Preclinical models for bladder cancer therapy research

Purpose of review Bladder cancer (BC) is a highly heterogenous disease comprising tumours of various molecular subtypes and histologic variants. This heterogeneity represents a major challenge for the development of novel therapeutics. Preclinical models that closely mimic in vivo tumours and reflect their diverse biology are indispensable for the identification of therapies with specific activity in various BC subtypes. In this review, we summarize efforts and progress made in this context during the last 24 months. Recent findings In recent years, one main focus was laid on the development of patient-derived BC models. Patient-derived organoids (PDOs) and patient-derived xenografts (PDXs) were demonstrated to widely recapitulate the molecular and histopathological characteristics, as well as the drug response profiles of the corresponding tumours of origin. These models, thus, represent promising tools for drug development and personalized medicine. Besides PDXs, syngenic in vivo models are of growing importance. Since these models are generated using immunocompetent hosts, they can, amongst others, be used to develop novel immunotherapeutics and to evaluate the impact of the immune system on drug response and resistance. Summary In the past two years, various in vivo and in vitro models closely recapitulating the biology and heterogeneity of human bladder tumours were developed.


INTRODUCTION
With an estimated 550.000 new cases and 200.000 deaths annually, bladder cancer (BC) ranks amongst the most common malignancies [1].Despite recent breakthroughs, such as the approval of the panfibroblast growth factor receptor (FGFR) inhibitor erdafitinib and several immune checkpoint inhibitors (e.g.pembrolizumab, nivolumab, avelumab), as well as the development of antibody-drug conjugates (e.g.enfortumab vedotin), patients suffering from advanced and metastatic BC still have a dire prognosis [2][3][4].
A major hindrance for the development of novel therapies is the high heterogeneity of bladder tumours.While approx.75% represent pure urothelial carcinoma (UC), the remaining 25% comprise various histologic variants such as squamous cell carcinoma (SCC), adenocarcinoma, or sarcomatoid carcinoma (SaC) [5].Moreover, numerous studies showed that BC can be subdivided into various molecular subtypes with different sensitivities to currently available therapies [6,7].
For the establishment of novel therapy schemes with specific activity in various BC subtypes, preclinical models that closely mimic in vivo tumours are indispensable.In this review, we summarize efforts and progress made in this context during the last 24 months.Given the literature published during this time period, we will mainly focus on 3-dimensional (3D) in vitro cultures, as well as on patient-derived xenograft and syngenic in vivo models (Fig. 1).

IN VITRO MODELS
For several decades, in vitro studies for drug development were mainly conducted using 2D cultures of well established, commercially available BC cell lines.Even though these models represent powerful tools, they have considerable limitations affecting their clinical representativeness.On the one hand,

KEY POINTS
Preclinial models that accurately reflect the biology and heterogeneity of bladder tumours are indispensable for the development of novel therapies.
Patient-derived organoids (PDOs) and patient-derived xenografts (PDXs) were shown to closely mimic their tumours origin and, thus, represent powerful tools for drug development.
To circumvent the necessity of invasive tumour sampling, protocols for the establishment of PDOs using cells isolated from urine were established.
Besides rodents, other species such as zebrafish were successfully employed for the establishment of PDX models.Syngenic/allogenic animal models that are generated using immunocompetent hosts are required for the development of novel immunotherapeutics and to assess the influence of the immune system on drug response and resistance.FIGURE 1. Overview of preclinical models for bladder cancer therapy research.Patient-derived in vitro and in vivo models recapitulate the molecular and histopathological characteristics, as well as the drug response profiles of the corresponding tumours of origin.Therefore, these models represent promising tools for drug development and personalized medicine.Syngenic/allogenic in vivo models that are generated using immunocompetent hosts are valuable tools for the development of novel immunotherapeutics.Thus, recently developed preclinical models have the potential to enable the development of novel BC treatments resulting in increased survival rates, decreased recurrence rates and enhanced health related quality of life (HRQL).
Preclinical models for bladder cancer therapy research Ertl et al.
classical BC cell lines have been cultured for many years, causing an accumulation of genetic and epigenetic alterations that arise with every passage [8,9].One the other hand, traditional 2D models cannot recapitulate interactions between different cell types or cancer cells and the tumour microenvironment, that significantly influence drug response [10].
In the last 2 years, most research groups focused on the establishment of 'organoids' that are defined as 3D structures consisting of multiple types of cells [11].Berndt-Paetz et al. developed organoids containing either of the classical BC cell lines RT4, RT112, T24 or CAL-29, as well as primary human bladder fibroblasts and smooth muscle cells.The organoids closely mimicked an inverse bladder wall, with a core of supportive cells surrounded by urothelial cells.Amongst other findings, the research group demonstrated significant differences in drug-response between organoids and conventional 2D cultures [12  & ].The superiority of 3-dimensional BC models in the context of drug development was also demonstrated by Wei et al., who compared the sensitivities of BC cell lines and of patient-derived organoids (PDOs) to cisplatin, the Bcl-2 inhibitor venetoclax and the MCL1 inhibitor S63845 [13  & ].In an attempt to establish tools for a personalized treatment approach, Minoli et al. established an extensive collection of PDOs deriving from tumours of various disease stages and grades.The PDOs were found to accurately represent the molecular and histopathological characteristics and multiclonal landscapes of their tumours of origin.Testing the sensitivities of the PDOs to standard of care therapies, treatment outcomes observed in BC patient cohorts were accurately recapitulated.PDOs were, thus, shown to be a promising tool for the stratification of patients according to drug response sensitivity profiles

