Ion channels are membrane proteins that create ionic concentration gradients by regulating the flow of ions across the plasma membrane. The primary function of ion channels is to maintain cellular homeostasis by regulating the inward and outward ion flux, but they are also higher order regulators of many downstream molecular signaling pathways (7). The four main ions that play a role in establishing the resting V_mem of a cell are: Ca²⁺, Na⁺, K⁺, and Cl^-. The Goldman equation links the overall transmembrane potential to the concentrations and permeabilities of various ion species. The resting potential V_mem depends on the internal and external K⁺, Na⁺, and Cl^- concentrations, ambient temperature, and permeability of each ion specie. Alterations in ion channel expression and activity are associated with the initiation, proliferation, and metastasis of cancer cells (21, 25). For instance, there is a host of ion channels whose expression is dysregulated in cancer cells and have been found to be associated with a metastatic phenotype (7). Here, we summarize major ion channels responsible for the disruption of homeostasis and aberrant activation of downstream signaling pathways in cancer including voltage-gated cation channels (Ca_V, Na_V, K_V), mechanosensitive cation channels, transient receptor potential (TRP) channels, and chloride channels (CLCs). Several review papers focusing extensively on ion channels implicated in cancer can be found in literature (26–29). It is worthwhile to note that disruption in expression of these ion channels leads to deregulation in a host of different signaling pathways in cancer (27–30). Prominent ones include the mitogen-activated protein kinase (MAPK) pathways, ERK and JNK signaling pathways, Wnt/ß-catenin pathway, PI3K/Akt pathway, Notch signaling, and the Rac and Rho pathways.

Resting potential established by ion channel and pump proteins is important for determination of differentiation state and proliferation. One way that carcinogenesis occurs is due to the disruption of electrical gradients, or the mechanisms by which they are perceived by cells (24). V_mem is an important non-genetic biophysical aspect of the microenvironment that regulates the balance between normally patterned growth and carcinogenesis (7). Cancerous and proliferative tissues are generally more positively charged or depolarized than non-proliferative cells (90, 91). V_mem values from -10 to -30 mV correspond to more undifferentiated, proliferative, and stem-like cells (92). For instance, the resting membrane potential in normal human mammary epithelial cells (HMEC) is -60 mV. This value goes up to -13 mV in breast cancer cells isolated from patients (93). Berzingi et al. experimentally compared V_mem in HMEC and two different invasive ductal human carcinoma cell lines, MCF7 (estrogen-receptor-positive) and MDA-MB-231 (estrogen-receptor-negative). The results indicated that MCF7 and MDA-MB-231 cells are 30.4 mV and 27.3 mV more depolarized in comparison to HMEC cells, respectively. It was also seen that HMEC grew at a much slower rate compared to MCF7 and MDA-MB-231 (32).

Lobikin et al. used a Xenopus tadpole model to confirm the role of ion flow in oncogenesis in vivo by investigating the consequences of depolarizing select cell groups (67). Embryos were exposed to glycine-gated chloride channel (GlyCl) activator ivermectin to control the membrane potential of a widely distributed, sparse population of cells expressing the GlyCl channel. The membrane potential of these specific cells could be set to any desired level by manipulating external chloride levels following ivermectin treatment. Tadpoles whose cells were depolarized were seen to exhibit excess melanocytes with a much more arborized appearance and colonize areas normally devoid of melanocytes, such as around the eyes and mouth. It was also shown that depolarization induces the up regulation of cancer relevant genes such as Sox10 and SLUG (94). Furthermore, susceptibility to oncogene-induced tumorigenesis was shown to be significantly reduced by forced prior expression of hyperpolarizing ion channels indicating that bioelectric signaling of the cellular microenvironment can both, induce and suppress, cancer-like cell behavior.

V_mem has been suggested as a cancer biomarker due to its role as an early indicator of tumorigenesis and is associated with tumors of diverse molecular origin (95–98). Induced tumor like structures (ITLS) can be formed in Xenopus and zebrafish embryos by mis-expressing mammalian oncogenes (Gli1, Xrel3 and KRASG12D) and mutant tumor suppressors (p53Trp248). ITLS’s are formed as a result of genetic interference with signaling pathways altered in several types of cancer including basal cell carcinoma, lung cancer, leukemia, melanoma, and rhabdomyosarcoma (99–102). Fluorescence reporters of V_mem in the injected animals have been found to reveal unique depolarization of tumors and increased sodium content compared to healthy tissues (7, 103). Moreover, depolarized foci have a higher success rate in predicting tumor formation as compared to cancer specific antigen level in the serum. For instance, Chernet and Levin found that depolarized foci, while present in only 19-30% of oncogene-injected embryos, predict tumor formation with 50-56% success rate as compared to prostate specific antigen level in the serum, which when used as a biomarker for prostate cancer, has a 29% predictive value (104, 105).

