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Glutathione-S-transferases (GST) enzymes detoxify xenobiotics and are implicated in response to anticancer therapy. This study evaluated the association of GST theta 1 (GSTT1), GSTT2, and GSTT2B with Mycobacterium bovis Bacillus Calmette-Guérin (BCG) response in non-muscle-invasive bladder cancer treatment. In vitro assessments of GSTT2 knockout (KO) effects were performed using cell lines and dendritic cells (DCs) from GSTT2KO mice. Deletion of GSTT2B, GSTT1, and single-nucleotide polymorphisms in the promoter region of GSTT2 was analysed in patients ( n = 205) and healthy controls ( n = 150). Silencing GSTT2 expression in MGH cells (GSTT2B FL/FL ) resulted in increased BCG survival ( p < 0.05) and decreased cellular reactive oxygen species. In our population, there are 24.2% with GSTT2B Del/Del and 24.5% with GSTT2B FL/FL . With ≤ 8 instillations of BCG therapy ( n = 51), 12.5% of GSTT2B Del/Del and 53.8% of GSTT2B FL/FL patients had a recurrence ( p = 0.041). With ≥9 instillations ( n = 153), the disease recurred in 45.5% of GSTT2B Del/Del and 50% of GSTT2B FL/FL . GSTT2 FL/FL patients had an increased likelihood of recurrence post-BCG therapy (HR 5.5 [1.87-16.69] p < 0.002). DCs from GSTT2KO mice produced three-fold more IL6 than wild-type DCs, indicating a robust inflammatory response. To summarise, GSTT2B Del/Del patients respond better to less BCG therapy and could be candidates for a reduced surveillance regimen.

Objectives: To investigate plasma and urinary kynurenine (KYN)-tryptophan (TRP) ratios in bladder cancer, expression of indoleamine 2,3-dioxygenase 1 (IDO1) in relation to tryptophan 2,3-dioxygenase (TDO2) in bladder tumour, and the correlation of KYN-TRP ratio with bladder tumour burden., Methods: Metabotyping of the TRP-KYN metabolic axis was performed via a clinical case-control study. Expression of IDO1 and TDO2 was measured in human biopsied tissues. Correlational experiments between KYN-TRP ratio and bladder tumour were performed using a murine orthotopic prostate-specific antigen (PSA)-secreting MB49 bladder cancer model., Results: We established for the first time that plasma TRP level was significantly decreased, while both plasma and urinary KYN-TRP ratios were significantly higher in bladder cancer patients, and expression level of IDO1 but not TDO2 was increased in human bladder tumour. We reported the positive correlation between IDO1 expression, KYN-TRP ratio, normalized PSA to creatinine, and bladder tumour burden in the murine model., Conclusion: Kynurenine-tryptophan ratio is a promising surveillance biomarker for bladder cancer, but would require further validation before clinical translation., (© 2020 The Authors BJU International © 2020 BJU International Published by John Wiley & Sons Ltd.)

Aim: To develop a strategy to improve response to bacillus Calmette-Gueri (BCG) using cytokine gene therapy ( Gmcsf + Ifnα ). Materials & methods: MB49-PSA tumor-bearing C57BL/6N mice were assigned into four groups: control; Gmcsf + Ifnα therapy; BCG therapy or combined therapy ( Gmcsf + Ifnα and BCG). In schedule 1, cytokine gene therapy was delivered before BCG therapy (eight instillations). In schedule 2, cytokine gene and BCG therapy were instilled alternatively (eight instillations). Tumors were analyzed by immunohistochemistry and mRNA analysis and urinary immune cells by flow cytometry. Results: Combined therapy in schedule 2 reduced tumor growth, increased immune cell recruitment and was associated with reduced inflammation when compared with BCG therapy. Conclusion: Alternating cytokine gene delivery with BCG therapy modulates the tumor environment increasing receptivity to BCG.

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified to secrete human Prostate Specific Antigen (PSA), and the procedure for the confirmation of tumor implantation. In brief, mice are anesthetized using injectable drugs and are made to lay in the dorsal position. Urine is vacated from the bladder and 50 µL of poly-L-lysine (PLL) is slowly instilled at a rate of 10 µL/20 s using a 24 G IV catheter. It is left in the bladder for 20 min by stoppering the catheter. The catheter is removed and PLL is vacated by gentle pressure on the bladder. This is followed by instillation of the murine bladder cancer cell line (1 x 10 5 cells/50 µL) at a rate of 10 µL/20 s. The catheter is stoppered to prevent premature evacuation. After 1 h, the mice are revived with a reversal drug, and the bladder is vacated. The slow instillation rate is important, as it reduces vesico-ureteral reflux, which can cause tumors to occur in the upper urinary tract and in the kidneys. The cell line should be well re-suspended to reduce clumping of cells, as this can lead to uneven tumor sizes after implantation. This technique induces tumors with high efficiency. Tumor growth is monitored by urinary PSA secretion. PSA marker monitoring is more reliable than ultrasound or fluorescence imaging for the detection of the presence of tumors in the bladder. Tumors in mice generally reach a maximum size that negatively impacts health by about 3 - 4 weeks if left untreated. By monitoring tumor growth, it is possible to differentiate mice that were cured from those that were not successfully implanted with tumors. With only end-point analysis, the latter may be mistakenly assumed to have been cured by therapy.

The aim of this study was to monitor changes in the expression of immune-related genes in the bladder after tumor implantation. Mice were orthotopically implanted with MB49-PSA cells (C57BL/6 mice) on day 1 and terminated on days 7, 14, 21, and 28. Another mouse model (MBT-2/C3H mice) was examined at day 7. Gene expression analysis was performed using a TaqMan Low Density Mouse Immune Panel (Applied Biosystems, USA) on RNA extracted from the bladders. Selected genes were reconfirmed by real-time PCR analysis and RT-PCR on the mRNA from other animals. Immune suppressive (IL13, IL1β, PTGS2, NOS2, IL10, CTLA4, and CCL22) and immune stimulatory genes (CSF2, GZMB, IFNγ, CXCL10, TNFα, CD80, IL12a, and IL6) and AGTR2 were increased by day 7. By day 28, IL10, CCL2, CCL5, CXCL11, CTLA4, GZMB, IFNγ, CSF2, and IL6 were significantly increased. Therapeutic strategies involving TH1 induction and TH2 dampening may improve responses to immunotherapy.