Induced Electric Fields Inhibit Breast Cancer Growth and Metastasis and Modulate the Immune Tumor Microenvironment

Abstract

Introduction

Metastatic triple-negative breast cancer (TNBC) remains highly challenging to treat despite advances in oncology. This study investigated whether non-invasive alternating, low-intensity induced electric fields (iEFs) could suppress tumor growth and metastasis while modulating anti-tumor immunity in TNBC.

Methods

Orthotopic TNBC mouse models were treated with alternating (100 kHz) and low-intensity (2.7 mV/cm peak), induced electric fields (iEFs), which were delivered non-invasively via a solenoid coil system. Tumor growth and lung metastasis were assessed following treatment. Immune profiling was performed to evaluate changes in T cell states and myeloid cell populations within both primary tumors and metastatic lung tissue.

Results

iEF treatment significantly reduced primary tumor growth (n=9) and lung metastases (n=5). Within the tumor microenvironment, iEFs decreased infiltration of immunosuppressive myeloid cells (n=8). In the lung metastatic niche, iEF therapy increased CD8+ T cell abundance while reducing immunosuppressive myeloid populations (n=8). We also observed that iEFs reduced the metastatic potential of cancer cells by inhibiting epithelial-to-mesenchymal transition (n=3).

Conclusion

Induced electric field therapy enhances anti-tumor immunity and suppresses metastatic progression in TNBC by enhancing T cell activity and remodeling immunosuppressive myeloid environments. These findings support iEFs as a promising non-invasive immunomodulatory strategy for metastatic TNBC.

Introduction

Triple-negative breast cancer (TNBC) represents 10–15% of all breast cancers reported¹ and is the most challenging to treat due to its aggressive phenotype and lack of clinically validated molecular targets.² Current treatments are limited and often ineffective, highlighting the need for new therapies. As breast cancer progresses, the tumor microenvironment (TME) becomes increasingly immunosuppressive,³ by the recruitment of immunosuppressive myeloid cells like myeloid-derived suppressor cells (MDSCs) and T regulatory cells (Tregs).^4,5 This type of TME is referred to as a “cold” immune landscape, characterized by an abundance of immunosuppressive cells and poor infiltration of CD8⁺ T cells.^6–9 Targeting these immunosuppressive elements may improve immunotherapy outcomes in metastatic breast cancer, including TNBC.

Low intensity induced electric fields (iEFs) produced by exogenously applied temporally varying magnetic fields offer great promise in treating cancer in a non-invasive, non-contact, and non-pharmacological manner.^10–12 Induced electric field therapy falls in the general class of non-pharmacological bioelectric cancer therapies, among which are Tumor Treating Fields (TTFs), which have been FDA approved for glioblastoma multiforme, lung cancer, and are in extensive trials for pancreatic cancer.^13–15 However, unlike iEF therapy, TTFs require physical contact with patients’ skin and have higher voltages and power requirements with accompanying electrical currents injected into the skin and tissue.¹⁶ In contrast to existing bioelectric, non-pharmacological therapies, iEF therapy utilizes low-intensity (~µT), rapidly varying (100,000 times per second) magnetic induction (B) fields produced by current-carrying insulated wires to induce electric fields by Faraday’s Law.^10–12 Since iEFs are at least 1000 times less intense than TTFs (mV/cm for iEF versus V/cm for TTFs),^10–12 and there is no electrical current injected into the skin, there is no heating, and devices can be made compact, non-contact, and non-invasive.

It has been previously shown that iEFs act selectively on human TNBC cell lines (MDA-MB-231 and MCF10CA1a) compared to normal epithelial breast cells (MCF10A).^10–12 These selective effects of iEF treatment include hindering cancer cell migration in the presence of chemokines and growth factors,^10–12 rendering their actin cytoskeleton diffuse and unable to form filopodia, and improving the efficacy of Akt inhibitor MK2206 in hindering their migration.¹⁰ iEF treatment also significantly reduced the activity of succinate dehydrogenase in the mitochondria of cancer cells, thereby interfering with their ability to produce ATP via oxidative phosphorylation.¹¹ While iEFs have demonstrated direct and selective tumor suppressive signaling responses in cancer cells in vitro, their effects in the context of an in vivo TME with an intact immune system are unknown.