IN VIVO MODELS
According to the location the tumour, in vivo models are classified as heterotopic and orthotopic [23,24].Orthotopic models can be generated by intravesical instillation or intramural injection of cancer cells, resulting in tumours located in the bladder.In heterotopic models, in contrast, tumour formation is achieved by subcutaneous injection of tumour cells [23].Beyond that, in vivo models can further be categorized depending on the origin of the implanted cancer cells.While xenograft models are established by the implantation of human cancer cells into immunocompromised hosts, tumour formation in allogenic or syngenic models is induced by inoculation of tumour cells of the same species/strain in immunocompetent animals [24].Besides the implantation of cancer cells, orthotopic tumours can also be induced by chemical carcinogenesis and genetic engineering [23].

Xenograft models
Traditionally, xenograft animals used for drug development were established by implantation of classical BC cell lines.However, the clinical representativeness of cell line-based xenograft models is, amongst others, limited because they lack the heterogeneity of in situ patient tumours.In addition, cell lines lack their original stroma representing an important factor for tumour growth and drug response.This issue can be circumvented by usage of patient-derived xenograft (PDX) models that are generated by direct engraftment of tumour fragments into immunocompromised animals [24].
With the aim to facilitate the identification of novel therapies for muscle-invasive BC (MIBC) and upper tract urothelial carcinoma (UTUC) Lang et al. established an extensive panel of PDXs.Besides UTUC and conventional UC, the PDX collection also included various BC variants, such as SCC and SaC.Comparing patient tumours and PDXs, the researchers found a high resemblance concerning histological and genomic features, but alterations of the transcriptomic profiles that resulted -in some cases -in changes of intrinsic subtypes.However, since actionable mutations were retained, the PDX models widely recapitulated drug response observed in the clinics [

Syngenic in vivo models
Xenograft models represent valuable tools for drug development.However, since they are generated using immunocompromised hosts, they cannot be used for the development of immunotherapies [24].Given the increasing significance of immunotherapeutics for the treatment of BC, numerous studies using syngenic in vivo models were recently conducted.
Using the murine cell line MB49 and female C57BL/6 wild-type mice, Domingos-Pereira et al.To study the effects of intravesical adoptive cell therapy (ACT) with tumour infiltrating lymphocytes (TIL), Bazargan et al. generated an orthotopic model using C57BL/6 mice and ovalbumin (OVA) expressing MB49 cells (MB49OVA).Profiling the immune microenvironment, it was confirmed that the model recapitulated key characteristics of human tumours.Intravesical administration of gemcitabine was demonstrated to have lymphodepleting effects on the bladder microenvironment, thereby reducing immunosuppressive populations, such as myeloid derived suppressor cells (MDSCs).Pretreatment with gemcitabine was shown to result in enhanced antitumor effects of ACT [36  & ].To assess the effects of anti-CD40 immunotherapy on BC, Wong et al. generated orthotopic BC models using C57BL/6J mice and the cell lines MB49 and UPPL1541, of which the latter recapitulates the luminal subtype of high-grade UC.In the MB49 model, it was shown that intravesical treatment with anti-CD40 agonist antibodies caused reduced tumour burden, whereby the therapeutic effect was reduced by blockage of IL-15.In both the MB49 and UPPL1541 models, anti-CD40 agonist antibodies caused upregulation of IL-15Ra on dendritic cells (DCs) and other myeloid populations.Moreover, using CD40-and FcgR-humanized C57BL/6J mice harbouring MB49 or UPPL1541 tumours, it was shown that the human anti-CD40 agonist antibody 2141-V11 caused reduced tumour size and that this effect was enhanced by cotreatment with IL-15

