Tissue acidosis plays a pivotal role in tumor progression: in particular, interstitial acidosis promotes tumor cell invasion, and is a major contributor to the dysregulation of tumor immunity and tumor stromal cells. The cell membrane and integral membrane proteins commonly act as important sensors and transducers of altered pH. Cell adhesion molecules and cation channels are prominent membrane proteins, the majority of which is regulated by protons. The pathophysiological consequences of proton-sensitive ion channel function in cancer, however, are scarcely considered in the literature. Thus, the main focus of this review is to highlight possible events in tumor progression and tumor immunity where the pH sensitivity of cation channels could be of great importance.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most challenging types of cancer with little or no response to immune checkpoint inhibitors (ICI). Photodynamic therapy (PDT) has been shown to ablate tumors and induce an immune response. In our study, we investigated the effect of PDT using the photosensitizer Bremachlorin, in its ability to reduce tumor burden and immunologically sensitize T-cell–high and T-cell–low murine PDAC tumors to the ICIs that blocks PD-1 immune checkpoint. In addition, we monitored the effect on survival and investigated if there was a response in PDT-treated and non–PDT-treated distant tumors. Our results showed that Bremachlorin PDT induces direct tumor killing that increased survival in both “hot” T-cell–high and “cold” T-cell–low PDAC tumors and that it can make T-cell–high tumors more sensitive to ICIs blocking PD-1. We found that T-cell–high tumor-bearing mice had an overall greater response to therapy than did T-cell–low tumor-bearing mice. One mouse with T-cell–high tumors exhibited complete tumor regression in both the treated and nontreated distant tumor 90 days after treatment. These results indicate that combining ICIs with Bremachlorin PDT could be a promising therapeutic intervention for enhancing PDAC’s response to therapy.

Supplementary Figure 1. Confocal 40x images of control conditions: negative control - no Bremachlorin, no Mitotracker® Green (left hand side); 100 µM Bremachlorin alone and no Mitotracker® Green (middle), no Bremachlorin and MitoTracker® Green alone (right hand side). From top to bottom: Bremachlorin, MitoTracker® Green, composite of Bremachlorin and MitoTracker® Green and bright field in T-cell low clones.

Supplementary Figure 6. Representative hematoxylin and eosin (H&E) images of whole tissue slice from T-cell low tumors from the different treatment groups, top row (left to right): control, anti-PD-1 alone, PDT alone TS and PDT alone NTS. Bottom row (left to right): PDT and anti-PD-1 TS and PDT and anti-PD-1 NTS. TS= treated side, NTS= non-treated side.

Supplementary Figure 2. Pre-screening the photosensitizer in vitro PDT dose. PDT was performed at four different concentrations of Bremachlorin: 100, 10, 1 and 0.1 µM as well as a control (just media) at 20mW/cm2 at 0 (dark control), 5, 10, and 20 J/cm2 (biological replicate n=1) in T-cell high (top) and T-cell low (bottom). MTS assay was performed 24 hours after PDT. Dark Ctrl = dark control.

Supplementary Figure 7. Representative images at 20x magnification of immunofluorescence analysis of CD8+ cells of T-cell high 500% tumor tissue of PDT and anti-PD-1 treated side (left) and non-treated side (right) (A). Absolute number of CD8+ cells quantified in T-cell high (B). TS= treated side, NTS= non-treated side.