Here, we describe the first in vivo application of iEFs in a mouse model of TNBC. We adopted a previously described non-contact method of applying iEFs in vitro¹⁰ for in vivo studies by inserting a mouse cage inside a Plexiglas open-ended box with a single-turn coil wound around the box. We demonstrate that iEF treatment inhibits primary tumor growth and distant metastases to the lung in vivo. We further show that iEF treatment enhances the anti-tumor immune responses in TNBC by reducing the immunosuppressive TME while demonstrating no adverse effects in treated mice. These results suggest that iEF treatment is a novel intervention for shaping an effective immune response against TNBC.

Materials and Methods

Cell Culture

4T1 cells were purchased from ATCC. 4T1 cells were cultured in RPMI-1640 (ATCC) supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 1% penicillin/streptomycin (Lonza). Cell lines were routinely checked and were free of mycoplasma.

Animal Studies

All experiments were approved by the Institutional Animal Care and Use Committee of The Ohio State University (2007A0233-R5) and followed the Guide for the Care and Use of Laboratory Animals. Animals were housed according to University Laboratory Animal Resources guidelines. Six-week-old female BALB/c mice were purchased from Jackson Laboratory. Mice were injected with 4T1 cells (1x10⁵ cells in 100 µL of 1:1 PBS and Matrigel, Corning 354234) directly into the fat pad of the 4^th mammary gland. Tumor-bearing mice were allocated to either the iEF or the sham treatment (control) groups. To ensure experimental rigor, mice were allocated using stratified randomization based on initial tumor volumes, achieving a statistically equivalent baseline tumor burden between the control and treatment groups. For tumor growth and metastases studies, n = 9 mice were placed in either the sham or iEF-treated groups. For immune profiling studies, n = 8 mice were placed in either group. Treatment began once palpable tumors had formed and continued until reaching endpoints. Palpable tumors were measured weekly using calipers. Tumor volume was calculated using the formula V = 4π/3 x (D_L/2) x (D_S/2)^2, where D_L is the largest and D_S is the smallest superficial Feret diameter. After treatment, mice were euthanized by CO₂ asphyxiation followed by cervical dislocation, and primary tumors and lungs were harvested for further analysis.

Application of iEF

The iEF apparatus comprises an open-ended, transparent acrylic box, around which a single-layer coil is wound (Figure 1A and Supplementary Figure 1A). A 20 V_pp, sawtooth-shaped, 100 kHz waveform was applied to the coil using a function generator to produce an iEF throughout the interior of the mouse cage. In this configuration, all mice in the cage receive simultaneous, systemic treatment. Figure 1B shows the contours of calculated iEF in the y-z plane (long side of the box/cage) with magnitudes on the order of 1 mV/cm. In comparison, these iEF magnitudes are about 1–2 orders of magnitude larger than the maximum iEF reported in previous in vitro experiments,^10–12 and 3 orders of magnitude smaller than the ~1 V/cm EFs reported with tumor-treating fields (TTFs).^17,18 As a further benchmark, the static electric field at the surface of the Earth is on the order of 1 V/cm.¹⁹ The non-negligible components of the iEF inside the box are in the x and y directions, with the iEF relevant to the location of the mice being primarily in the x direction toward the center of the cage and comparable in magnitude in both x and y directions near the edges of the cage floor (Figure 1B). In the region of the cage occupied by mice, the calculated RMS values of the magnitude of iEF range from 0.67 mV/cm to 1.21 mV/cm with a spatial average value of 0.97 mV/cm (Supplementary Table 1). For in vivo experiments, once tumors were palpable, mice were transferred to sterile cages with a bottled water supply, and the entire cage was slid into the bore of the coil for treatment. The coil housing was constructed of ¼-thick acrylic with outer dimensions of 20×21.5x39.5 cm (height x width x length). The housing was wrapped with 32 AWG magnetic wire with a total of 62 equally spaced turns. The DC resistance of the coil was 28 Ω. The inductance of the coil was measured by an LCR meter (Keysight U1733C) to be 437 µH at 100 kHz. The applied fields were generated by applying a 100kHz, 20Vpp sawtooth waveform (Hewlett Packard 33120A), which generated a peak magnetic and electric field strength of 5.5 µT and 2.7 mV/cm, respectively, in the plane where the mice are free to move. It is important to note that due to the low current through the windings (< 36mA peak), there was no heating of the coils.

