Breast cancer is the most common cancer among women in the world, in which 1 in 8 women in the world is at risk of developing breast cancer ¹ . Furthermore, about 2.1 million new cancer cases in 2018 are breast cancer cases and this type of cancer is the leading cause of death in women with cancer ² . Commonly, the treatment of breast cancer is carried out in two ways. The first, termed local therapy, controls or removes the tumor in specific areas, such as through breast-conserving therapy and mastectomy that may be followed by radiotherapy. The second, called systemic therapy, uses the circulatory system in the body to inhibit cancer cells growth that spread in parts of the body. This type of treatment includes chemotherapy, targeted therapy, and endocrine therapy ³ . However, breast cancer therapy applied so far often causes negative effects, such as the presence of pathophysiological mechanisms that cause chronic pain ⁴ , or the mechanism of multidrug resistance (MDR) in breast cancer cells as a reaction to chemotherapy ^{5, 6} . Moreover, breast cancer therapies may attack certain normal cells or tissues that result in toxicity symptoms ⁷ . A novel cancer therapy based on electric field exposure with intermediate frequency and low intensity called Tumor Treating Fields (TTFields) has been developed to prevent such negative effects of cancer therapy and still inhibit cancer growth. No toxic side effects or adverse events were found on animal tumor models nor patients under the exposure to this electric field-based cancer therapy, except for skin toxicity or dermatitis of low severity ^{8, 9} .

Since skin toxicity or dermatitis arises in electric field therapy due to prolonged direct contact between the electrodes and the skin ¹⁰ , we have developed a type of non-contact electric field therapy using capacitive modality to avoid such injuries. In our previous study in preclinical setting, cancer therapy namely Electro-Capacitive Cancer Therapy (ECCT) using the non-contact electric fields with intermediate frequency (100 kHz) and low intensity (18 peak-to-peak Voltage/Vpp) could reduce the size of breast tumors in mice significantly without causing histological damage to mammary and skin tissues. In addition, lymphocyte and macrophage infiltration was found around the tumor area through the blood vessels ¹¹ . One of the mechanisms of tumor cells death can be carried out with the act of anti-tumor mechanism of various immune cells, including helper and cytotoxic T cells of lymphocytes, as well as macrophages ¹² . Cytotoxic CD8 ⁺ T cells which is activated by helper CD4 ⁺ T cells, may enter the tumor microenvironment and induce apoptosis, resulting in shrinkage of the tumor lesions ^{12, 13} . Macrophages with the M1 phenotype play an important role in the recognition and destruction of cancer cells and their presence usually indicates a favorable prognosis. Whereas macrophages with the M2 phenotype are involved in anti-inflammation, helping the angiogenesis process and the expression of scavenger receptors, and have a significant role in tumor development and metastasis ^{12, 13} , including breast tumor ^{14, 15} . One of the marker proteins to observe the infiltration of macrophages in a tissue, including mammary tumor tissue, is the CD68 protein ^{14– 16} .

Cancer therapy that can re-induce immune response to destroy and clean up tumor cells is a therapy that is being developed because of its positive effects. By activating immune cells, the immune cells may reach the tumor-specific areas, including metastatic area, and specifically attacking only tumor cells, not the normal cells in the vicinity ¹⁷ . Petri et al. ¹⁸ reported that static electric fields increase the immune response in mice. Moreover, electric fields modulate the activation and polarisation of some immune cells, including T cells ¹⁹ and macrophages ²⁰ . Voloshin et al. ²¹ reported that TTFields exposure combined with anti-PD-1 therapy induced immunogenic cell death in lung and colon tumors. In addition, Pratiwi et al. ²² reported the increased expression of CD68 and caspase-3 in rat breast tumor tissues under the exposure to 150 kHz non-contact electric fields while reducing the expression of PCNA and ErbB2 proteins. However, the study by Pratiwi et al. ²² showed an increase of the tumor size during therapy. Using different intermediate frequency (100 kHz), we examined the effect of non-contact electric fields exposure on tumor growth in mammary tumors-induced rats and observed the activation of immune cells in fighting the tumor cells. We hypothesized that the increase in the size of mammary tumors would not happen under exposure to this non-contact electric fields with the induction of immune cells, especially lymphocytes and macrophages.

The outcome of this study was the growth rate of mammary tumors under exposure to non-contact electric fields. Comparison of histological characteristics between breast tissue and breast cancer in the animal tumor model, the distribution of molecular markers that described the proliferation and the mechanism of cell death, and the response of immune cells under exposure, as will be explained coherently below.

In the present study, the significantly lower growth rate of rat mammary tumors in the IT group with a negative mean value of SGR compared to INT group with a positive mean value of SGR ( Figure 2a) showed the inhibitory effect of non-contact electric fields of ECCT against mammary tumor cells that are actively dividing in the animals, as we have previously reported in our preliminary study in mice ¹¹ . In addition, the development of mammary tumors exposed to 100 kHz non-contact electricfields of ECCT was decreased, whereas in the study of Pratiwi et al. ²² which used a frequency of 150 kHz, it was increased. This may suggest that differences in the frequency of the electric fields can give different therapeutic results in cancer ^{37, 38} and the 100 kHz was the appropriate frequency of non-contact electric fields in inhibiting the proliferation of mammary tumor cells.

