Abstract 2852: Targeting NPEPPS overcomes cisplatin resistance in patient-derived bladder cancer tumoroids
Authors: Scholtes Mathijs P. , Akbarzadeh Maryam , Romal Shahla , van Dijk Miranda , Kan Tsung Wai , Mahmoudi Tokameh , Zuiverloon Tahlita C. 
Published: June 2023

Introduction and Objectives: Cisplatin-based neoadjuvant chemotherapy (NAC) is the recommended treatment for muscle-invasive bladder cancer (MIBC) patients. Patients with a pathological complete response after NAC (pCR), have a good 5-yr OS of 80%. However, due to NAC resistance, only 25% of patients achieve a pCR. Our aim is to increase the number of patients reaching a pCR by overcoming cisplatin resistance. Previously we identified NPEPPS as a key player in cisplatin resistance that prevents cisplatin uptake via volume-regulated anion channels (VRAC) in cisplatin-resistant cell lines. Studies have demonstrated that patient-derived bladder cancer tumoroids recapitulate the originating tumor at the molecular level, presenting a model system to study chemotherapy resistance ex vivo. Here, we used MIBC tumoroids to functionally interrogate NPEPPS’ role in cisplatin resistance.

Material and Methods: Ex vivo interrogation of NPEPPS function in cisplatin resistance was performed in tumoroid cultures generated from MIBC patients (N=10). Tumor tissue was obtained either at transurethral resection of bladder tumor (TURBT, N=5) or RC (N=5), and clinical resistance to NAC was defined as ypT≥2, nodal positive disease at RC or progression during NAC. Molecular characterization of tumor specimens and corresponding tumoroids was performed by bladder cancer-specific SNaPshot mutation analysis, copy-number aberrations analysis, immunohistochemistry (IHC), and hematoxylin & eosinophilic staining (HE). The effect of NPEPPS on cisplatin uptake was investigated using cisplatin treatment combined with the pharmacological NPEPPS-inhibitor tosedostat, shRNA-mediated NPEPPS knock-down or lentiviral NPEPPS over-e

xpression followed by cytometry by time of flight (CyTOF) analysis. CellTiter-Glo cell viability assays and caspase 3/7 apoptosis assays were used to investigate whether NPEPPS inhibition overcomes cisplatin resistance ex vivo.

Results: Five out of ten patients received NAC, all whom had tumors that were clinically resistant to NAC. Tumoroid cultures from MIBC patients unresponsive to NAC showed resistance to serum concentrations of cisplatin as well as increased NPEPPS mRNA expression. Meanwhile, molecular characterization of tumor and tumoroid pairs confirmed that patient-specific tumor traits were maintained in tumoroids. Pharmacological NPEPPS inhibition or NPEPPS depletion by shRNA-mediated knock-down increased cisplatin influx into the cell and resensitized NAC resistant tumoroids to serum concentrations of cisplatin. Furthermore, NPEPPS overexpression increased cisplatin resistance in cisplatin-sensitive tumoroids.

Conclusion: We confirm that NPEPPS is associated with cisplatin-resistance in an ex vivo MIBC tumoroid model. These findings have potential for rapid translation into the clinic and invite trials investigating tosedostat to overcome chemoresistance.

Citation Format: Mathijs P. Scholtes, Maryam Akbarzadeh, Shahla Romal, Miranda van Dijk, Tsung Wai Kan, Tokameh Mahmoudi, Tahlita C. Zuiverloon. Targeting NPEPPS overcomes cisplatin resistance in patient-derived bladder cancer tumoroids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2852.