Figure 1.

iEF treatment of mice. (A) Schematic of experimental setup. A function generator powers the electromagnetic coil with the plastic mouse cage sitting in the bore of the coil. Mice are free to move around within the cage (image created using BioRender). (B) Contour plot of side- and front-view of the electric field generated by the coil. The black boundary represents the coil edges, while the red, dashed boundary represents the region the mice primarily move around. Black arrows in the front-view indicate the direction of the induced electric field during peak field strength.

Quantification of iEF

While the magnitude of the electric field component of iEF cannot be directly and non-intrusively measured in vivo, it can be calculated from Maxwell’s equations (see Supplementary Calculation 1). The intensity and distribution of the iEF was calculated using a custom MATLAB script. The vector potential of the coil was calculated at equally spaced points separated by 4 mm in the x and y (width and height of the coil) and 8 mm in the z dimension (axis of the coil). The vector potential is a function of the geometry of the coil and the coil’s winding current. The electric field component is then the partial derivative of this term with respect to time, while the magnetic field component is the curl. For reported field values, the maximum iEF intensity is that value in the plane where the mice are, and the root mean squared (RMS) is taken of the electric field over time to obtain a temporal average. The average, maximum, and minimum field strength in Supplementary Table 1 are with respect to the space the mice occupy.

The calculated iEF properties were validated experimentally by comparing calculated values of the magnetic induction and comparing with point-wise measurements taken from inside the box using a flux gate magnetic sensor (Magnetic Sciences, Model#MC162) (Supplementary Figure 1B). The variation of field strength throughout the cage can be seen in Figure 1B and Supplementary Figure 1C.

Flow Cytometry

Mouse tumor or lung samples were mechanically disrupted and subjected to digestion with collagenase-IV (1.5 mg/mL; Gibco) for 45 min at 37°C with intermittent vortexing. The digested tissues were subsequently passed through 70 μm filters and layered on a discontinuous Percoll gradient (Cytiva). Centrifugation was carried out at 1800 rpm for 20 minutes without braking, resulting in the collection of immune cells at the gradient interface. These isolated cells were stained at 4°C with Live/Dead Blue viability dye for 10 minutes (Invitrogen) to exclude the nonviable cells. The cells were stained for extracellular surface markers for 30 minutes. All intracellular staining was performed using the Foxp3 TF staining kit (Invitrogen) according to the manufacturer’s instructions. All samples were recorded on a Cytek Aurora high-dimensional flow cytometer. All fluorochrome-conjugated primary antibodies used for flow cytometry are provided in Supplementary Table 3. The gating strategy for isolating cell populations is shown in Supplementary Figure 2. The data was analyzed using FlowJo software. Flow cytometry data were analyzed by creating groups. The same gates were applied consistently across all samples to ensure unbiased and standardized quantification.

Histopathology

All lungs and tumors were harvested at the same endpoint from both control and iEF-treated mouse groups. For the quantification of lung metastases, entire lungs were collected, processed for histology, and Hematoxylin and Eosin (H&E)–stained. Lung sections were mounted on microscope slides and digitally scanned by the Comparative Pathology & Digital Imaging Shared Resource at OSU. Single sections spanning all lung lobes were examined for each mouse, ensuring that metastases throughout the lungs were captured. All sections were analyzed in a blinded manner such that the mouse and condition were censored during analysis. The entire slide containing all lobes was included in the quantification. Metastatic sites were counted manually by visual inspection, with a minimum diameter of 40 µm constituting a metastatic site.

Safety Study

For safety studies, n = 5 mice were placed in either sham or iEF treated cages. Mice were exposed to iEF treatment for 30 days. Blood was collected at the time of euthanasia via cardiac puncture. Blood was either collected into an EDTA coated vial for complete blood count (CBC) or a serum separator tube for serum chemistry. The CBC was automated using a Genesis Veterinary Hematology Analyzer from Oxford Science. Serum chemistry was automated using a Vet Axcel Chemistry Analyzer from Alfa Wasserman. Organs were also collected at the time of euthanasia and were processed for histology and Hematoxylin and Eosin (H&E) staining. Liver, lung, and spleen sections were mounted on a microscope and digitally scanned by the Comparative Pathology & Digital Imaging Shared Resource at OSU.