The absence of tumors in the placebo (NIT) group ( Figure 4b) demonstrated the safety of non-contact electric fields of ECCT to healthy or normal animal models, as we have previously reported ¹¹ . In addition to its safety, this electric fields exposure did not produce acute damage to rat skin tissue or chronic pain which usually occurs after radiation therapy, including breast cancer radiation therapy ^{4, 39, 40} . Instead, it helped wound healing after cancer treatment in rats of IT group, indicated by the drying wound and the reduction of wound size ( Figure 3b and Figure 3c). Endogenous electric fields are known to play as a signal that direct cell migration in epithelial wound healing and external electric fields exposure may accelerate wound healing by directing the migration of T cells ¹⁹ and fibroblast, as well as their proliferation and transdifferentiation ^{41, 42} . In addition, Hoare et al. ²⁰ reported that external electric fields may contribute to the coordination and regulation of macrophage functions, including wound healing. The effect of non-contact electric fields of ECCT in directing immune cells was also seen in the fluid contained in the mammary tumors of the IT group as indicated by the presence of macrophages and lymphocytes infiltration into the tumor area ( Figure 7) as we have previously reported ^{11, 22} . This immune cell infiltration can promote the destruction of cancer cells ^{12, 21, 43} as demonstrated in the IT group ( Figure 2a and Figure 2c).

The cellular effects of electric fields exposure on PCNA expression during S-phase may indicate the incidence and the growth rate of mammary tumors ⁴⁴ . Since PCNA is a S-phase marker in interphase that has a central role in DNA replication and repair ⁴⁵ , the decrease of PCNA expression in mammary tumor cells in IT group ( Figure 6b) may indicate a decrease in the incidence and the growth rate of mammary tumors ⁴⁴ . In addition, alternating electric fields exposure with intermediate frequency and low intensity used in this study can cause tumor cells that are dividing to experience uneven segregation of chromosomes and mitotic catastrophe which followed by programmed cell death ⁴⁶ . This is supported by the study of Mujib et al. ⁴⁷ which reported that p53 protein can serve as a marker for cancer cell death via apoptosis due to non-contact electric fields exposure. Moreover, the hollow cavities, also called dead cell islands, with cells debris, may be the spaces that used to be the place for mammary tumor cells that then died through apoptosis ^{48, 49} , since caspase-3 was found in this place in the IT group ( Figure 5f). In addition, within these dead cell islands, the expression level of caspase-3 was very low ⁴⁹ , as we found in the IT group ( Figure 6d).

The lower expression of caspase-3 in the IT group ( Figure 6d) may suggest the mechanism of mammary tumor cell death under exposure to non-contact electric fields of ECCT through caspase-independent cell death. Meggyeshaz et al. ⁵⁰ reported tumor destruction by electric fields exposure and concomitant heat with elevated DNA fragmentation and low level of caspase-3 expression localized to inflammatory cells which dominantly follows a caspase-independent pathway. Meanwhile, higher caspase-3 expression in INT group ( Figure 6d) suggested that higher apoptosis events may have occurred in this group, resulted from oxidative stress due to elevated reactive oxygen species in mammary tumors ^{51, 52} . Although apoptosis occurred in the mammary tumors, the tumor cell death rate was relatively much lower than the proliferation rate, so that mammary tumor size increased ⁵³ , as seen in the INT group ( Figure 2a and Figure 2b).

