, Xe4MeCF3 reduces tumour growth in a chemoresistant CRPC patient-derived xenograft. In addition, this treatment targets signalling pathways associated with cancer progression in the remaining cells. CONCLUSION AND IMPLICATIONS:Taken together, these results unravel the potency of VDR agonists to overcome chemoresistance in CRPC and open new avenues for the clinical management of PCa.

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology III (MP65)1 May 2024MP65-09 BLADDER CANCER ORGANOIDS: A RELIABLE TOOL FOR VALIDATION OF IN VITRO NEW THERAPEUTIC APPROACHES Emilie Decaup, Claire Béraud, Anne Sophie Bajeot, Xavier Gamé, Nicolas Monjotin, Pascal Rischmann, Philippe Lluel, and Mathieu Roumiguié Emilie DecaupEmilie Decaup , Claire BéraudClaire Béraud , Anne Sophie BajeotAnne Sophie Bajeot , Xavier GaméXavier Gamé , Nicolas MonjotinNicolas Monjotin , Pascal RischmannPascal Rischmann , Philippe LluelPhilippe Lluel , and Mathieu RoumiguiéMathieu Roumiguié View All Author Informationhttps://doi.org/10.1097/01.JU.0001008756.24343.22.09AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: In patients with bladder cancer (BC) immune therapies have recently demonstrated an improvement for overall survival rates compared to chemotherapy alone. However, it remains an unmet need to develop novel therapeutic strategies. In this context, organoids have emerged as a promising animal-free tool revealing patients' heterogeneity and allowing large preclinical therapeutic screening. Urosphere developed a BC organoids biobank currently containing 8 models with phenotypic, pharmacological and molecular characterization. The aim of this study was to demonstrate the relevance of our organoids models to validate therapeutic approaches such as targeted or CAR-T cell therapies using EGFR as target. METHODS: Urothelial progenitors were isolated from tumour samples and seeded in Matrigel®. Omic data allowed us to select models with high transcriptomic EGFR expressions. To evaluate targeted-therapy, organoids were treated with erlotinib (0.1 to 10 µM) for 5 days. Cell viability was measured with CellTiter-Glo3D® assay. To validate CAR-T cell approach, organoids were co-cultured with anti-EGFR or control CAR-T cells at 4 effector:target ratios. Cell viability was measured with CellTiter-Glo3D® and Caspase-Glo® 3/7 3D assays. Pro-inflammatory and Granzyme B release were analysed by multiplex assay. RESULTS: Organoids from 2 models with high transcriptomic EGFR expression were selected and protein expression was confirmed by immunofluorescence. For targeted therapy, erlotinib presented significant efficacy in both models. In the model with high EGFR expression, erlotinib induced cell death up to 80% with an IC50 of 2.5 µM whereas in the lower-expressing model the IC50 was 21.4 µM. Concerning CAR-T cell therapy, after 48 h of co-culture with anti-EGFR CAR-T cells, a decrease of cell viability associated with an increase of apoptosis was observed. After 72 h of co-culture, an increase in the secretion of pro-inflammatory cytokines (IFN-g and TNF-a) and Granzyme B by anti-EGFR CAR-T cells was also observed. In co-cultures with organoids presenting high EGFR expression, CAR-T cells activation appeared higher with 14 fold release of IFN-g and TNF-a by anti-EGFR CAR-T cells compared to those co-cultured with organoids lower expressing EGFR. CONCLUSIONS: In an EGFR targeting approach, the use of organoids to evaluate both targeted- and CAR-T cell therapies was validated with concordant results. This robust translational model is fully transposable to other therapeutic approaches, whether in a traditional culture model or in a co-culture system. Source of Funding: Urosphere © 2024 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 211Issue 5SMay 2024Page: e1080 Advertisement Copyright & Permissions© 2024 by American Urological Association Education and Research, Inc.Metrics Author Information Emilie Decaup More articles by this author Claire Béraud More articles by this author Anne Sophie Bajeot More articles by this author Xavier Gamé More articles by this author Nicolas Monjotin More articles by this author Pascal Rischmann More articles by this author Philippe Lluel More articles by this author Mathieu Roumiguié More articles by this author Expand All Advertisement PDF downloadLoading ...