Immunoblotting

Immunoblotting was performed following the method as described.²⁰ Briefly, cell lysates were electrophoresed on NuPAGE 4–12% gradient precast gels (Invitrogen) and subsequently transferred onto nitrocellulose membranes with a pore size of 0.45 μm (BioRad). These membranes were then incubated with primary antibodies at specific dilutions (Supplementary Table 4). We used HRP-conjugated secondary antibodies, specifically goat anti-rabbit IgG for detection, and a chemiluminescent substrate (Millipore) was used for protein visualization. β-actin was used for loading controls for the blots.

Statistical Analysis

Due to the lack of prior data on induced electric field effects in vivo, a resource equation was employed to estimate the number of animals per group.²¹ For a degree of freedom between 10 and 20, we calculated a minimum and maximum number of animals per group to be 6 and 11 respectively. This informed our use of n = 8–9 mice per group for our tumor growth and immune panel experiments.

Statistical analyses were performed with Prism software (GraphPad Software Inc). The data for safety studies, tumor growth, metastases, and flow cytometry data are reported as median ± interquartile range. Significance was calculated using the Mann–Whitney U-test to assess the differences and associated p-values between control and iEF-treated groups. Both tumor growth and flow cytometry data were further corrected for multiple comparisons (tumor growth across time points and flow data across cell subtypes) with the Benjamini-Hochberg procedure at a level of 5% for false discovery rate. Statistical significance was noted in the figures as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and ns: non-significant.

Results

Induced Electric Fields (iEFs) are Safe and Show No Adverse Events in Mice

We first evaluated the safety profile of iEF treatment in mice. Immunocompetent and non-tumor-bearing BALB/c mice were exposed to either iEF or sham (unpowered) coils for 30 days. This treatment duration was chosen based on the expected lifetime of BALB/c mice orthotopically injected with 4T1 cells, where humane endpoints are commonly observed by ~30 days post‑inoculation. We did not observe any significant changes in body weight between the iEF-treated and control groups (Figure 2A). In addition, we did not observe any significant changes in behavior, such as hunching and aggressive behavior, in iEF-treated mice compared to the control.

Figure 2.

iEF treatment is well tolerated and safe in mice. Healthy BALB/c mice were continuously treated under iEF or in unpowered sham coils (control) for 30 days (n = 5). (A) Body weight was measured every seven days in control and iEF treated mice. (B) Representative H&E sections of liver, lung, and spleen from control or iEF treated mice. Organs were collected at endpoint. No abnormalities were observed from control or iEF treated mice. (C) Comparison of blood and serum markers for control and iEF treated mice. Blood and serum were extracted at endpoint via cardiac puncture and processed by automated counter and chemical analyzer. Shaded region is normally observed range in 8–10-week, female BALB/c mice.²² All plots show median and interquartile range. p values were calculated by the Mann–Whitney U-test with Benjamini-Hochberg procedure to correct for multiple comparisons. (ns, non-significant: p > 0.05).

We also analyzed the histopathological features of vital organs like the liver, lungs, and spleen, and observed no significant morphological and pathological changes in organs derived from iEF-treated mice compared to the control (Figure 2B). Furthermore, no major significant differences were observed in whole blood count and blood biochemistry in iEF-treated mice compared to the control (Figure 2C). These results demonstrate that iEFs (2.7 mV/cm peak) can be safely applied to healthy mice without any adverse effects.

iEFs Inhibit Tumor Growth and Lung Metastasis

To determine whether iEF treatment suppresses TNBC growth in vivo, we used a well-established 4T1 orthotopic mouse breast tumor model. 4T1 cells were injected into the mammary fat pads of BALB/c mice. These mice were divided into either control (unpowered coils) or iEF treatment once all mice developed palpable tumors. Tumor-bearing mice were placed in cages inside the iEF apparatus as described earlier (Figure 3A). Treatment continued until one mouse from either group met the predefined criteria for removal. To maintain a consistent study endpoint, all mice from both groups were euthanized on the same day. Tumor tissues and lung samples were harvested simultaneously from all animals. iEF-treated mice exhibited a significant reduction in both tumor volume and tumor weight compared with control mice (Figure 3B and C). Moreover, lung metastasis was significantly decreased in the iEF-treated group relative to controls (Figure 3D–E). Consistently, the maximum Feret diameter of lung metastatic lesions was also significantly reduced in iEF-treated mice compared with control animals (Figure 3F).

