Local HT treatment has been widely used to treat solid tumors in experimental models 32–34 and in cancer patients 35–38. Heat may come from various external sources including infrared radiation, radiofrequency, microwave and ultrasound, where these approaches could give different effects in eradicating the tumor. Potentially, NIR was reported to be efficient in killing the tumor cells selectively without causing disruption on surrounding normal tissues. In this study, heat was applied using wIRA with a 750 watt halogen lamp and a 780 nm high pass filter, resulting a peak output at 820 nm. This device is commonly used to treat skin diseases, wound and many other areas of medicines. WIRA is easy to operate, gentle to skin and allows deeper penetration compared to conventional infrared therapy 39. As breast cancer is a superficial tumor, the use of local HT is clinically appropriate and has potential to treat the tumor effectively. While conducting the local HT procedure, it is very important to monitor the tumor temperature precisely because even the slightest temperature change could give a different level of heat-stress to the tumor and stromal cells. The treatment efficacy could also be temperature dependent. For instance, heating tumor at 42oC may improve vasculature permeability thus better immune cells trafficking into the tumor 6. This scenario however, was not observed at temperature more than 43oC due to hemorrhage 6. We selected 43oC for our HT approach because this fever to moderate temperature is believed to be immunotherapeutic 6. The treatment was repeated three times for 30 minutes each to ensure the optimum effect was achieved.
Using this approach, HT was able to inhibit the growth of subcutaneous EMT6 tumor, with 36% of treated mice showing complete tumor regression. These mice also survived for 50 days after the treatment, demonstrating that HT could provide a therapeutic benefit that prolongs mice survival. HT caused tumor cells to undergo necrosis and apoptosis which affects the cell number and tumor size and subsequently led to inhibition of tumor growth. Consistent with tumor inhibition, HT also decreased cell proliferation, which was observed through the reduction of PCNA expression. Therefore, we reasoned that NIR not only induce cell death but also retarded the division of new cells by arresting the cell cycle. In particular, NIR induces heat stress to the cells. It was reported that the stress response pathway always elicits cell cycle arrest to circumvent the transmission of damaged macromolecules and provides the time for cells to repair themselves, resulting in suppression of cell proliferation 40.Accordingly, a more pronounced proliferating activity was found in our untreated breast tumor, which correlates with the findings of other clinical studies that demonstrated PCNA is greatly involved in breast cancer initiation and development 41,42
Heat shock proteins have been recognized as an important participant in immune system 27,43. Hsps are present in normal cells conditions but will be highly expressed under stress condition including heat-stress. Necrotic cancer cells destroyed by HT will release antigenic tumor peptides that includes Hsp into extracellular matrix. These extracellular Hsps then will act as danger signal to activate dendritic cells and subsequently T cells to generate adaptive immune response. The antitumor effect is dominated by Th1 CD4+ T lymphocytes. Generally, CD4 T cells perform immunosurveillance and regulate immune system, alongside with Th1 and Th2 cytokines to balance the cell-mediated immunity 44. Th1 CD4+ T lymphocytes secretes Th1 cytokines including IFN-γ, TNF-α, IL-2 which in turn act as marker for Th1 immune response. In response to cancer, CD4 T cells provide help in the priming of tumor-specific CD8 T cells, thus permitting the differentiation and expansion of tumor antigen-specific CTLs 45. Subsequently, effector CD8 T cells migrate into the tumor and perform their killing activity accordingly. Similarly, we reported a high level of Hsp in the heated tumor (Fig. 8a), accompanied by increased DC activation in dLN of treated mice (Fig. 8b). Our findings also demonstrated a significant increase of tumor infiltrated-CD8 T cells with greater CD8 T cells infiltrates tumor over time (Fig. 8c). This scenario suggests HT treatment likely stays effective for a longer period, which may be related to higher antigen-specific CTLs produced as a result from increased Hsp-peptide complex cross presentation by DCs 46.
The upregulation of IFN-γ in tumor also supports these findings (Fig. 8d), where IFN-γ is able to inhibit tumor cell proliferation and differentiation 45. Besides orchestrating the innate immune response, IFN-γ, which is mainly produced by activated T cells and NK cells, also mediates the adaptive anti-tumor response. For instance, IFN-γ increases the expression of MHC, making them more susceptible to immune recognition and destruction by CD8+ T cells 47. Other than IFN-γ, IL-2 also plays a critical role in the induction of the immune response. However, the non-significant increase in IL-2 reported here maybe due to the dual role played by this cytokine. IL-2 is responsible for regulating CD8 effector function as well as promoting the differentiation of CD4+ T lymphocytes into Tregs. On another note, these intratumoral cytokines were analyzed 2 weeks post-treatment, which is considerably late as they usually secreted at the early stage to help initiate the immune response. This may also explain the non-significant different in expression between both groups. Hence, future study is suggested to be conducted at day 4–7 post-treatment.
Tregs have long been known to suppress the immune response and favor the growth of tumor cells, causing the cancer to progress and to become metastatic. The recruitment of CD4+CD25+FoxP3+ T cells to the tumor distorts T cells responses from an effector to a regulatory subtype, causing protective antitumor immunity to diminish 48. As been reviewed by Plitas and Rudensky (2020), the presence of this population in tumor microenvironment is linked with the suppression of T cells, NK cells and DC in animal models and in cancer patients 49. Similar situations observed in our data correlate with those findings, suggesting the effectiveness of NIR in limiting the immunosuppressive activity (Fig. 8e). The CD8:Treg ratio is commonly used to predict prognosis in breast cancer 50,51. NIR also causes higher CD8:Treg ratios at D21 post-inoculation, which implies a favorable prognosis. In conjunction with these results, HT is believed to disrupt the function of Tregs and interfere with their migratory properties 52. Additionally, heat stress enhances the cytotoxic activity of NK cells against Tregs 53, which also explains our result showing that the Tregs population in the tumor is conversely proportional to NK cells. Taken together, these findings reflect the dampening of immunosuppressive activity mediated by HT by NIR.
Collectively, our data suggest that a monotherapy of NIR is able to activate and promote the infiltration of immune cells in breast tumor, thus may be an effective in the treatment of breast cancer. With easy administration in a controlled manner, the application of local hyperthermia could potentially offer clinical benefit for the treatment of breast cancer.