Photodynamic therapy (PDT) is an anticancer strategy utilizing light-mediated activation of a photosensitizer (PS) which has accumulated in tumor and/or surrounding vasculature. Upon activation, the PS mediates tumor destruction through the generation of reactive oxygen species and tumor-associated vasculature damage, generally resulting in high tumor cure rates. In addition, a PDT-induced immune response against the tumor has been documented in several studies. However, some contradictory results have been reported as well. With the aim of improving the understanding and awareness of the immunological events triggered by PDT, this review focuses on the immunological effects post-PDT, described in preclinical and clinical studies. The reviewed preclinical evidence indicates that PDT is able to elicit a local inflammatory response in the treated site, which can develop into systemic antitumor immunity, providing long-term tumor growth control. Nevertheless, this aspect of PDT has barely been explored in clinical studies. It is clear that further understanding of these events can impact the design of more potent PDT treatments. Based on the available preclinical knowledge, recommendations are given to guide future clinical research to gain valuable information on the immune response induced by PDT. Such insights directly obtained from cancer patients can only improve the success of PDT treatment, either alone or in combination with immunomodulatory approaches.

Chronic inflammation drives the development of colorectal cancer (CRC), where tumor-infiltrating immune cells interact with cancer cells in a dynamic crosstalk. Mast cells (MC), one of earliest recruited immune cells, accumulate in CRC tissues and their density is correlated with cancer progression. However, the exact contribution of MC in CRC and their interaction with colon cancer cells is poorly understood. Here, we investigated the impact of primary human MC and their mediators on colon cancer growth using 2D and 3D coculture models. Primary human MC were generated from peripheral CD34⁺ stem cells. Transwell chambers were used to analyze MC chemotaxis to colon cancer. Colon cancer cells HT29 and Caco2 differentially recruited MC by releasing CCL15 or SCF, respectively. Using BrdU proliferation assays, we demonstrated that MC can directly support colon cancer proliferation and this effect was mediated by their cellular crosstalk. 3D coculture models with cancer spheroids further confirmed the pro-tumor effect of MC on colon cancer growth, where direct cell-cell contact is dispensable and increased production of multiple soluble mediators was detected. Moreover, TLR2 stimulation of MC promoted stronger growth of colon cancer spheroids. By examining the transcriptome profile of colon cancer-cocultured MC versus control MC, we identified several MC marker genes, which were deregulated in expression. Our study provides an advanced in vitro model to investigate the role of human MC in cancer. Our data support the detrimental role of MC in CRC development and provide a molecular insight into the cellular crosstalk between MC and colon cancer cells.

Mast cells (MC) accumulate in colorectal cancer (CRC) and the relationship between MC density and cancer progression has been well recognized. MC can be either pro-tumor or anti-tumor players, depending on the local factors present in the tumor microenvironment. Upon malignant transformation, cancer cells express high levels of sialic acids on cell membrane or by secretion. Siglecs are a family of immunoglobulin-like receptors that bind sialic acids and each subtype has a distinct pattern of expression on immune cells. Among them, Siglec-6 is expressed predominately by MC. However, the function of Siglec-6 in MC is largely unexplored and whether it is expressed by CRC-associated MC remains unknown. In this study, we explored the function of Siglec-6 in CD34⁺ derived human MC. MC activation was initiated by IgE crosslinking with or without preincubation of anti-Siglec-6 Ab. Siglec-6 engagement significantly attenuated IgE-dependent MC degranulation as measured by ß-hexosaminidase release and CD63 expression. Interestingly, the production of GM-CSF was also shown reduced upon Siglec-6 engagement. To mimic the milieu of CRC, we cultured primary human MC with colon cancer cells or under hypoxia and Siglec-6 was then measured on these conditioned MC. Coculture with colon cancer cells (HT29 and Caco2) induced upregulation of Siglec-6 on MC. In comparison, normal colon cells (CCD841) had no effect. Also, a time-dependent increase of Siglec-6 by MC was observed under 1% O₂. Immunohistochemistry of CRC tissue showed expression of Siglec-6 by MC in submucosa. Lectin immunochemistry revealed the presence of actual ligands for Siglec-6 in human CRC tissues. Together, our findings illustrate that Siglec-6 is a functionally inhibitory receptor on MC and suggest that Siglec-6 expression may be relevant for MC activity in the tumor microenvironment of CRC.

Nanobody-targeted photodynamic therapy (NB-PDT) has been recently developed as a more tumor-selective approach rather than conventional photodynamic therapy (PDT). NB-PDT uses nanobodies that bind to tumor cells with high affinity, to selectively deliver a photosensitizer, i.e., a chemical which becomes cytotoxic when excited with light of a particular wavelength. Conventional PDT has been reported to be able to induce immunogenic cell death, characterized by the exposure/release of damage-associated molecular patterns (DAMPs) from dying cells, which can lead to antitumor immunity. We explored this aspect in the context of NB-PDT, targeting the epidermal growth factor receptor (EGFR), using high and moderate EGFR-expressing cells. Here we report that, after NB-PDT, the cytoplasmic DAMP HSP70 was detected on the cell membrane of tumor cells and the nuclear DAMP HMGB1 was found in the cell cytoplasm. Furthermore, it was shown that NB-PDT induced the release of the DAMPs HSP70 and ATP, as well as the pro- inflammatory cytokines IL- 1β and IL-6. Conditioned medium from high EGFR-expressing tumor cells treated with NB-PDT led to the maturation of human dendritic cells, as indicated by the upregulation of CD86 and MHC II on their cell surface, and the increased release of IL-12p40 and IL-1β. Subsequently, these dendritic cells induced CD4+ T cell proliferation, accompanied by IFNγ release. Altogether, the initial steps reported here point towards the potential of NB-PDT to stimulate the immune system, thus giving this selective-local therapy a systemic reach.