Figure 3.

iEF inhibits TNBC tumor growth and metastasis. (A) Graphical protocol for in vivo experiments. 4T1 cells were injected in the mammary fat pad of female 6- to 8-week-old BALB/c mice and placed in either untreated control (n=9) or iEF treated (n=9) groups. Treatment began once palpable tumors had formed (red arrow). Once the endpoint was reached, organs and primary tumor were either digested into a single cell suspension for flow cytometry or formalin fixed and sectioned for histology analysis. (B) Tumor volume was measured over the course of iEF treatment, and (C) tumor weights were measured at the end of the experiment (n=9). (D) Representative H&E-stained sections of mouse lungs harvested at the end of treatment. Black arrows indicate examples of metastases. Higher resolution images of metastatic sites are taken from the macroscopic views indicated by the dashed boxes. Scale bars are 5 mm, 1mm, and 100 µm for the left, middle, and right images, respectively. (E) Number of metastases identified in H&E sections of lungs (n=5). (F) Maximum Feret diameter of metastatic sites in H and E sections of lungs. Tumor volume is plotted as median and interquartile ranges. All box plots show median, interquartile range, and data limits. p values were calculated by the Mann–Whitney U-test. (** p < 0.01, **** p<0.0001).

The observations that iEF treatment reduced lung metastases prompted us to analyze the effect of iEF on epithelial-to-mesenchymal transition (EMT) in 4T1 cells in vivo. EMT plays a key role in breast cancer cell invasion and metastasis.²³ The hallmark of EMT is downregulation of epithelial markers such as E-cadherin and upregulation of mesenchymal markers such as N-cadherin.²⁴ Strikingly, we observed that iEF treatment significantly reduced the expression of N-cadherin and increased the expression of E-cadherin (Supplementary Figure 3). These results suggest that iEF treatment limits metastatic outgrowth in vivo by inhibiting EMT in breast cancer cells.

iEF Reduces Immunosuppressive Features of TNBC TME

The dynamic interplay between tumor and immune cells significantly influences the metastatic potential of tumor cells.^25,26 Importantly, endpoint-only immune analyses may fail to capture the dynamic and evolving nature of antitumor immunity, potentially missing early immunological events that shape disease progression and metastasis. At more advanced stages of tumor progression, many immune populations exhibit functional exhaustion and obscure early modulatory effects of iEF treatment. Therefore, we selected an earlier time point (Day 8) to evaluate tumor progression and metastatic dissemination. This approach enables a more accurate assessment of the direct impact of iEF on the immune TME. 4T1 tumor-bearing mice were treated with sham coils or iEF for 8 days following the development of palpable tumors (Supplementary Figure 4A). Primary tumors were harvested and analyzed using high-dimensional multispectral flow cytometry to evaluate the early immune cell alterations.

We observed that iEF treatment resulted in a significant reduction in CD39 expression on PD-1⁺TIM3⁺ CD8⁺ T cells within the tumor (Figure 4A). CD39 is an ectonucleotidase associated with terminal T cell exhaustion and immunosuppressive function.²⁷ CD8⁺ T cells play a crucial role in targeting and eliminating tumor cells, and reduced CD39 expression in this subset suggests a shift toward a less exhausted and potentially more functional CD8⁺ T cell phenotype, which may enhance anti-tumor immune responses.

Figure 4.

iEF treatment reduces immunosuppressive tumor microenvironment and promotes antitumor immunity in an orthotopic breast cancer model. 4T1 cells were injected in the mammary fat pad of female 6- to 8-week-old BALB/c mice and placed in either untreated control or iEF treated groups (n = 8 per group). After 8 days, all tumors were harvested and analyzed for the recruitment of various immune cells by flow cytometry. Representative flow cytometry plots for the tumors are illustrated. Flow cytometry results revealed changes in the levels of (A) CD45+CD3+CD8+TIM3+PD-1+CD39, (B) CD45+CD11c+CD24+MHCII+, (C) CD45+CD11b-CD103+ (DC1) and CD45+CD11b+CD103- (DC2), (D) CD45+CD11b+Ly6G+ (granulocytic). All box plots show median, interquartile range, and data limits. p values were calculated by the Mann–Whitney U-test with Benjamini-Hochberg procedure to correct for multiple comparisons. (* p < 0.05).