Carcinogen-induced in vivo models
A major factor affecting the pathogenesis of BC is chemical carcinogenesis, for instance due to smoking or occupational exposure [38].Consequently, it is also possible to generate orthotopic BC models using carcinogenic agents [24].Shah et al., for instance, induced invasive tumours in male and female C57BL/6 mice by continuous exposure to N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN).Subsequently, tumours were passaged by subcutaneous implantation into sex matched C57BL/6 hosts.Histopathologic and molecular characterization showed that the eight established tumour lines represented basal/squamous (BaSq), stromal-rich (SR) and neuroendocrine (NE)-like molecular subtypes.As reported for human tumours, cisplatin exhibited antineoplastic effects in a SR tumour line, while a BaSq line was found to be resistant

CONCLUSION
The landscape of BC research is marked by significant advances in recent years, yet clear challenges persist.Despite advancements like FGFR inhibitors, ICIs and ADCs, the prognosis for advanced and metastatic BC remains bleak, with high heterogeneity further complicating therapeutic development.Efforts have intensified address this complexity, with a particular focus on refining preclinical models to better mimic patient tumour biology.
While the discussed models offer promising avenues for bladder cancer research, they are not without limitations.Preclinical models, such as organoids and PDXs, may still inadequately capture the complexity of human tumours or long-term treatment effects, potentially leading to discrepancies in drug responses.Moreover, these models often require invasive procedures for tumour sampling, limiting their applicability for longitudinal studies.Furthermore, in vivo models face challenges in recapitulating e.g., human immune responses, limiting their utility in immunotherapy development.Addressing these limitations through ongoing refinement of models is crucial to improving their predictive accuracy and clinical utility.
Overall, the convergence of advanced preclinical models and innovative therapeutic strategies holds great promise for overcoming the challenges posed by BC heterogeneity and resistance mechanisms.Leveraging 3D organoids, PDXs, and syngenic/allogenic models promises to enhance drug discovery, personalized treatment approaches, and immunotherapy development.Continued interdisciplinary efforts in this direction are crucial for translating preclinical findings into clinically effective treatments, ultimately improving outcomes for patients battling this highly prevalent disease.

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Viergever BJ, Raats DAE, Geurts V, et al.Urine-derived bladder cancer organoids (urinoids) as a tool for cancer longitudinal response monitoring and therapy adaptation.Br J Cancer 2024; 130:369-379.To circumvent the necessity of invasive tumour sampling, a protocol for the establishment of BC organoids ('urinoids') using cells isolated from urine of BC patients was established.Urinoids were shown to be highly similar to the original tumour and to exhibit comparable sensitivities to various chemotherapeutics as organoids of the same patient.

20.
& Walz S, Pollehne P, Geng R, et al.A protocol for organoids from the urine of bladder cancer patients.Cells 2023; 12:2188.A protocol for the establishment of BC organoids using cells isolated from urine of BC patients was established.The formation and primary expansion of urinoids was successful in 83% of cases, whereby the success rate was independent from factors such as sex and age of the patient, disease status and prior cancer-specific treatment.Syngenic BC models were generated and used to study resistance against anti-PD-1 and/or PD-L1 therapy.Amongst other findings, the gene Serpinf1 was identified as a relevant factor in the resistance to anti-PD-1antibodies.Two different syngenic BC models were established to assess the effects of the bacterial minicell-based integrin-targeted oncolytic agent VAX014.In both models, intravesical administration resulted in significantly prolonged survival.Synergies between VAX014 and anti-PD-L1 therapy were observed.

35.
& Huang C-P, Lu H-L, Shyr C-R.Antitumor activity of intratumoral xenogeneic urothelial cell monotherapy or in combination with chemotherapy in syngeneic murine models of bladder cancer.Am J Cancer Res 2023; 13:2285-2306.Two different syngenic mouse models were generated to study the effects of intratumoral xenogeneic urothelial cell (XUC) immunotherapy.In both models, intratumoural injection of porcine urothelial cells resulted in the suppression of tumour growth that was further enhanced by chemotherapy.