BackgroundDespite the significant advances in the management of advanced prostate cancer (PCa), metastatic PCa is currently considered incurable. For further investigations in precision treatment, the development of preclinical models representing the complex prostate tumor heterogeneity are mandatory. Accordingly, we aimed to establish a resource of patient-derived xenograft (PDX) models that exemplify each phase of this multistage disease for accurate and rapid evaluation of candidate therapies.MethodsFresh tumor samples along with normal corresponding tissues were obtained directly from patients at surgery. To ensure that the established models reproduce the main features of patient’s tumor, both PDX tumors at multiple passages and patient’s primary tumors, were processed for histological characteristics. STR profile analyses were also performed to confirm patient identity. Finally, the responses of the PDX models to androgen deprivation, PARP inhibitors and chemotherapy were also evaluated.ResultsIn this study, we described the development and characterization of 5 new PDX models of PCa. Within this collection, hormone-naïve, androgen-sensitive and castration-resistant (CRPC) primary tumors as well as prostate carcinoma with neuroendocrine differentiation (CRPC-NE) were represented. Interestingly, the comprehensive genomic characterization of the models identified recurrent cancer driver alterations in androgen signaling, DNA repair and PI3K, among others. Results were supported by expression patterns highlighting new potential targets among gene drivers and the metabolic pathway. In addition, in vivo results showed heterogeneity of response to androgen deprivation and chemotherapy, like the responses of patients to these treatments. Importantly, the neuroendocrine model has been shown to be responsive to PARP inhibitor.ConclusionWe have developed a biobank of 5 PDX models from hormone-naïve, androgen-sensitive to CRPC primary tumors and CRPC-NE. Increased copy-number alterations and accumulation of mutations within cancer driver genes as well as the metabolism shift are consistent with the increased resistance mechanisms to treatment. The pharmacological characterization suggested that the CRPC-NE could benefit from the PARP inhibitor treatment. Given the difficulties in developing such models, this relevant panel of PDX models of PCa will provide the scientific community with an additional resource for the further development of PDAC research.

BackgroundMuscle-invasive bladder cancer (MIBC) and upper urinary tract urothelial carcinoma (UTUC) are molecularly heterogeneous. Despite chemotherapies, immunotherapies, or anti-fibroblast growth factor receptor (FGFR) treatments, these tumors are still of a poor outcome. Our objective was to develop a bank of patient-derived xenografts (PDXs) recapitulating the molecular heterogeneity of MIBC and UTUC, to facilitate the preclinical identification of therapies. MethodsFresh tumors were obtained from patients and subcutaneously engrafted into immune-compromised mice. Patient tumors and matched PDXs were compared regarding histopathology, transcriptomic (microarrays), and genomic profiles [targeted Next-Generation Sequencing (NGS)]. Several PDXs were treated with chemotherapy (cisplatin/gemcitabine) or targeted therapies [FGFR and epidermal growth factor (EGFR) inhibitors]. ResultsA total of 31 PDXs were established from 1 non-MIBC, 25 MIBC, and 5 upper urinary tract tumors, including 28 urothelial (UC) and 3 squamous cell carcinomas (SCCs). Integrated genomic and transcriptomic profiling identified the PDXs of three different consensus molecular subtypes [basal/squamous (Ba/Sq), luminal papillary, and luminal unstable] and included FGFR3-mutated PDXs. High histological and genomic concordance was found between matched patient tumor/PDX. Discordance in molecular subtypes, such as a Ba/Sq patient tumor giving rise to a luminal papillary PDX, was observed (n=5) at molecular and histological levels. Ten models were treated with cisplatin-based chemotherapy, and we did not observe any association between subtypes and the response. Of the three Ba/Sq models treated with anti-EGFR therapy, two models were sensitive, and one model, of the sarcomatoid variant, was resistant. The treatment of three FGFR3-mutant PDXs with combined FGFR/EGFR inhibitors was more efficient than anti-FGFR3 treatment alone. ConclusionsWe developed preclinical PDX models that recapitulate the molecular heterogeneity of MIBCs and UTUC, including actionable mutations, which will represent an essential tool in therapy development. The pharmacological characterization of the PDXs suggested that the upper urinary tract and MIBCs, not only UC but also SCC, with similar molecular characteristics could benefit from the same treatments including anti-FGFR for FGFR3-mutated tumors and anti-EGFR for basal ones and showed a benefit for combined FGFR/EGFR inhibition in FGFR3-mutant PDXs, compared to FGFR inhibition alone.

