Although more novel cancer regimens, such as targeted therapy and immunotherapy, have entered the clinic in recent years, chemotherapy remains the primary means of clinical anti-cancer treatment. Nevertheless, most tumors eventually develop resistance to chemotherapy, leading to recurrence and progression. Currently, the molecular mechanism of cancer chemotherapy tolerance is yet to be fully understood. Muscle-invasive bladder cancer (MIBC) is the most common and malignant urologic neoplasm, and cisplatin-based chemotherapy is the first-line treatment for unresectable and metastatic MIBCs. However, due to the emergence of chemoresistance, a significant proportion of chemotherapy treatments fail, resulting in tumor recurrence and progression.

The State Key Laboratory of Biotherapy, West China Hospital, Sichuan University discovered that semi-squamous differentiation is a novel characteristic of chemotherapy-resistant bladder cancer. A differentiation therapy targeting Cathepsin H (CTSH) promoting the terminal differentiation of drug-resistant tumor cells has thus been proposed to give a new approach to the differentiation therapy of solid tumors.

With a lack of animal models of MIBC, this study established a primary and orthotopic MIBC mouse model with P53 and PTEN deletion and MYC overexpression, in which clinicopathological characteristics of MIBC patients can be accurately characterized. This newly established model was utilized to test the response of MIBC to the clinical chemotherapy regimen (cisplatin + gemcitabine). The study revealed that the model could accurately replicate the complete process from a clinically similar response to relapse and drug resistance.

To analyze the molecular mechanism of chemoresistance in MIBC, the molecular path of acquired resistance in MIBC was constructed using single-cell omics analysis. Experiments such as barcoding tracing confirmed that tumor cell lineage plasticity is the primary pathway of acquired resistance in MIBC. In combination with multi-omics and pathological analyses such as transcriptome and proteome, it was found that progressive squamous differentiation was a significant feature of acquired resistance in MIBC. Clinically, squamous differentiation also exits in chemo-resistant MIBCs, and squamous differentiation score is closely related to poor prognosis of patients. Furthermore, multi-omics analyses showed that cathepsin H (CTSH) is a therapeutic target for squamous differentiation of chemoresistant MIBCs. CTSH is found to gradually increase with chemotherapy in both mice and human MIBCs. Furthermore, the knockout of CTSH can specifically restrain in vitro and in vivo growth of chemoresistant MIBCs. Also, E64 (CTSH inhibitor) can effectively inhibit the growth of resistant mouse MIBC and patient PDX, with no significant effect observed on chemosensitive MIBC.

Through pathological analysis, differentiation marker staining and analyses such as single-cell omics, transcriptome, and proteomics, MIBC cells found with a certain degree of squamous differentiation present underwent terminal differentiation after CTSH knockout or E64 treatment. Mechanistically, after CTSH inhibition, tumor necrosis factor (TNF) is up-regulated, promoting terminal squamous differentiation of tumor cells through TNF receptor 1 (TNFR1) and downstream CASPASE-8. This ultimately leads to pyroptosis of chemoresistant MIBC cells.

This study, based on a novel precise mouse model of bladder cancer, revealed that semi-squamatization is a type of lineage plasticity associated with acquired chemoresistance in MIBC. This suggests that differentiation via targeting of CTSH can be a potential therapeutic strategy for treating chemoresistant MIBCs. Although differentiation therapy has been successfully applied in leukemia treatment, particularly in the all-trans retinoic acid + arsenicals treatment of acute promyelocytic leukemia, most attempts to use it in solid tumors have failed. This study is expected to provide new ideas for differentiation therapy of solid tumors.

In this study, the main innovations include: 1) Developing a new primary, orthotopic bladder cancer model created from gene-edited normal organoids, which accurately recapitulates the full course of bladder cancer chemotherapy; 2) Discovering a new type of lineage plasticity called "semi-squamatization" associated with chemoresistance in bladder cancers; 3) Identifying CTSH as a new therapeutic target for drug-resistant bladder cancers; 4) Elucidating the molecular and cellular mechanisms of targeting CTSH in the treatment of drug-resistant bladder cancer, and proposing differentiation treatment strategies for solid tumors.

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