Clemens W. G. M. Löwik

Supplementary Figure 4. Bremachlorin’s ex-vivo bio-distribution of non-homogenized organs. Ex-vivo bio-distribution of Bremachlorin from mice with T-cell high (magenta) and T-cell low (blue) tumors and their organs shown as µg of Bremachlorin/g, 6 and 24 hours after administration (n=4 per group).

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.

Supplementary Figure 3. Positive control X-ray. (A) Confocal 40x images of T-cell high (left hand side) and T-cell low (right hand side) KPC clones nucleus (DAPI) and yH2AX, 1 hour after X-ray radiation (+) or without (-). (B) Average yH2AX foci per nucleus in in T-cell high (magenta) and T-cell low (blue) KPC clones. All the statistics produced here were calculated using two-way ANOVA (analysis of variance) followed by the Tukey’s method for multiple mean comparison * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.0001.

Supplementary Figure 5. Representative hematoxylin and eosin (H&E) images of whole tissue slice from T-cell high 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, PDT and anti-PD-1 NTS and Bremachlorin control. TS= treated side, NTS= non-treated side.

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 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.

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 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.

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.