Abstract Background: Muscle-invasive bladder cancers (MIBCs) constitute a heterogeneous group of tumors with poor outcome. Recently, MIBC molecular subtyping efforts from an international consortium led to the identification of six subtypes, improving prediction of clinical outcomes and treatment responses. FGFR3 alterations (mutations and translocations), observed in 20% of MIBCs, are found mainly in the luminal papillary subtype that respond poorly to chemo- and immunotherapy. Basal tumors represent 35% of MIBCs and were shown to be better responders to chemotherapy. Here, we describe the development and characterization of patient-derived primary MIBC xenografts (PDX) belonging to these main subtypes. Methods: Bladder tumors were obtained from patients at surgery. Tumor fragments were subcutaneously engrafted into immune-compromised mice. Primary tumors and matched PDX tumors at multiple passages were analyzed regarding growth characteristics, histopathology (H&E staining, CK5/6, FOXA1, and GATA3 immunohistochemistry), gene expression (Affymetrix U133 plus 2.0 microarray), and genetic stability (STR profiling). Hotspot oncogenic mutations for FGFR3, PIK3CA, HRAS, KRAS, NRAS, PPARG, and RXRA were also assessed. Additionally, pharmacologic responses to standard-of-care and targeted therapies were characterized. Findings: From 152 MIBC tumors at all stages and grades, 32 PDX models were successfully established (21.1% success rate). This take rate did not seem correlated to any classical tumor characteristics. Importantly, transcriptomic analysis allowed us to identify PDX models belonging to different molecular subtypes, notably the basal-like and luminal papillary subtypes, including PDXs with FGFR3 mutations. All histologic, genetic, and molecular features validated the stability of the PDX models compared to the parental tumors. Histologic analyses correlated with the molecular classification. These models reproduced the response to cisplatin-based therapies observed in the clinic. Basal models, except one harboring a FGFR3 mutation, were sensitive to anti-EGFR therapies but to a lesser extent than to chemotherapy. FGFR3-mutated PDX models, including a basal model, were highly responsive to FGFR3 inhibitors and less responsive to chemotherapy. Conclusion: We have developed and characterized highly relevant preclinical models for MIBCs, including basal and FGFR3-mutated tumors, recapitulating molecular heterogeneity and drug responses as observed in patients with MIBCs. They represent essential tools for developing new, efficient therapies against this deadly disease. Citation Format: Claire Béraud, Hervé Lang, Myriam Lassalle, Véronique Lindner, Aurélie Kamoun, Michel Soulié, Elodie Guillon, Clémentine Krucker, Xavier Gamé, Pascal Rischmann, Aurélien De Reynies, Yves Allory, François Radvanyi, Philippe Lluel, Thierry Massfelder, Isabelle Bernard-Pierrot. Establishment of a panel of patient-derived tumor xenograft models recapitulating molecular heterogeneity and drug response of muscle-invasive bladder tumors [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr A24.

Abstract Prostate Cancer (PCa) is the second most frequent cancer in men worldwide and the fifth leading cause of cancer death with an incidence rate of 13.5%. PCa is driven by multiple genomic alterations, with distinct patterns and clinical implications. These genomic alterations occurring both early and later in the natural history of the disease (ranging from localized disease, initially responsive to androgen deprivation therapy, to Castrate Resistant Prostate Cancers -CRPC) allow classification of PCa in several molecular subtypes with potential clinical relevance. Patient-Derived Xenograft (PDX) models have become the most reliable in vivo human cancer models. Developing such models that capture the biological heterogeneity and mutational landscape of PCa, remains a challenge, but is essential for delivery of precision medicine in metastatic castrate resistant stages. In this study, we present the genomic and transcriptomic landscapes, as well as the pharmacological status of an established bank of seven (7) prostate PDX models ranging from hormone naïve to hormone-resistance PCa specimens. Samples of PCa along with normal corresponding tissues were obtained directly from patients at surgery. Fragments were subcutaneously xenografted into immunocompromised mice to establish PDX models. After the first growth in mice, they were serially passaged in vivo and considered to be established from P3. To ensure model stability, PDX tumors at multiple passages and patients' primary tumors were processed for histological, transcriptomic (Affymetrix U133 plus 2.0 microarray) and STR profile analyses. Genomic characteristics (WES, CNA) were also investigated. Finally, the responses of the PDX models to androgen deprivation and docetaxel were also evaluated. 7 PDX models were successfully established (> P3 in mice) out of 253 primary prostatic tumors collected from surgery. Within those models, one matched pair of responsive adenocarcinoma and neuroendocrine castration-resistant (NE-CRPC) models from the same patient was generated. Histological, transcriptomic and STR profiling validated the stability of the models compared to the parental tumor. The genomic analyses revealed i) the mutational burden rise with the resistance to treatments of the models, correlating with clinical results ii) an increase of metastatic genes loss in the NE-CRPC compared to the corresponding hormone sensitive adenocarcinoma. Furthermore, for all the PDX models generated, genomic and mutational analyses revealed specific molecular features and allowed molecular classification depending on tumor stage. Based on the molecular taxonomy of primary prostate cancers, the presented panel covers the different progression steps of the pathology. Considering the scarcity of useful models for PCa and the difficulties to develop such models, the prostate PDX models collection presented here should clearly help understanding disease progression and supporting precision medicine approaches for patients with advanced PCa. Citation Format: Myriam Lassalle, Claire Béraud, Hervé Lang, Véronique Lindner, Yves Allory, Eric Potiron, Thierry Massfelder, Philippe Lluel, Yolande Misseri. Mutational landscape and pharmacological profiling of a panel of prostate PDX models including hormone-naïve, hormone-sensitive and castrate-resistant prostate cancer specimens [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1668.

