Lysosomal accumulation of sunitinib has been suggested as an underlying mechanism of resistance. Here, we investigated if photochemical internalization (PCI), a technology for cytosolic release of drugs entrapped in endosomes and lysosomes, would activate lysosomal sequestered sunitinib. By super-resolution fluorescence microscopy, sunitinib was found to accumulate in the membrane of endo/lysosomal compartments together with the photosensitizer disulfonated tetraphenylchlorin (TPCS_2a). Furthermore, the treatment effect was potentiated by PCI in the human HT-29 and the mouse CT26.WT colon cancer cell lines. The cytotoxic outcome of sunitinib-PCI was, however, highly dependent on the treatment protocol. Thus, neoadjuvant PCI inhibited lysosomal accumulation of sunitinib. PCI also inhibited lysosomal sequestering of sunitinib in HT29/SR cells with acquired sunitinib resistance, but did not reverse the resistance. The mechanism of acquired sunitinib resistance in HT29/SR cells was therefore not related to lysosomal sequestering. Sunitinib-PCI was further evaluated on HT-29 xenografts in athymic mice, but was found to induce only a minor effect on tumor growth delay. In immunocompetent mice sunitinib-PCI enhanced areas of treatment-induced necrosis compared to the monotherapy groups. However, the tumor growth was not delayed, and decreased infiltration of CD3-positive T cells was indicated as a possible mechanism behind the failed overall response.

The programmed death ligand-1 (PD-L1), also known as CD274 or B7-H1, is mainly expressed on cancer cells and/or immunosuppressive cells in the tumor microenvironment (TME) and plays an essential role in tumor progression and immune escape. Immune checkpoint inhibitors (ICIs) of the PD-1/PD-L1 axis have shown impressive clinical success, however, the majority of the patients do not respond to immune checkpoint therapy (ICT). Thus, to overcome ICT resistance there is a high need for potent and novel strategies that simultaneously target both tumor cells and immunosuppressive cells in the TME. In this study, we show that the intracellular light-controlled drug delivery method photochemical internalization (PCI) induce specific and strongly enhanced cytotoxic effects of the PD-L1-targeting immunotoxin, anti-PD-L1-saporin (Anti-PDL1-SAP), in the PD-L1+ triple-negative breast cancer MDA-MB-231 cell line, while no enhanced efficacy was obtained in the PD-L1 negative control cell line MDA-MB-453. Using fluorescence microscopy, we reveal that the anti-PD-L1 antibody binds to PD-L1 on the surface of the MDA-MD-231 cells and overnight accumulates in late endosomes and lysosomes where it co-localizes with the PCI photosensitizer fimaporfin (TPCS_2a). Moreover, light-controlled endosomal/lysosomal escape of the anti-PD-L1 antibody and fimaporfin into the cytosol was obtained. We also confirm that the breast MDA-MB-468 and the prostate PC-3 and DU-145 cancer cell lines have subpopulations with PD-L1 expression. In addition, we show that interferon-gamma strongly induce PD-L1 expression in the per se PD-L1 negative CT26.WT cells and enhance the PD-L1 expression in MC-38 cells, of which both are murine colon cancer cell lines. In conclusion, our work provides an in vitro proof-of-concept of PCI-enhanced targeting and eradication of PD-L1 positive immunosuppressive cells. This light-controlled combinatorial strategy has a potential to advance cancer immunotherapy and should be explored in preclinical studies.