We also found that iEF treatment increased the abundance of dendritic cells (DCs) expressing high levels of major histocompatibility complex class II (MHCII) molecules (Figure 4B). Additionally, iEF treatment increased the enrichment of Type 1 DCs (DC1s) compared to the control group (Figure 4C). We further observed a reduction in Ly6G+ granulocytic myeloid cells in the iEF treatment group compared to the control group (Figure 4D). However, we did not observe any significant changes in Ly6C+ monocytic myeloid cells (Supplementary Figure 4B).

iEF Treatment Regulates the Immune Infiltrate at the Metastatic Site in the Lung

Immune remodeling in distant organ microenvironments plays a critical role in regulating lung metastasis. To investigate this, matched lung samples from 4T1 tumor-bearing mice treated with iEF for 8 days were analyzed using multi-spectral flow cytometry. We found that iEF-treated mice showed increased recruitment of CD8+ T cells at the metastatic site in the lungs (Figure 5A). The CD8+ T cells also showed high Ki-67 expression (Figure 5B), which suggests that the CD8+ T cells in the lung microenvironment are actively dividing and potentially exerting a more robust immune response.²⁸ However, we did not observe any significant changes in the frequency of TIM3 and PD-1 co-expressing exhausted CD8+ T cells (Supplementary Figure 5A). Moreover, iEF treatment caused an increased infiltration of B cells (CD19+) in the lung microenvironment (Figure 5C). Of note, these CD19+ B cells in the lung microenvironment show reduced PD-L1 (programmed death ligand 1) expression (Figure 5D). iEF treatment also decreased the enrichment of CD11b+Ly6G+ granulocytic myeloid cells in the lung microenvironment (Figure 5E). However, the Ly6C+ monocytic immune cells were unchanged (Supplementary Figure 5B).

Figure 5.

iEF regulates immune cell recruitment to the metastatic site in the lung. 4T1 cells were injected in the mammary fat pad of female 6- to 8-week-old BALB/c mice and placed in either untreated control (n=8) or iEF treated (n=8) groups. After 8 days, all lungs were harvested and analyzed for the recruitment of various immune cells by flow cytometry. Representative flow cytometry plots for the lungs are illustrated. Flow cytometry results revealed changes in the levels of (A) CD45+CD3+CD8+, (B) CD45+CD3+CD8+Ki-67+, (C) CD45+CD19+, (D) CD45+CD19+PD-L1+, (E) CD45+CD11b+Ly6G+ (granulocytic). All box plots show median, interquartile range, and data limits. p values were calculated by the Mann–Whitney U-test with Benjamini-Hochberg procedure to correct for multiple comparisons. (* p < 0.05, *** p < 0.001).

Discussion

In this study, we developed iEF technology as a non-invasive strategy that suppressed both primary tumor growth and metastatic to the lungs in a murine TNBC model. Our findings demonstrate that iEF treatment inhibits EMT and elicits robust anti-tumor immune responses not only within the primary tumor but also at the lung metastatic site, highlighting its systemic immunomodulatory potential. These effects are particularly relevant for TNBC, a subtype characterized by limited targeted treatment options and highly immunosuppressive TME. While initial results focus on TNBC, extending this modality to ER+ and HER2+ subtypes warrants further investigation, particularly whether iEFs could synergize with endocrine therapies or HER2-targeted agents by reducing immunosuppressive immune cells as observed in TNBC models. Importantly, iEF treatment was well tolerated in vivo, with no detectable adverse effects on body weight, hematologic parameters, or histopathological features of major visceral organs.