Prostate cancer (PCa) is a highly heterogeneous and complex disease, with evolving treatment options over the course of disease progression. Preclinical PCa research is hampered by a lack of predictive models fully capturing all phases of this multistage disease. Despite progresses in the development of genetically-engineered animal models, these ones do not recapitulate faithfully (i) human disease and (ii) tumor heterogeneity. Models obtained by xenografting human tumors in immunodeficient animals (PDX models, for patient-derived tumor xenografts) remain unavoidable tools in PCa translational and preclinical research since they closely conserve cancer characteristics observed in patients. PDX models are thus invaluable tools to evaluate new potential therapeutic agents. We are presenting here the characteristics of two PDX models derived from the same patient before and after acquisition of the hormone-resistance status. Samples of PCa were obtained from patients at surgery and then subcutaneously xenografted into immunocompromised mice to establish PDX models. After the first growth in mice, they were serially passaged in vivo, considering a model established from P3. PDX tumors at multiple passages and patients' primary tumors from which they are derived were processed for further analyses. Specifically, we performed histological, genetic (AR, PTEN, P53 and ERG status), transcriptomic (Affymetrix U133 plus 2.0 microarray) and STR profiles analyses. In addition, we also evaluated the responses of the PDX models to androgen deprivation and docetaxel. Since 9 years, 252 prostatic tumors have been collected at all stages. Up to now, 7 PDX models were successfully established (> P3 in mice), i.e. 2.7 % success rate. All histological, genetic and molecular analyses validated the stability of the models compared to the parental tumor. Interestingly, we were able to generate one matched pair of responsive and castration resistant models from the same patient. These two PDX models displayed the major molecular features of the disease in humans including PTEN, TP53 and AR modifications. In addition, in vivo results show heterogeneity of response to androgen deprivation and docetaxel, similar to the responses of patients to these treatments. Considering the scarcity of useful PDX models for PCa and the difficulties to develop such models, the PDX models collection presented here should clearly help to open the road of cure for patients with advanced PCa.Citation Format: Hervé Lang, Claire Beraud, Myriam Lassalle, Véronique Lindner, Eric Potiron, Philippe Lluel, Thierry Massfelder. Characterization of hormone-sensitive and castrate-resistant phenotypes in prostate cancer patient-derived PDX models generated from the same patient [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 92.