Recently, Carvalho developed a computational model of cancer initiation, including the propagation of a cell depolarization wave in the tissue under consideration (106). This model looks at an electrically connected single layer tissue in two and three dimensions and simulates ion exchange between cells as well as between cells and the extracellular environment. It was seen that a polarized tissue with cells in quiescent state tends to change state if a large enough perturbation changes its homeostatic conditions, such as a carcinogenic event. The induced depolarized state is able to then propagate to neighboring cells in a wave like manner. The developed model shows the importance of community effects associated with cell electrical communication leading to both, short- and long-range influences and ultimately, cancer.

The microenvironment functions to guide the cell through space and to direct tissue growth through time. It also plays a significant role in the physiological outcome of a given V_mem input. The tumor microenvironment (TME) is a complex entity and consists of multiple cell types embedded in the extracellular matrix (ECM), including immune cells, endothelial cells and cancer associated fibroblasts (CAFs) which communicate with cancer cells and with other CAFs during tumor progression (107). One way this communication is mediated is by a plethora of bioactive molecules, including proteins, lipids, coding and non-coding RNAs, and metabolites, which are secreted into extracellular vesicles (EVs) (108, 109).

The mechanical microenvironment impacts bioelectric regulation and cell proliferation (9). An early indication of this were studies in the late 1900s which found that cells within a low cell density (fewer cell-cell contacts) exhibited reduced proliferation (110) and that cells in a confluent monolayer are more hyperpolarized than individual cells (111, 112). Similarly, chemical components of the cellular microenvironment have the ability to impact cell phenotype. Factors such as hypoxia (113) and pH (114) have been demonstrated to drive cancer progression. Moreover, hypoxic tumors exhibit more aggressive phenotypes. Tumor cells under hypoxia can produce a secretion partly in the form of EVs that modulates the microenvironment to facilitate tumor angiogenesis and metastasis (115). V_mem thus functions at the interface of chemical and mechanical signals by creating an electrical gradient across cells, which in turn gates voltage-sensitive channels. This creates a tightly connected communication pathway between a cell and its microenvironment (9, 116–119).

The key components of the mechanical microenvironment ( Figure 2 ) are solid and fluid pressure, substratum stiffness (120–128) tissue geometry, and mechanical stress (129–131). These components of the physical microenvironment are primarily dependent on mechanosensitive calcium channels Ca_V3.3 (132, 133). Cells have the ability to sense the surrounding substratum by applying force through actomyosin motors in stress fibers linked to focal adhesions (134). Varying the substratum stiffness has been demonstrated to influence cellular behaviors including differentiation (122), apoptosis (126), proliferation (125), gene expression (135–137), migration (138), cell stiffness (139), and epithelial-mesenchymal transition (EMT) (127). Along with microenvironments of varying rigidity, cells also experience mechanical stress due to the dense packing of neighboring cells. Cell-cell contacts are critical for propagation of bioelectric signals via the transport of ions through gap junctions (140–142). The normal breast epithelium cell line MCF10A was demonstrated to respond differently to an EF in vitro depending on the confluency of the cell culture (143). The study observed that clustered cells are more sensitive to an EF due to increased cell-cell contacts.

Physical signals from the V_mem of the microenvironment also contribute to tumorigenesis (9). Furthermore, pressure activates oncogenic factors such as p38, ERK, and c-Src which are involved in the regulation of cell proliferation, differentiation, and apoptosis (132). Tumors in vivo are under higher pressure and are also stiffer than the surrounding tissue which creates a microenvironment that promotes cell proliferation (133). Increased pressure also enhances the invasiveness of tumor cells (121). Additionally, a key communication pathway between cells and their ECM is Integrin signaling pathway which regulates cytosolic Ca²⁺ levels (144). These cytosolic Ca²⁺ concentrations play an important role in cancer-related processes such as EMT (38), metastasis (21), and apoptosis (126, 145). For instance, inducing EMT in human breast cancer cells has been shown to upregulate cytosolic calcium levels (38).

The dissemination of primary tumor cells to secondary organs is called metastasis. This involves cancer cells breaking away from the primary tumor, traveling through blood or lymphatic systems, and forming secondary or metastatic tumors in other parts of the body. Metastasis is a multi-step process ( Figure 3 ) and involves the following events: local invasion to surrounding tissues, intravasation into the vasculature or lymphatics (where they are called circulating tumor cells or CTCs), survival and circulation in the vessels, and extravasation and colonization in a secondary organ (where they are called disseminated tumor cells or DTCs) (146, 147). Bioelectricity mediates many of the normal cell functions which are disrupted in metastasis. Factors such as ion channel expression, V_mem, and external EFs have been determined to regulate invasion and metastasis. Furthermore, the migration of cancer cells out of the primary tumors into local tissues through various physical barriers is driven by components of the local tumor microenvironment and executed by complex signaling pathways in the cell (10).

