Effects of combined radiotherapy with immune checkpoint blockade on immunological memory in luminal-like subtype murine bladder cancer model

Abstract

MIBC is a highly lethal disease, and the patient survival rate has not improved significantly over the last decades. UPPL is a cell line that can be used to recapitulate the luminal-like molecular subtype of bladder cancer and to discover effective treatments to be translated in patients. Here, we investigate the effects of combinational treatments of radiotherapy and immunotherapy in this recently characterized UPPL tumor-bearing mice. We first characterized the baseline tumor microenvironment and the effect of radiation, anti-PD-L1, and combinatorial treatments. Then, the mice were re-challenged with a second tumor (rechallenged tumor) in the contralateral flank of the first tumor to assess the immunological memory. Radiation slowed down the tumor growth. All treatments also decreased the neutrophil population and increased the T cell population. Anti-PD-L1 therapy was not able to synergize with radiation to further delay tumor growth. Furthermore, none of the treatments were able to generate immune memory. The treatments were not sufficient to induce a significant and lasting pool of memory cells. We show here that anti-PD-L1 treatment added to radiotherapy was not enough to achieve T cell-mediated memory in UPPL tumors. Stronger T cell activation signals may be required to enhance radiation efficacy in luminal-like bladder cancer.

Keywords: Bladder cancer; combination therapy; immune checkpoint inhibitors; immune memory; immunotherapy; radiotherapy.

Figure 1.

Immune profile of UPPL tumors and PD-L1/PD-1 expression within the tumor micro-environment. A. Percent of CD45⁺ of neutrophils, dendritic cells, monocytes, CD4+ T cells and CD8+ T cells (n=6) B. Percentage of cells expressing PD-L1 within their respective populations (neutrophils, dendritic cells, and monocytes). C. Percentage of cells expressing PD-L1 within CD4+ T cells and CD8+ T cells compartments. D. percentage of tumor cells (gated on CD45⁻ cells) expressing PD-L1. Number of columns indicate mean frequency.

Figure 2.

Radiation slows tumor growth, modifies the TME and does not impact re-challenge. A. Schematic of the treatment plane for each group (n=6-8). B. Tumor growth upon tumor reaching the size of 0.1cm³ for each different treatment arm. C. Kaplan-Meier survival curve analysis for each treatment group starting from the day the reaching the size of 0.1cm³. D. Monocytes, neutrophils, and dendritic cells frequencies (gated on CD45⁺cells) in the TIME following two weeks of treatment. E. CD4+ and CD8+ T cells frequencies in the TME following two weeks of treatment. F. Primary tumor TME effort and central memory T cells frequencies in the TME. G. Re-challenge tumor growth, on the opposite flank of the primary tumor upon reaching 0.1cm³ for each different treatment group.*= P < 0.05; **= p < 0.01.

Figure 3.

Combinatorial treatments affect the TME of re-challenge tumors differently than that of the primary tumor. A. Frequencies of monocytes, neutrophils, and dendritic cells in the primary tumors and in the re-challenge tumors (gated on CD45⁺ cells). B. Comparison of the T cell frequencies in the first tumors and the re-challenge tumors(gated on CD45⁺CD⁺). *= p < 0.05; **= p < 0.01; ***= p < 0.01.