Abstract Muscle-invasive bladder cancers (MIBCs) constitute a heterogeneous group of tumors with a poor outcome. Recently, MIBC molecular subtyping efforts from an international consortium led to the identification of six subtypes to improve prediction of clinical outcomes and treatment responses. These subtypes can be schematically divided into luminal (differentiated) and non-luminal subtypes. FGFR3 alterations (mutations and translocations) are among the most frequent genetic events in bladder carcinoma and are found mainly in one subtype, the luminal papillary that respond poorly to chemo- and immuno-therapy. Here we describe the development and characterization of patient-derived primary MIBC xenografts (PDX) belonging to these different subtypes. Bladder primary tumors and normal corresponding tissues were directly obtained from patients at surgery. Tumor fragments were subcutaneously xenografted into immune-compromised mice. After the first growth in mice, they were serially passaged. PDXs tumors at multiple passages and patients’ primary tumors from which they are derived were processed for analyses including growth characteristics, histopathology (H&E, CK5/6, FOXA1 and GATA3), gene expression (Affymetrix U133 plus 2.0 microarray), genetic stability (STR profiling). Specifically, hotspot oncogenic mutations including FGFR3, PIK3CA, HRAS, KRAS, NRAS, and PPARG were also explored. Additionally, pharmacological responses to standards of care and targeted therapies were characterized. Since 10 years, we have collected 152 MIBC tumors at all stages and grades. Up to now, 32 PDX models have been successfully established (> P3 in mice), i.e. 21.1 % success rate. This take rate seems not to be correlated to any classical tumor characteristics. Importantly, transcriptomic analysis allowed us to identify PDX models belonging to the different molecular subtypes including the basal-like and the luminal papillary subtypes (which include several PDX with FGFR3 mutations). All histological, genetic and molecular features validated the stability of the PDX models compared to the parental tumors. Histological analyses were correlated with the molecular classification. These models reproduced the response to cisplatin-based therapies observed in the clinic and FGFR3-mutated PDX models were shown to be highly responder to FGFR3 inhibitors. We have developed highly relevant preclinical models for MIBCs corresponding to the main subtypes which have been described. They represent essential tools for developing adapted and efficient therapies against this deadly disease. Citation Format: Hervé Lang, Claire Beraud, Myriam Lassalle, Véronique Lindner, Michel Soulié, Xavier Gamé, Pascal Rischmann, Yves Allory, François Radvanyi, Isabelle Bernard-Pierrot, Philippe Lluel, Thierry Massfelder. High specific characterization of patient-derived tumor xenograft models for accelerating drug development in muscle-invasive bladder cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1930.

The upregulation of PPARγ/RXRα transcriptional activity has emerged as a key event in luminal bladder tumors. It renders tumor cell growth PPARγ-dependent and modulates the tumor microenvironment to favor escape from immuno-surveillance. The activation of the pathway has been linked to PPARG gains/amplifications resulting in PPARγ overexpression and to recurrent activating point mutations of RXRα. Here, we report recurrent mutations of PPARγ that also activate the PPARγ/RXRα pathway, conferring PPARγ-dependency and supporting a crucial role of PPARγ in luminal bladder cancer. These mutations are found throughout the protein—including N-terminal, DNA-binding and ligand-binding domains—and most of them enhance protein activity. Structure-function studies of PPARγ variants with mutations in the ligand-binding domain allow identifying structural elements that underpin their gain-of-function. Our study reveals genomic alterations of PPARG that lead to pro-tumorigenic PPARγ/RXRα pathway activation in luminal bladder tumors and may open the way towards alternative options for treatment.

FGFR3 alterations (mutations or translocation) are among the most frequent genetic events in bladder carcinoma. They lead to an aberrant activation of FGFR3 signaling, conferring an oncogenic dependence, which we studied here. We discovered a positive feedback loop, in which the activation of p38 and AKT downstream from the altered FGFR3 upregulates MYC mRNA levels and stabilizes MYC protein, respectively, leading to the accumulation of MYC, which directly upregulates FGFR3 expression by binding to active enhancers upstream from FGFR3. Disruption of this FGFR3/MYC loop in bladder cancer cell lines by treatment with FGFR3, p38, AKT, or BET bromodomain inhibitors (JQ1) preventing MYC transcription decreased cell viability in vitro and tumor growth in vivo. A relevance of this loop to human bladder tumors was supported by the positive correlation between FGFR3 and MYC levels in tumors bearing FGFR3 mutations, and the decrease in FGFR3 and MYC levels following anti‐FGFR treatment in a PDX model bearing an FGFR3 mutation. These findings open up new possibilities for the treatment of bladder tumors displaying aberrant FGFR3 activation. In bladder carcinoma, alterations of FGFR3 receptor are often observed and lead to constitutive activation and oncogene addiction, which can be targeted with a pan‐FGFR inhibitor. Our identification and characterization of a FGFR3/MYC positive feedback loop opens new avenues for targeted therapies. In bladder carcinoma, alterations of FGFR3 receptor are often observed and lead to constitutive activation and oncogene addiction, which can be targeted with a pan‐FGFR inhibitor. Our identification and characterization of a FGFR3/MYC positive feedback loop opens new avenues for targeted therapies.