The type of receptor possessed by breast cancer cells in this study was the ErbB2 receptor ( Figure 5c and Figure 5d). The generation of mammary tumors with ErbB2 receptors were the results of carcinogenesis due to DMBA administration into the animal tumor model ⁵⁴ . Despite such insignificant results in the expression level of ErbB2 between INT and IT groups ( Figure 6c), tumor size in the INT group continued to increase from baseline, and its tumor growth rate was higher than that in the IT group ( Figure 2a). In addition, the number of mitotic figures and the percentage of PCNA-positive tumor cells in the INT group were also higher than those in the IT group ( Figure 6a and Figure 6b, respectively). This suggests that INT group experienced higher tumor cell proliferation than IT group. Moreover, the higher number of CD68 ⁺ macrophages as one of inflammatory cells seen in the tumor microenvironment of INT group ( Figure 7a and Figure 8a) was capable to induce tumor growth by contributing to cellular migration of tumor cells and metastasis ^{12, 14, 55} . Ong et al. ⁵⁶ also reported that rat mammary carcinoma had significantly higher percentage of CD68 ⁺ macrophages compared to benign mammary tumors with a lower growth rate. CD68 ⁺ macrophages that infiltrate to the necrotic areas of INT group ( Figure 7a) may be considered as M2 macrophages which play a role in tumor growth and provide a poor prognosis ^{12, 56, 57} . In addition, increased M2 macrophages or Tumor-associated macrophages (TAMs) infiltration in mammary tumors would be accompanied by an increase in tumor size ^{14, 15, 58} , as shown in the INT group ( Figure 2a and Figure 8a). Conversely, the lower the number of infiltrating TAMs in rat mammary tumors, the smaller the size of the tumors ^{12, 15} , as shown in the IT group ( Figure 2a and Figure 8a). Moreover, the expressions of chemokine (C-C motif) ligand 2 (CCL2) and interleukin 18 (IL18) that have main roles in the development of metastatic breast cancer and excessive angiogenesis by amplifying M2 macrophage polarization ^{59, 60} were down-regulated under the exposure to non-contact electric fields of ECCT at 150 kHz frequency ²² . Similarly, non-contact electric field (100 kHz) exposure in our study may also inhibit the amplification of M2 macrophage polarization resulted in the negative mammary tumor growth rate in rats of IT group ( Figure 2a). The infiltration of CD68 ⁺ macrophages in mammary tumors of IT group suggested that exposure to non-contact electric fields of ECCT may direct macrophages, likely of M1 phenotype, to the tumor areas ^{11, 21} and then engulf and digest dead cell, debris and tumor cells ^{12, 61} that eventually produced hollow cavities ^{48, 49} as seen in Figure 7b. This macrophage-mediated clearance of dead cells was occurred in caspase-3-independent death ⁶² , since the IT group had lower expression of caspase-3 than the INT group ( Figure 6d).

Immunogenic tumor cell death induced by the electric fields exposure in the IT group can also be caused by the interaction of lymphocyte with receptors on membrane of mammary tumor cells with the production of cytokines ^{21, 46} . The distribution of CD4 ⁺ and CD8 ⁺ T cells within blood vessels and mammary tumors of IT group revealed that non-contact electric fields of ECCT may direct the spreading of these lymphocytes from blood vessels to tumor areas ^{11, 19, 21} , including necrotic areas in it ( Figure 7d and Figure 7f). The presence of CD4 ⁺ T cells in mammary tumors would induce the activation of CD8 ⁺ T cells to kill the tumors ^{12, 63, 64} as an adaptive immune response ^{12, 13, 65} . However, since the percentage of CD4 ⁺ T cells in INT and IT groups was not significantly different ( Figure 8b), the presence of CD4 ⁺ T cells in tumor areas may not always indicate tumor elimination, instead they may indicate tumor progression ^{12, 30, 66} . Moreover, electric field exposure can decrease CD4 ⁺ T cells polarisation ¹⁹ , resulted in the lower percentage of CD4 ⁺ T cells in IT group compared to INT group ( Figure 8b). Conversely, the presence of CD8 ⁺ T cells in the necrotic areas which characterized by inflammation ^{33, 34} , indicated their role in the inflammatory response to clear dead cells ^{12, 67} recognized as foreign agents ¹³ . In addition, a higher percentage of CD8 ⁺ T cells in IT group ( Figure 8c) indicated increased anti-tumor immunity and immunological ability against tumor progression ^{66, 68, 69} . CD8 ⁺ T cells may release granzymes during granule exocytosis when tumor cells are marked for elimination ^{12, 13, 70} , and the granzymes may induce caspase-independent apoptotic pathways ^{71, 72} which support the presence of hollow cavities in the tumor areas with low caspase-3 expression ⁴⁹ as seen in the IT group ( Figure 5f and Figure 6d).

The ratio of CD4/CD8 is correlated to clinical aspects and could be used as prognostic factor for breast cancer, where higher CD4/CD8 ratio in INT group ( Figure 8d) was correlated to mammary tumor progression ⁷³ . Conversely, the higher positive area of CD8 ⁺ T cells and the lower ratio of CD4/CD8 in IT group suggested a good prognosis in rat breast cancer ^{12, 66, 73} with non-contact electric field exposure. Further study will be conducted to investigate the potency of non-contact electric fields exposure in wound healing upon breast cancer treatment by observing the presence of cytokines, as well as the migration of immune cells and fibroblast that regulate wound healing ^{20, 41, 42} by using tissue samples from the IT group. Based on the evidence of its efficacy and safety on preclinical studies, the 100 kHz non-contact electric field has been used in phase I clinical trials of ECCT for healthy volunteers.

In summary, low intensity (18 Vpp) and intermediate frequency (100 kHz) non-contact electric fields exposure showed inhibition of mammary tumor growth in rats by inducing CD8 ⁺ T cells that lead to tumor cells death supported by decreased CD4/CD8 ratio. It also showed inhibition of M2 macrophage polarization resulted in the negative tumor growth rate. The proposed therapy gives good prognosis in rats with mammary tumors and potentially helps in wound healing of the tumor aftermath during the treatment.