Concurrent activation of voltage-gated sodium channels (VGSCs) and blockade of Na⁺ pumps causes a targeted osmotic lysis (TOL) of carcinomas that over-express the VGSCs. Unfortunately, electrical current bypasses tumors or tumor sections because of the variable resistance of the extracellular microenvironment. This study assesses pulsed magnetic fields (PMFs) as a potential source for activating VGSCs to initiate TOL in vitro and in vivo as PMFs are unaffected by nonconductive tissues. In vitro, PMFs (0–80 mT, 10 msec pulses, 15 pps for 10 min) combined with digoxin-lysed (500 nM) MDA-MB-231 breast cancer cells stimulus-dependently. Untreated, stimulation-only, and digoxin-only control cells did not lyse. MCF-10a normal breast cells were also unaffected. MDA-MB-231 cells did not lyse in a Na⁺-free buffer. In vivo, 30 min of PMF stimulation of MDA-MB-231 xenografts in J/Nu mice or 4T1 homografts in BALB/c mice, concurrently treated with 7 mg/kg digoxin reduced tumor size by 60–100%. Kidney, spleen, skin and muscle from these animals were unaffected. Stimulation-only and digoxin-only controls were similar to untreated tumors. BALB/C mice with 4T1 homografts survived significantly longer than mice in the three control groups. The data presented is evidence that the PMFs to activate VGSCs in TOL provide sufficient energy to lyse highly malignant cells in vitro and to reduce tumor growth of highly malignant grafts and improve host survival in vivo, thus supporting targeted osmotic lysis of cancer as a possible method for treating late-stage carcinomas without compromising noncancerous tissues.

Upregulation of voltage-gated sodium channels (VGSCs) and Na⁺/K⁺-ATPase (sodium pumps) is common across most malignant carcinomas. Targeted osmotic lysis (TOL) is a developing technology in which the concomitant stimulation of VGSCs and pharmacological blockade of sodium pumps causes rapid selective osmotic lysis of carcinoma cells. This treatment of cervical carcinoma is evidence that TOL is a safe, well-tolerated and effective treatment for aggressive advanced carcinomas that has the potential to extend life without compromising its quality. TOL is likely to have broad application for the treatment of advanced-stage carcinomas.

Background. Targeted osmotic lysis (TOL) is a novel technology that involves concomitant stimulation of voltage-gated sodium channels (VGSCs) and the pharmacological blockade of Na⁺, K⁺-ATPase causing lysis of highly malignant cancer cells. Hypothesis/Objectives. TOL offers an option for treating advanced carcinomas in companion animals. Animals. Two cats and 2 dogs that presented to veterinary hospitals for evaluation and treatment of one of several forms of carcinoma. Methods. Digoxin was administered to achieve steady-state, therapeutic concentrations. The animals were then exposed to pulsed electric field stimulation. Pre- and posttreatment assessments of tumor size and quality of life were compared. The treatment frequency and survivability varied, based on the patients premorbid functioning and response to treatment. Results. Regardless of cancer type, TOL consistently increased survival beyond expected, often improving, but without compromising of quality of life. Conclusions and Clinical Importance. TOL warrants consideration as an option for managing advanced carcinomas.

Sodium regulation is a potentially major driver of cancer metastasis. Voltage-gated sodium channels (VGSCs) and Na,K-ATPase are sodium transporters that are upregulated in many advanced carcinomas and are implicated as metastatic drivers. However, little is known about what drives this overexpression, how these proteins influence metastatic behavior, or whether these complementary sodium transporters are co-regulated in cancer. Using sodium transporter regulation in healthy neurons as a model, the present study demonstrated that the inflammatory mediator tumor necrosis factor alpha (TNFα) affects the expression of VGSCs and Na,K-ATPase in an in vitro model of metastatic breast cancer. Acute TNFα challenge increased RNA for sodium transporter subtypes by 20–100%, TNFα reduced the overall expression of VGSCs by 20–30% at all time-points examined, and long-term administration increased nuclear localization of the α1 subtype of Na,K-ATPase while increasing the overall expression of the α3 subtype. This study established that VGSCs and Na,K-ATPase are co-regulated by TNFα at the RNA level, and it was demonstrated that both TNFα and sodium transport-blocking drugs can significantly impact cellular metastasis-like behavior. Together these data are evidence that inflammation in metastatic breast cancer co-regulates the expression of VGSCs and Na,K-ATPase, and this regulatory system may contribute to carcinogenesis.

In vitro cancer research predominantly utilizes established cell lines of a chosen cancer type as phenotypic representations of various disease states, whether they be non-cancerous, non-invasive, highly metastatic, or derived from distinct cellular origins within an organ or tissue. While such methodology is useful to study cancer cells from diverse disease states, when one considers the tremendous complexity of intertumoral heterogeneity, it becomes problematic to use cell lines derived from distinct individuals with diverse malignancies to study the progression of tumorigenesis in a single cancer type. To address this need, a novel murine model was developed, composed of six cell lines isolated from progressively advanced tumors all derived from a single parental line (Ray et al., Exp. Cell Res. 340, 1-11). Each cell line should represent a snapshot of a particular stage of this cancer’s development, with the entire model providing a platform to study tumorigenic changes incurred as the transformed cells progressed from a localized tumor to an aggressively malignant growth. However, isolating primary cell lines can be an inefficient process, and a successfully isolated line may not accurately represent the dominant neoplastic cell type from a tumor. For that reason, it is necessary to characterize the cell lines from this model to confirm that the observed in vitro behavior of the cells matches the expected phenotype of the tissue from which it was derived. To this end, we performed an in vitro characterization of each cell line in the model. The three lines derived from distant metastatic tumors displayed significantly higher rates of two dimensional motility and invasive capacity as compared to the parental cell line, the line derived from a primary tumor or from a local metastasis. The distant metastatic lines also displayed elevated rates of extracellular acidification, an indication of increased rates of aerobic glycolysis. These measures demonstrated convincingly that the behavior of the cells in culture mirrored the expected phenotype of the tissue from which they were derived, thus validating the utility of the model. We then performed a comprehensive proteomic analysis via orbitrap tandem mass spectrometry, yielding expression data on over 3,000 proteins across all cell lines. This analysis yielded expected results that further validated the model, such as showing an increasing trend of expression of cancer-associated proteins like transcription factor AP-1 and decreasing trends of expression of tumor suppressors like protein NDRG1. However, it also revealed striking changes in protein expression in targets not currently implicated in tumorigenesis, thus providing novel targets for further examination. In total, this study validated a novel murine cell model as a valuable tool in the study of tumorigenesis, while providing a robust proteomic data set to guide future research into cancerous progression. Citation Format: Steven D. Scahill, Kelly Jean Sherman, Jessie Guidry, Harry J. Gould, Dennis Paul. In vitro characterization of a novel murine model of cancerous progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1684.