Loss of Glutathione-S-Transferase Theta 2 (GSTT2) Modulates the Tumor Microenvironment and Response to BCG Immunotherapy in a Murine Orthotopic Model of Bladder Cancer

Loss of the glutathione-S-transferases Theta 2 (Gstt2) expression is associated with an improved response to intravesical <i>Mycobacterium bovis</i>, Bacillus Calmette-Guérin (BCG) immunotherapy for non-muscle-invasive bladder cancer (NMIBC) patients who receive fewer BCG instillations. To delineate the cause, Gstt2 knockout (KO) and wildtype (WT) C57Bl/6J mice were implanted with tumors before treatment with BCG or saline. RNA was analyzed via single-cell RNA sequencing (scRNA-seq) and real-time polymerase chain reaction (RT-PCR). BCG induced PD-L1 expression in WT mice bladders, while pro-inflammatory TNF-α was upregulated in KO bladders. ScRNA-seq analysis showed that Gstt2 WT mice bladders had a higher proportion of matrix remodeling fibroblasts, M2 macrophages, and neuronal cells. In KO mice, distinct tumor cell types, activated fibroblasts, and M1 macrophages were enriched in the bladders. In WT bladders, the genes expressed supported tumorigenesis and immunosuppressive PD-L1 expression. In contrast, Gstt2 KO bladders expressed genes involved in inflammation, immune activation, and tumor suppression. An 11-gene signature (Hmga2, Peak 1, Kras, Slc2a1, Ankfn1, Ahnak, Cmss1, Fmo5, Gphn, Plec, Gstt2), derived from the scRNA-seq analysis predicted response in NMIBC patients (The Cancer Genome Atlas (TCGA) database). In conclusion, our results indicate that patients with WT Gstt2 may benefit from anti-PD-L1 checkpoint inhibition therapy.