While most of the previous studies mainly assessed
While most of the previous studies mainly assessed the effect of laser light on cancer THZ1 in culture, here we intentionally explored whether PBM exerted the same activity ex vivo and in vivo. We exposed various primary cells and cancer cell lines to different laser protocols (L1, L2 and L3) and noticed that in most instances, the net effect was a significant increase in cell metabolism and proliferation, in agreement with available literature data (Frigo et al., 2009). Of notice, not all cell types responded in the same manner to each protocol, possibly indicating a different content of endogenous porphyrins and cytochromes, which are generally acclaimed as the primary laser light receptors (Farivar et al., 2014). Indeed, primary cells tended to respond better to PBM than cancer cells, which often carry abnormalities in their mitochondria. Most relevant, when irradiating the same B16F10 cells ex vivo and in vivo, we observed an opposite effect. Despite a modest, but significant increase in ATP content was detected in cultured cells upon exposure to the L3, all 3 protocols markedly reduced tumor growth in vivo, as well as the number of neoplastic cells infiltrating and invading of surrounding tissues. These results are consistent with a few published reports assessing the in vivo behavior of tumors exposed to laser light, and showing that low power and energy doses have no influence on tumor growth, or rather they can inhibit it (Abe et al., 1993). The lower index of tumor growth and invasiveness in laser treated animals, and especially in the L3 group, was accompanied by a notable presence of immune cells (essentially T lymphocytes and DCs) around the tumor mass. These findings are in line with the recent observation that laser light can be exploited to activate dermal DCs to effectively migrate to draining lymph nodes and to induce antigen-specific CD8+ and CD4+ T cell responses in the same melanoma mouse model (Terhorst et al., 2015). Besides opening the way to completely novel strategies to enhance cancer immunotherapy and vaccination in general, these data also shed some light on the mechanisms by which PBM could inhibit tumor growth in vivo, and thus be considered a safe procedure in cancer patients. Notably, we showed that the direct irradiation of primary DCs significantly increased the secretion of type I IFNs and a consistent up-regulation of these cytokines was detected in vivo in B16F10 tumors upon exposure to any of the 3 tested laser protocols. Type I IFNs are well known for their peculiar anti-proliferative, pro-apoptotic and anti-angiogenic properties, which are essential for immunosurveillance of cancer, and already exploited clinically in a variety of malignancies (reviewed in Zitvogel et al., 2015). The observation that laser treatment completely lost its effect in IFNAR KO mice is a clear indication that IFNα and IFNβ are essential mediators of the anti-cancer activity of laser light in vivo. At what extent the activation of immune cells, and in particularly DCs, might also be responsible of the clearance od dysplastic lesions observed in the oral carcinogenesis model, similarly to what observed in human oral SCC (Upadhyay et al., 2012), still remains an open question. Since it is known that mild hyperthermia can exert immune-modulatory activities (Knippertz et al., 2011), we cannot formally exclude that the observed effects derive, at least in part, from cell and tissue heating. However, the evidence that the most effective protocol (L3) did not increase the temperature of either cultured cells or body surface, points toward a minimal, if not null contribution of hyperthermia to the anti-cancer effect of PBM. From our analysis of intra-tumoral immune cell content by flow cytometry, we invariably detected an increased number of cells in not perfused compared to perfused animals in the L3 group. This is an indirect, albeit strong indication that laser treatment enhanced the perfusion of the tumor mass, thus increasing the number of cells within its intravascular compartment. Of notice, whereas most of the immune cell types were equally abundant within the extravascular space in control and laser-treated animals, L3 therapy resulted in a significant reduction on the number of F4/80+Ly6C+ cells, which most probably represent pro-angiogenic macrophages. This observation led us to analyze the tumor vasculature in both the B16F10 and the oral carcinogenesis models. In both cases, we observed that laser irradiation resulted in a significant increase in the number of α-SMA+ arterial vessels and a more regular and structured vessel pattern, as assessed by immunofluorescence and in vivo perfusion with fluorescent lectin. The ultimate outcome of these effects was the reduction of tumor progression, fitting the concept of “vessel normalization”, originally put forward as a goal to improve blood supply and enhance drug delivery by low-dose anti-angiogenic agents and more recently emerging as an innovative strategy to inhibit tumor growth rate (Carrer et al., 2012).