The complex interactions between cancer cells, immune cells, and their microenvironment are pivotal for mediating tumor progression and metastasis.²⁹ A key finding from our study is that iEFs enhanced host immune responses at both the primary mammary tumor and the metastatic lung sites (Figures 4 and 5). In mammary tumors, iEFs reduced the enrichment of immunosuppressive cells such as Ly6G+ granulocytic myeloid cells. Granulocytic myeloid cells contribute to enhancing the immunosuppressive microenvironment to support tumor progression.³⁰

In addition, iEF treatment is associated with a less exhausted CD8⁺ T cell phenotype, as evidenced by a reduced frequency of CD39-expressing PD-1⁺TIM3⁺ CD8⁺ T cells within the tumor. This subset has been previously characterized as a highly exhausted population.²⁷ Given the central role of CD8⁺ T cells in tumor cell clearance, attenuation of exhaustion may contribute to enhanced effector function and strengthened anti-tumor immunity. Notably, iEF treatment also expanded the DC1 compartment, which is essential for cross-presenting tumor antigens to CD8⁺ T cells and initiating potent cytotoxic immune responses.³¹ In parallel, increased MHC class II expression suggests improved antigen-presenting capacity toward CD4⁺ T cells, thereby facilitating coordinated adaptive immune activation. Collectively, the results indicate a shift toward a more immunostimulatory, anti-tumor microenvironment.

Beyond the primary breast tumor, iEF treatment elicited a strong immune response at lung metastatic sites. In the lungs, iEFs expanded the population of proliferating (Ki-67+) CD8+ T cells, indicating rapid local expansion and exerting a more robust immune response within the metastatic microenvironment.²⁸ CD8⁺ T cells have been shown to directly inhibit TNBC metastasis.²⁸ Consistently, a decline in the pro-tumor CD11b+Gr1+ granulocytes in the pulmonary microenvironment was also observed, signifying a shift towards an anti-tumorigenic milieu. Granulocytic myeloid cells facilitate TNBC metastasis by both inhibiting CD8⁺ T cell function and establishing a supportive pre-metastatic lung microenvironment.^28,32,33

Furthermore, iEF treatment reduced the expression of PD-L1 in B cells in the lung microenvironment. B cells are central to adaptive immunity.³⁴ PD-L1 is a ligand that binds to the PD-1 receptor on T cells, inhibiting T cell activation and proliferation.³⁵ PD-L1 is known to be an immune checkpoint molecule, and reduced expression suggests a potentially more favorable immune environment.³⁶ PD-L1-expressing B cells may directly interact with PD-1-expressing T cells and inhibit their function, thereby diminishing the anti-tumor response. This may contribute to immune tolerance within the lung microenvironment, allowing cancer cells to proliferate and evade immune surveillance. The enrichment of PD-L1+ B cells correlates with higher tumor grade in breast cancer³⁷ and is associated with impaired T cell activity at metastatic sites in melanoma patients.³⁸ However, the mechanisms driving PD-L1 upregulation in B cells remain poorly understood.

Beyond preclinical application of iEFs, one must consider the challenges of adapting the treatment for human patients. Translation of iEF treatment to therapeutic delivery in human patients is readily doable via wearable garments with embedded coils. As mentioned above, since the magnetic permeability of tissues and cells is very near that of a vacuum, the applied magnetic induction permeates everywhere through the body with negligible field attenuation. Although safety has now been demonstrated in mouse models as a result of this work, a similar demonstration of adverse-free effects in humans may be necessary. It is also possible that higher iEF intensities may be necessary when scaling up from mice to humans to reproduce the strong anti-tumor and anti-metastasis response observed in mice. Additionally, there is also a question as to the persistence of the effects after cessation of the iEF treatment. This aspect will be critically important to determine treatment schedules for patients in the clinic.

To our knowledge, this is the first study to demonstrate the in vivo anti-tumor potential of iEF treatment against TNBC. As this was a proof-of-principle study, we utilized a singular, peak electric field strength of 2.7 mV/cm, which is the maximum producible intensity with the custom iEF apparatus used. Since the safety studies revealed that this maximum intensity was free of any adverse effects or toxicity (see Induced Electric Fields (iEFs) are Safe and Show No Adverse Events in Mice), no dose-response studies were conducted, and the experiments reported here were performed with the maximum producible dose of iEF with our apparatus. Future studies are needed to elucidate the more detailed mechanisms underlying immune cell changes at both primary and metastatic sites. While our current work establishes in vivo efficacy and broad immune modulation, it remains unclear whether these effects result from a direct impact on immune cells or are mediated indirectly via cancer cell-derived signals. In vitro studies using diverse subsets of T cells, dendritic cells, and myeloid cells are warranted to better define the underlying mechanisms. Importantly, the emerging immunomodulatory properties of iEFs open new possibilities for their use in combination with immunotherapy or chemotherapy. Future studies are warranted for assessing these combinations to establish iEFs as a versatile and effective modality in precision immuno-oncology.