Cues within the TME can promote local invasion (148). For instance, an ECM protein fibronectin can attract breast cancer tumor cells to the vasculature via haptotaxis (directional migration in response to substrate-bound cues) to promote dissemination. During tumor invasion, constant communication occurs between tumor cells and surrounding stromal cells via extracellular vesicles (EVs) (115). Even upon entering a secondary tissue, the transition of a DTC into an overt metastasis is highly dependent on the local microenvironment of this organ (10). Hence, the formation of a supportive premetastatic niche, composed of ECM and resident immune cells is essential to provide nutrients and survival signals that drive DTC survival and outgrowth. Recent work suggests that tumor cells may be able to prime the premetastatic site from a distance before colonization to create a more favorable niche, for example, by secreting exosomes, a subpopulation of EVs (6). Furthermore, even within cancer cells, there is variability in the amount of depolarization. A more depolarized V_mem is associated with a higher metastatic potential and forced hyperpolarization of cells can reduce their migration and invasiveness (24, 141, 149, 150).

To study the effect of EFs on cell galvanotaxis, Zhu et al. employed unique probe systems to characterize cancer cell electrical properties and their migratory ability under an EF (151). It was found that tumors established from 4T1, a triple-negative murine breast cancer cell line, produced heterogeneous intratumor potentials causing a flow of endogenous EFs inside and outside of the tumors, which may in turn affect cell migration behavior and ultimately contribute to cancer metastasis. Moreover, tumor electric potentials were found to increase with increase in tumor size, which is an important factor since the primary tumor size has been reported to be linked to the metastatic potential (152, 153). Finally, it was also found that metastatic sublines (m4T1) from lung, heart, axillary lymph node and spleen showed different galvanotaxis thresholds. For instance, parental 4T1 and lung metastatic cell lines were found to respond to EFs as low as 50 mV/mm, while other metastatic sublines showed an anodal migration in a field of 100 mV/mm or higher. Additionally, the migration speeds also varied among different metastatic sublines. Cancer cell monolayers were found to have a higher migration persistence (defined as the ratio of displacement to trajectory length) under EFs than that of isolated cells, suggesting that cancer cells migrated more linearly in a certain direction when responding to EFs collectively.

Interestingly, bioelectric factors override most chemical gradients and other cues in a multi-cue environment during cell migration (8). Lobikin et al. investigated a cell population termed as “instructor” cells which when depolarized, is able to direct the activity of an entirely different set of cells (7). The “instructor” cells induce metastatic phenotype in normal melanocytes by serotonergic signaling, a mechanism which mediates long-range bioelectric signaling. Furthermore, instructor cells also disrupt blood vessel patterning upon depolarization. The melanocytes were then found to acquire three properties commonly associated with metastasis – hyper-proliferation, a highly dendritic morphology, and invasion into tissues such as blood vessels, gut, and neural tube. This data illustrated the power of depolarized V_mem as an epigenetic initiator of widespread metastatic behavior in the absence of a centralized tumor.

Molecular-resolution tools have recently been developed for real-time detection and manipulation of bioelectric properties in vivo (91, 154). An important component of such investigations is the ability to track spatio-temporal distribution of V_mem gradients in vivo, over significant periods of time.

Extracellular vesicles (EVs) facilitate inter-cellular communication via delivery of proteins and nucleic acids, including microRNA (miRNA) and mRNA (180). EVs-mediated communication is vital during the establishment of planar cell polarity and the developmental patterning of tissues (181). EVs are particularly enriched in the tumor microenvironment (182, 183) and as mentioned in the previous sections of this paper, they play a special role in cancer development and progression. In a recent study, Fukuta et al. demonstrated that external stimuli such as low levels of electric field treatment that activate intracellular signaling would likely increase exosome secretion from the cells. It was seen that an electric field of 0.34 mA/cm² increases the secretion of these EVs from cultured cells of murine melanoma B16F1 and murine fibroblast 3T3 Swiss Albino without compromising their quality (180). These results together indicate that the bioelectric dysregulation or depolarization of cells that occurs during cancer may be responsible for the upregulation of EVs in the cancer tumor microenvironment. At the same time, the increase in production of EVs plays a role in disrupting the bioelectric homeostasis, forming a feedback loop. The change in cell state and EV production along with the interdependence of the two are major mechanisms responsible for the bioelectric dysregulation of cancer.