In the treatment of breast cancer, radiotherapy (RT) is an integral part and particularly aims to eliminate cancer cells and stop tumor growth. Besides its local effects, RT can also stimulate the immune system by causing immunogenic cancer cell death which consecutively leads to an anti-tumor immune response. However, due to the immune suppressive tumor microenvironment, and RT’s own immune suppressive effects the anti-tumor immune response often fails to eliminate tumor masses or their distant metastases when RT is used alone. The development of multi-modal treatments for cancer has shown that combining RT with other immunomodulatory treatments, such as hyperthermia (HT), can enhance RT’s effects and successfully stimulate anti-tumor immunity. HT is an anti-cancer treatment that heats the tumor locally to 39-44°C for at least 60 min. HT is a potent radiosensitizer, and for the treatment of recurrent and advanced breast cancer, HT is commonly used in combination with RT and chemotherapy (CT). HT has various effects on cancer, such as inhibiting the DNA damage repair system, inducing cancer cell death, and improving blood flow and oxygenation. Hyperthermia-induced immunomodulation has been shown in numerous preclinical models but is still often under-appreciated clinically. HT can increase the infiltration of immune cells into the tumor due to improved blood flow. By inducing the release of danger signals such as heat shock protein 70 (Hsp70) mostly in combination with RT, HT fosters the transport of tumor antigens and direct activation of dendritic cells (DCs) which consecutively leads to the priming of cytotoxic T cells against the tumor. However, little is known about how the combination of HT with RT affects the effector phase of anti-tumor immune responses and thereby how it impacts the immune phenotype of cancer cells. It is also unclear whether HT-modulated anti-tumor responses are influenced by the DNA damage response and whether the sequence of the combination of RT and HT modulates the cancer cell phenotype differently. Our work focused on exploring these questions, and we have used a custom-made conventional heating chamber to perform HT in breast cancer cells and patient-derived breast cancer organoids. HT was delivered in different temperatures (constant 39°C; 41°C, and 44°C) for 60 min. HT was combined with hypo-fractionated radiation of two times 5 Gy in different sequences (either pre-irradiation or post-irradiation). Two human breast cancer cell lines (MDA-MB-231 and MCF-7), and one murine breast cancer cell line (4T1) were investigated. To compare the results in a more physiological setting, patient-derived breast cancer organoids, and normal tissue organoids were investigated as well. HT combined with RT induced significantly more cell death in the MCF-7 cell line, apoptosis being the most prominent cell death form and both apoptosis and necrosis in the MDA-MB-231 cell line. Similar findings were observed in the murine 4T1 cell line with apoptosis being the most prominent cell death form, and significantly more cell death was observed after being treated with HT and RT. There was no significant difference between the treatment sequences in regards to inducing cancer cell death. When HT with higher (41°C and 44°C) temperatures was combined with RT, the effects on breast cancer cell death were more prominent. HT at any temperature alone did not impact on cancer cell death. Analyses of the breast cancer immune phenotype revealed a high dynamic of immune checkpoint molecules (ICMs) expression after treatment with HT and RT. Combinations of RT and HT, irrespective of the treatment sequence, induced increased expression of both immune inhibitory (PD-L1, PD-L2, and HVEM) and immune stimulatory ICMs (OX40-L) on breast cancer cells. In MCF-7 cells, high expression of PD-L2, PD-L1, and HVEM was observed at later time points after the treatments with HT and RT combination (72h, 120h). Especially when higher temperatures of HT (41°C, 44°C) were applied together with RT a significantly higher expression of ICMs compared to RT alone was observed in MCF-7 cells. Regarding the second human breast cancer cell line MDA-MB-231, PD-L2 was the most prominent ICM being upregulated after a combination of HT and RT. In MDA-MB-231 cells at earlier time points after the treatments (24h, 48h), HT in combination with RT significantly increased the expression of PD-L1 and HVEM. Similar findings were observed with the murine 4T1 breast cancer cells as well. In all of the examined cell lines, the sequence of the treatments did not significantly impact on the immune phenotype of the breast cancer cells. In a more physiological setting, the combination of HT of 41°C and RT, again irrespective of the sequence, significantly reduced the viability of the patient-derived breast cancer organoids (PDOs). On the contrary, the treatments (HT, RT alone or in combination) did not show any significant effect on normal breast tissue. The treatment-induced alterations in the immune phenotype of the breast cancer organoids were weaker compared to the 2D tumor cell line cultures and dependent on the tumor characteristics and stage. In T-71 organoids, HVEM was significantly upregulated on the cancer cells at 72h after a combination of HT with RT. Further, there was a tendency for increased expression of PD-L1 after combinational treatments as well. Co-culture of treated breast cancer cells with monocyte-derived dendritic cells showed no significant change concerning its activation. Similar findings were also observed in co-culture of treated breast cancer organoids and activated CD8+ T cells. Nevertheless, there was a slight tendency that when organoids were treated with RT and HT combination, the CD8+ T-cells killing was more effective. Furthermore, we aimed to investigate the effects of HT and RT on the DNA damage repair system of breast cancer cells since it has become evident that it is interconnected with immune alterations. We focused on γH2AX foci as an indicator of double-strand break (DSB) repair and micronuclei formation (MN) as an indicator of genetic instability. The analyses focused on HT of 41°C alone or in combination with RT, again applied in different sequences. In general, in all investigated cell lines (MCF-7, MDA-MB-231, and 4T1 cells) significantly higher amounts of γH2AX foci was detected in HT plus RT treated breast cancer cells, particularly at a later time point (120h after treatment). Here, a significant increase was only observed when HT was given before RT. This might indicate that HT inhibits the DNA damage repair of the cancer cells enabling RT-induced DNA damage to remain unrepaired even until a later time point. Furthermore, a higher frequency of cells with micronuclei was observed in the breast cancer cells treated with HT followed by RT at the later time point (120 h). In general, a higher frequency of micronuclei was observed in all breast cancer cell lines after combined HT and RT treatments at the later time point, again with the effect being more prominent in the setting where HT was applied before RT. To gain first insights into how DNA damage and related alterations might impact a key ICM in breast cancer, we analyzed how MN formation affects the expression of HVEM. For all of the investigated breast cancer cell lines, a higher expression of HVEM was significantly correlated with a high frequency of cells with micronuclei. Moreover, one of the signs of senescent cells is an enlarged nucleus, and our results showed that a larger nucleus area was also correlated with higher expression of HVEM and higher frequency of MN. However, the presence of MN in cells per se did not affect the expression of HVEM, suggesting that another pathway might be involved in this process. In summary, we revealed that HT is an effective treatment that sensitizes breast cancer cells and organoids to RT. The combination of HT and RT significantly induces breast cancer cell death and affects the expression of both immune inhibitory and immune stimulatory ICMs. HT followed by RT effectively inhibits the DNA damage repair system of breast cancer cells, significantly delaying DNA repair. Furthermore, DNA damage induced by HT and RT can potentially affect the immune response, although further research is needed, particularly with preclinical in vivo model systems. As a translational aspect, our results suggest when HT is combined with RT, the expression of ICMs should be closely monitored, and additional immune checkpoint inhibitor treatment may help to reduce the unwanted immunosuppressive effects of the HT and RT combination.
Azzaya Sengedorj (Fri,) studied this question.