Conclusion

This study demonstrates that iEFs represent a safe and promising non-invasive therapeutic approach for TNBC. Across comprehensive in vivo assessments, iEF exposure showed no evidence of systemic toxicity, adverse behavioral changes, or organ pathology, supporting a favorable safety profile in healthy mice. Importantly, iEF treatment significantly suppressed primary tumor growth and reduced lung metastatic burden in the 4T1 orthotopic TNBC model. Mechanistically, these anti-tumor effects were associated with inhibition of EMT, suggesting a direct role in limiting tumor invasiveness. In parallel, iEFs reshaped the tumor immune microenvironment by reducing immunosuppressive and exhausted immune cell phenotypes, including CD39-expresing PD-1⁺TIM3⁺ CD8⁺ T cells and granulocytic myeloid populations, while enhancing DC activation and promoting a more pro-inflammatory, antigen-presenting state. Furthermore, iEF treatment exerted immune-modulatory effects at distant metastatic sites, where increased proliferation and recruitment of CD8⁺ T cells, along with reduced immunosuppressive myeloid cells, collectively contributed to a less permissive metastatic niche in the lung. Overall, these findings position iEFs as a novel physical modality against TNBC without detectable toxicity. While further studies are required to define optimal dosing, mechanistic specificity, and translational scalability in humans, this work provides strong preclinical evidence supporting iEFs as a potential adjunct or combination strategy in precision immuno-oncology, particularly for aggressive and immunologically cold tumors such as TNBC.

Funding Statement

This work was supported by an Idea Grant from Pelotonia and the Ohio State University Comprehensive Cancer Center (OSUCCC), an Accelerator Award supported by the Keenan Center for Entrepreneurship at Ohio State University and the Ohio Third Frontier Technology Validation and Start-up Fund, an NCI SBIR Phase I Grant (R43CA287836), and the Cancer Biology Research Program from of the OSUCCC (P30CA016058). This work was also supported in part by the Mary Wieczynski Furnivall Cancer Research Fund (JWS) and NIH R01CA292020 and DOD Breast Cancer Breakthrough Level 2 HT9425-25-1-0355 (RKG).

Abbreviations

TNBC, Triple Negative Breast Cancer; iEF, Induced Electric Field; TME, Tumor Microenvironment; DC, Dendritic Cell; MDSC, Myeloid-Derived Suppressor Cell; TTF, Tumor Treating Field; RMS, Root Mean Squared; CBC, Complete Blood Count; EDTA, Ethylenediaminetetraacetic Acid; HRP, Horseradish Peroxidase; EMT, Epithelial-to-Mesenchymal Transition; MHCII, Major Histocompatibility Complex Class II (MHCII).

Data Sharing Statement

Data available upon request from corresponding author Jonathan W Song.

Author Contributions

All authors made significant contributions to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

Ramesh K. Ganju, Vish V. Subramaniam, and Jonathan W. Song are co-founders of and shareholders in EMBioSys, Inc. EMBioSys is in commercial development of an induced electric field therapy system for the treatment of cancer. Vish V. Subramaniam is Chief Scientific Officer for EMBioSys and worked as a research collaborator and contributor to this manuscript and the research reported herein. EMBioSys has issued a sub-Award for further work building on the work reported here to the co-authors via a Phase 1 SBIR NCI funded project (1R43CA287836-01 and 5R43CA287836-02). Ramesh K. Ganju, Vish V. Subramaniam, and Jonathan W. Song reports grants, non-financial support from EMBioSys, outside the submitted work. Manish Charan, Travis H. Jones, Dinesh K. Ahirwar, Vish V. Subramaniam, Ramesh K. Ganju, and Jonathan W. Song all report a patent EP4408521A1 granted and a patent US20240399161A1 pending to Ohio State University. In addition, Travis H Jones, Vish V. Subramaniam, and Jonathan W. Song report patent US12306176B2 pending to Ohio State University. In addition, Dr Vish Subramaniam has a patent US20240115872A1 pending to Ohio State University.

The authors report no other conflicts of interest in this work.