Proliferative glomerulonephritis is characterized by local inflammation and mesangial cell deterioration, followed by mesangial proliferation and glomerular healing. Parathyroid hormone related peptide (PTHrP) is a mesangial cytokine-like growth factor implicated in mesangial proliferation and survival. No data are available about its role in glomerulonephritis. Herein, we analyzed the expression and role of PTHrP in glomerular inflammation and healing in an experimental model of glomerulonephritis induced by i.v. injection of Habu snake venom in mice. The temporal analysis showed marked renal damage in the first days after venom injection and the beginning of recovery within 7 days. Glomerular expression of PTHrP (transcript and protein) was observed in the early phase after venom injection (from day 1 to day 3), along with an inflammatory environment. The inactivation of secreted PTHrP with PTHrP-neutralizing antibody (PTH2E11; 120 mu g i.p. daily) reduced the markers of local inflammation (expression of macrophage chemotactic protein-1; regulated upon activation, normal T cell expressed and secreted; cyclooxygenase 2; IL-6; and macrophage infiltration) and abolished the expression of PTHrP itself. Moreover, the glomerular cell proliferation was hampered, and the healing process was prevented on day 7 after venom injection. These results show that PTHrP has antinomic actions in glomerulonephritis, participating in both the proinflammatory condition and the healing process. Our work reveals the essential role of PTHrP in early glomerular repair in an experimental model of glomerulonephritis.

Abstract Kidney, prostate and bladder cancers (KCa, PCa and BCa, respectively) represent 1 700 000 cases and 450 000 deaths worldwide per year, with an incidence rising yearly by 1-10%. Surgery is usually curative at early and localized stages but there are no efficient therapies at advanced and metastatic stages for any of them. Although genetically-modified and/or chemically-induced avatar models do exist for these cancers and may help to identify new therapeutic targets, they suffer from a lack of an extended biological concordance with the natural history and heterogeneity of the diseases. Patient-derived tumor xenograft models are now well recognized as reliably reproducing tumor heterogeneity and have become over the past few years the preclinical tools of choice to test drugs and identify biomarkers. Since 10 years, we are continuously developing a unique panel of PDX models for these major urological cancers. Tumor tissues along with normal corresponding tissues were obtained from patients at surgery. Patient informed consent and clinical history are available for all patients. Tumor tissues pieces were xenografted subcutaneously in the interscapular space of nude mice, and serially passaged into mice after the first engraftment, up to passage 10. To ensure model stability between primary tumors and tumors growing in mice but also from passage to passage, we performed various analyses at histopathological, genetic (short tandem repeat fingerprinting) and molecular (cDNA profiling) levels. In addition, growth characteristics and responses to standards of care (SOCs) were examined. Finally, specific molecular characteristics were also explored including expression of the androgen receptor, PSA and pan-cytokeratin for PCa models and hotspot mutations of FGFR3, PIK3CA, K/N/H-RAS for BCa models. Up to now, we have xenografted 336 (on 569 samples), 247 and 152 KCa, PCa and BCa tumor tissues, and developed 30 (8.9% success rate), 6 (2.1%) and 30 (19.7%) PDX models, respectively. We recently published part of the KCa PDX models collection (Lang et al., Oncotarget, 2016). Characterization studies showed that PDX models are stable at all levels analyzed considering concordance to primary tumors and from passage to passage; and less than 5% of genes were differentially expressed between the primary tumors and PDX tumors at various passages. Responses to SOCs recapitulated the clinical state. Only for KCa PDX models, the take rate was correlated to tumor stage and grade, and sarcomatoid components. Importantly, several molecular subtypes were defined in our collection of BCa PDX models including PDXs with FGFR3 mutations and PDXs of basal subtype, the most aggressive one. Overall, this panel of PDX models for urological cancers should definitely help to find molecularly guided targeted therapies for these still incurable cancers at metastatic stages. Citation Format: Hervé Lang, Claire Béraud, Myriam Lassalle, Isabelle Bernard-Pierrot, Véronique Lindner, Yves Allory, Michel Soulié, Xavier Gamé, Pascal Rischmann, Eric Potiron, François Radvanyi, Philippe Lluel, Thierry Massfelder. A comprehensive patient-derived tumor xenograft (PDX) collection representing the heterogeneity of kidney, prostate and bladder cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1035.