36.
& Bazargan S, Bunch B, Ojwang' AME, et al.Targeting myeloid-derived suppressor cells with gemcitabine to enhance efficacy of adoptive cell therapy in bladder cancer.Front Immunol 2023; 14:.Syngenic mouse models were generated to study the effects of intravesical adoptive cell therapy (ACT) with tumor infiltrating lymphocytes (TIL).Profiling the immune microenvironment, it was confirmed that the model recapitulated key characteristics of human tumours.Pretreatment with gemcitabine was shown to result in enhanced antitumor effects of ACT.

&
Wong JL, Smith P, Angulo-Lozano J, et al.IL-15 synergizes with CD40 agonist antibodies to induce durable immunity against bladder cancer.Proc Natl Acad Sci USA 2023; 120:e2306782120.Syngenic mouse models were generated to assess the effects of anti-CD40 immunotherapy on BC.The human anti-CD40 agonist antibody 2141-V11 was shown to cause reduced tumour size.This effect was enhanced by cotreatment with IL-15.Orthotopic mouse models were generated by chemical carcinogenesis and the effects of inhibitors of the histone methyltransferase EZH2 were assessed.The EZH2 catalytic inhibitor EPZ0011989 was shown to cause a reduction of tumour progression and increased immune infiltration.The antineoplastic effects of EZH2 inhibition was demonstrated to depended on an intact adaptive immune system.
generated an orthotopic model for non-muscle-invasive BC.Amongst others, tumour infiltration by myeloid derived suppressor cells (MDSC) and tumour associated macrophages (TAM) was studied and the expression of various chemokines and PD-1/ PD-L1 was analysed.Thereby, it was shown that the established model represents a suitable tool for the development of ICIs and therapies targeting chemoattractants [30 & ].In order to study acquired resistance to ICIs, Denis et al. developed BC models by subcutaneous injection of MB49 and MBT-2 cells into C57BL6 or C3H/HeNRj mice, respectively.Resistance against anti-PD-1 and/or PD-L1 therapy was achieved by serial implantations and exposure to the respective antibodies.Amongst other findings, the gene Serpinf1 was identified as a relevant factor in the resistance to anti-PD-1 antibodies [31 & ].To study mechanisms contributing to an immunosuppressive tumour microenvironment, Dominguez-Gutierrez et al. used organotypic tumour tissue slice cultures that were obtained from orthotopic BC generated by subcutaneous injection of MBT-2 cells in C3/He mice.The approach revealed that hyaluronan (HA) produced in cell clusters in the stroma and tumour-draining lymph nodes supported the development of immunosuppressive PD-L1 þ macrophages, thereby mediating immune escape and resistance to immunotherapy [32 & ].Xu et al. developed murine triple knockout (TKO: Trp53, Pten, Rb1) organoids that were injected subcutaneously, intravesically, or orthotopically into C57BL/6J mice.TKO tumours exhibited basal-like features and expressed, in contrast to lesions generated by implantation of MB49 cells, urothelial lineage markers.Treatment of subcutaneous TKO tumours with anti-PD-1 antibodies resulted in a mixed pattern of response reflecting immunotherapy responses observed in the clinics [33 & ].Tsuji et al. generated orthotopic BC models using the cell lines MBT-2 and MB49 and female C3H/HeJ or C57BL/6J mice to assess the effects of the novel bacterial minicellbased integrin-targeted oncolytic agent VAX014.In both models, intravesical administration resulted in significantly prolonged survival.Assessing combined treatment of VAX014 and systemic anti-PD-L1 therapy, synergies of the two agents were observed [34 & ].To study the effects of intratumoral xenogeneic urothelial cell (XUC) immunotherapy, Huang et al. generated syngenic mouse models by subcutaneous implantation of MBT-2 and MB49 cells.In both models, intratumoural injection of porcine urothelial cells resulted in the suppression of tumour growth that was further enhanced by chemotherapy (i.e., gemcitabine and cisplatin) [35 & ].
[39 & ].To assess the effects of the histone deacetylase (HDAC) inhibitor chidamide on BC, Wang et al. generated orthotopic models by instillation of N-methyl-Nnitrosourea (MNU) in the bladders of female Sprague-Dawley rats.Intravesical application of mitomycin C and chidamide alone or in combination revealed synergistic effects of the two drugs [40 & ].To assess the effects of inhibitors of the histone methyltransferase EZH2, Piunti et al. generated orthotopic tumour models by exposure of C57BL/ 6 mice to BBN.Application of the EZH2 catalytic inhibitor EPZ0011989 by oral gavage caused a reduction of tumour progression and increase immune infiltration.Further, it was shown that the antineoplastic effects of EZH2 inhibition depended on an intact adaptive immune system [41 & ].
Berndt-Paetz M, Han S, Weimann A, et al. Cell line-based human bladder organoids with bladder-like self-organization-a new standardized approach in bladder cancer research.Biomedicines 2023; 11:2958.Classical BC cell lines, primary human bladder fibroblasts and smooth muscle cells were used to generate organoids.Differences in drug response between organoids and conventional 2D cultures were demonstrated.13. & Wei Y, Amend B, Todenh€ ofer T, et al.Urinary tract tumor organoids reveal eminent differences in drug sensitivities when compared to 2-dimensional culture systems.Int J Mol Sci 2022; 23:6305.Patient-derived organoids representing BC and UTUC were generated.The superiority of 3-dimensional BC models in the context of drug development was demonstrated.14. && Minoli M, Cantore T, Hanhart D, et al.Bladder cancer organoids as a functional system to model different disease stages and therapy response.Nat Commun 2023; 14:2214.An extensive collection of PDOs deriving from tumours of various disease stages and grades was established.The PDOs were shown to represent promising tools for the stratification of patients according to drug response sensitivity profiles.15. && Seiler R, Egger M, De Menna M, et al.Guidance of adjuvant instillation in intermediate-risk nonmuscle invasive bladder cancer by drug screens in patient derived organoids: a single center, open-label, phase II trial.BMC Urol 2023; 23:89.A clinical phase II trial with the aim to identify the most effective instillation therapy for individual NMIBC patients using PDOs is presently being conducted.16. & Garioni M, Tschan VJ, Blukacz L, et al.Patient-derived organoids identify tailored therapeutic options and determinants of plasticity in sarcomatoid urothelial bladder cancer.Npj Precis Oncol 2023; 7:112.A long-term organoid-like SaC model and organoids representing conventional UC were established.By high-throughput drug screening, compounds with specific activity against UC or SaC, as well as therapies with antineoplastic effects in both disease entities were identified.17. & Jiang Y, Sun X, Song X, et al.Patient-derived bladder cancer organoid model to predict sensitivity and feasibility of tailored precision therapy.Curr Urol 2023; 17:221-228.Chimeric antigen receptor (CAR)-T cells targeting the transmembrane protein B7H3 were generated.Using PDOs, specific antigen recognition and immune activation were confirmed.18. & Zhao Z, Zhang S, Jiang N, et al.Patient-derived immunocompetent tumor organoids: a platform for chemotherapy evaluation in the context of t-cell recognition.Angew Chem Int Ed Engl 2024; 63:e202317613.T-cell-retaining PDOs were generated to study the role of immune cells in the response to anticancer compounds.19.
PR, Kwenda EP, Donelan W, et al.Detection of PD-L1expressing myeloid cell clusters in the hyaluronan-enriched stroma in tumor tissue and tumor-draining lymph nodes.J Immunol 2022; 208:2829-2836.Organotypic tumor tissue slice cultures obtained from syngenic BC models were used to study mechanisms contributing to an immunosuppressive tumor microenvironment.Hyaluronan (HA) produced in cell clusters in the stroma and tumourdraining lymph nodes were shown to support the development of immunosuppressive PD-L1 þ macrophages, thereby mediating immune escape and resistance to immunotherapy.33.& Xu D, Wang L, Wieczorek K, et al.Single-cell analyses of a novel mouse urothelial carcinoma model reveal a role of tumor-associated macrophages in response to anti-PD-1 therapy.Cancers 2022; 14:2511.Murine triple knockout (TKO: Trp53, Pten, Rb1) organoids were generated injected into mice.Treatment with anti-PD1-antibodies resulted in a mixed pattern of response reflecting immunotherapy responses observed in the clinics.34.& Tsuji S, Reil K, Nelson K, et al.Intravesical VAX014 synergizes with PD-L1 blockade to enhance local and systemic control of bladder cancer.Cancer Immunol Res 2022; 10:978-995.
38. Alfred Witjes J, Max Bruins H, Carrio ´n A, et al.European Association of Urology guidelines on muscle-invasive and metastatic bladder cancer: summary of the 2023 guidelines.Eur Urol 2024; 85:17-31.39. & Shah SD, Gillard BM, Wrobel MM, et al.Syngeneic model of carcinogeninduced tumor mimics basal/squamous, stromal-rich, and neuroendocrine molecular and immunological features of muscle-invasive bladder cancer.Front Oncol 2023; 13:1120329.Syngenic mouse models were established by chemical carcinogenesis.The models were shown to represent basal/squamous (BaSq), stromal-rich (SR) and neuroendocrine (NE)-like molecular subtypes.Cisplatin exhibited antineoplastic effects in a SR tumour line, while a BaSq line was found to be resistant.40.& Wang S-C, Yu C-Y, Wu Y-C, et al.Chidamide and mitomycin C exert synergistic cytotoxic effects against bladder cancer cells in vitro and suppress tumor growth in a rat bladder cancer model.Cancer Lett 2022; 530:8-15.Orthotopic rat models were generated by chemical carcinogensis and the effects of the HDAC inhibitor chidamide were assessed.Intravesical application of mitomycin C and chidamide revealed synergistic effects of the two drugs.41. & Piunti A, Meghani K, Yu Y, et al.Immune activation is essential for the antitumor activity of EZH2 inhibition in urothelial carcinoma.Sci Adv 8: eabo8043.
Efforts were also made to develop novel methods for the accurate analysis of 3-dimensional BC models.Becker et al. established a label-free and noninvasive imaging procedure to monitor drug response combining Raman microspectroscopy (RMS) and fluorescence lifetime imaging microscopy (FLIM) [21].Zhang et al. developed a deep learning model for the evaluation of the growth status of organoids called AU2Net (Attention and Cross U2Net) [22].This algorithm is able to provide accurate segmentation results from organoid images, thus improving the analyses of drug screens using 3D cultures.
Steele et al. used PDXs to assess mechanisms of acquired cisplatin resistance.Thereby, the researchers found that treatment with the monoclonal ErbB3 antibody seribantumab resulted in significantly reduced tumour growth in both cisplatin resistant and sensitive models [26].Laranjeira et al. employed PDXs to evaluate the effects of DNA methyltransferase inhibitors (DNMTi) on BC.Amongst others, it was shown that treatment with decitabine and 5-aza-4 0 -thio-2 0 - 25 && ]. & ].
L, Fischer F, Fleck JL, et al.Data-driven identification of biomarkers for in situ monitoring of drug treatment in bladder cancer organoids.Int J Mol Sci 2022; 23:6956.22. Zhang S, Li L, Yu P, et al.A deep learning model for drug screening and evaluation in bladder cancer organoids.Front Oncol 2023; 13:1064548.23.J€ ager W, Moskalev I, Raven P, et al.Orthotopic mouse models of urothelial cancer.In: Schulz WA, Hoffmann MJ, Niegisch G, editors.Urothelial carcinoma: methods and protocols.Springer; 2018:177-197.24.Zhu S, Zhu Z, Ma A-H, et al.Preclinical models for bladder cancer research.Hematol Oncol Clin North Am 2021; 35:613-632.Preclinical models for bladder cancer therapy research Ertl et al. , B eraud C, Cabel L, et al.Integrated molecular and characterization of patient-derived xenografts from bladder and ureteral cancers identifies new potential therapies.Front Oncol 2022; 12:930731.An extensive panel of PDXs including models for UTUC, conventional UC and histologic variants such as SaC and SCC was established.A high resemblance of PDXs to the corresponding tumours of origin was demonstrated.Moreover, it was shown that the PDX models widely recapitulated drug response observed in the clinics.26.Steele TM, Tsamouri MM, Siddiqui S, et al.Cisplatin-induced increase in heregulin 1 and its attenuation by the monoclonal ErbB3 antibody seribantu-Villanueva H, Wells GA, Miller MT, et al.Characterizing treatment resistance in muscle invasive bladder cancer using the chicken egg chorioallantoic membrane patient-derived xenograft model.Heliyon 2022; 8:e12570.Chicken embryo chorioallantoic membrane (CAM) PDX models for MIBC were developed and a high resemblance to the tumours of origin was shown.Treatment of CAM-PDX with cisplatin confirmed that the models mirrored clinical therapy resistance.30.Domingos-Pereira S, Sathiyanadan K, Polak L, et al.Tumor-microenvironment characterization of the MB49 non-muscle-invasive bladder-cancer orthotopic model towards new therapeutic strategies.Int J Mol Sci 2022; 24:123.An orthotopic mouse model for nonmuscle invasive BC was generated and demonstrated to represent a suitable tool for the development of ICIs and therapies targeting chemoattractants.31.Denis M, Grasselly C, Choffour P-A, et al.In vivo syngeneic tumor models with acquired resistance to anti-PD-1/PD-L1 therapies.Cancer Immunol Res 2022; 10:1013-1027.