Journal: bioRxiv

Article Title: Ionic Regulation of Mechanosurveillance and Metastasis via the MRTFA/KCNMB1 Axis

doi: 10.64898/2026.01.13.699089

Figure Lengend Snippet: (A) Schematic demonstrating the functional differences in KCNMB1 WT and its gain-of-function germline variant KCNMB1 E65K . For the same amount of intracellular Ca 2+ , KCNMB1 E65K promotes a higher degree of K + efflux through the BK channels than the KCNMB1 WT . KCNMA1 is the pore forming subunit of BK channels. (B) Left: Confocal images of representative cells loaded with IPG-1 AM K + binding dye. Scale bar = 10 µm. Right: Graphs showing mean fluorescence intensity of intracellular K + bound dye. Each graph is representative of 3 independent experiments; AT-3: Control (pRetroX) = 82 cells, KCNMB1 WT = 101 cells, KCNMB1 E65K = 79 cells; E0771: Control (pRetroX) =63 cells, KCNMB1 WT = 96 cells, KCNMB1 E65K = 185 cells; Kruskal-Wallace with multiple comparisons. All error bars are mean±s.e.m. ns, not significant; * P <0.05; ** P <0.01; *** P <0.001. (C) Left: Confocal images of representative AT-3 and E0771 KCNMB1 WT and KCNMB1 E65K cells compared to controls. Cells are stained with phalloidin (F-actin). Scale bar = 10 µm. Right: Anisotropy measurements for corresponding cell lines. Each graph is a compilation of 3 independent experiments that are represented by different shades of colors. AT-3: Control (pRetroX) = 108 cells, KCNMB1 WT = 142 cells, KCNMB1 E65K = 95 cells; E0771: Control (pRetroX) = 181 cells, KCNMB1 WT = 176 cells, KCNMB1 E65K = 156 cells; Mann-Whitney test. All error bars are mean±s.e.m. ns, not significant; * P <0.05; ** P <0.01; *** P <0.001. (D) AFM stiffness measurements of AT-3 and E0771 KCNMB1 WT and KCNMB1 E65K cells compared to pRetrox controls. Results are from 3 combined experiments. AT-3: Control (pRetroX) = 14 cells, KCNMB1 WT = 13 cells, KCNMB1 E65K = 12 cells; E0771: Control (pRetroX) = 13 cells, KCNMB1 WT = 13 cells, KCNMB1 E65K = 13 cells; Mann-Whitney test. All error bars are mean±s.e.m. ns, not significant; * P <0.05; ** P <0.01; *** P <0.001. (E) Left: Confocal images of representative AT-3 and E0771 KCNMB1 knockdown cells compared to control. Cells are stained with phalloidin (F-actin). Scale bar=10 µm. Right: Anisotropy measurements for corresponding cell lines. Graphs are representative of 3 independent experiments. AT-3: Control (pLKO) = 45 cells, shKCNMB1 = 29 cells; E0771: Control (pLKO) = 68 cells, shKCNMB1 = 100 cells; Mann-Whitney test. All error bars are mean±s.e.m. ns, not significant; * P <0.05; ** P <0.01; *** P <0.001. (F) AFM stiffness measurements of AT-3 and E0771 KCNMB1 knockdown and AT-3 JPH2 knockdown cells compared to controls. AT-3 and E0771 graphs are compiled from 2 independent experiments. AT-3: Control (pLKO) = 20 cells, shKCNMB1 = 20 cells; E0771: Control (pLKO) = 17 cells, shKCNMB1 = 17 cells; AT-3: Control (pLKO) = 20 cells, shJPH2 = 20 cells; Mann-Whitney test. All error bars are mean±s.e.m. ns, not significant; *P<0.05; **P<0.01; ***P<0.001. (G) Left: Confocal images of representative pericytes with KCNMB1 knockdown compared to control. Cells are stained with phalloidin (F-actin). Scale bar = 10 µm. Right: Anisotropy measurements for corresponding cell lines. Results are a compilation of 3 independent experiments. Pericytes: Control (pLKO) = 59 cells, shKCNMB1 = 44 cells; Mann-Whitney test. All error bars are mean±s.e.m. ns, not significant; * P <0.05; ** P <0.01; *** P <0.001. (H) AFM stiffness measurements of representative pericytes with KCNMB1 knockdown compared to control. Results are from 3 combined experiments. Pericytes: pLKO = 14 cells, shKCNMB1 = 14 cells; Mann-Whitney test. All error bars are mean±s.e.m. ns, not significant; * P <0.05; ** P <0.01; *** P <0.001. (I) Model demonstrating how the role of KCNMB1 in cancer cells contrasts its role in primary pericytes that perform excitation-contraction coupling. KNCMB1 functions to stiffen cancer cells, while it functions to soften pericytes by shutting off voltage gated calcium channels (VGCC).

Article Snippet: Thus, we queried which cells of the tumor microenvironment expressed the molecular target of BMS-204352, KCNMA1 and its associated proteins KNCMB1 and JPH2 in cancer patients’ primary tumors and in healthy lung cells annotated in the Human Protein Atlas.

Techniques: Functional Assay, Variant Assay, Binding Assay, Fluorescence, Control, Staining, MANN-WHITNEY, Knockdown

Journal: bioRxiv

Article Title: Ionic Regulation of Mechanosurveillance and Metastasis via the MRTFA/KCNMB1 Axis

doi: 10.64898/2026.01.13.699089

Figure Lengend Snippet: (A) Association between high KCNMA1 (red), high KCNMB1 (purple) and high JPH2 (blue) mRNA expression and survival in breast cancer patients from all PAM50 subtypes (“a”) or Luminal A (“LA”), Luminal B (“LB”), Normal Like (“NL”), Basal (“B”), or Her2+ (“Her2”). Solid circles (“ ”), squares (“ ”), diamonds (“ ”) represent overall survival, distant metastasis free survival, and relapse free survival, respectively. Dark and light colors represent FDR ≤ 1% and FDR = 20%. (B) Examples of Kaplan-Meier plots for overall survival of breast cancer patients expressing high and low KCNMA1, KCNMB1, and JPH2 mRNA in the Kmplotter microarray datasets. Expression thresholds were set automatically, and false discovery rate is less than 1% for all inquiries. (C) Expression of listed genes in normal (non-cancerous), primary tumor and metastasis samples obtained from patients by using TNMplot tool. (D) Schematic of what KCNMB1 mediated cell stiffening could hypothetically regulate. (E) Representative images of wound healing assays to determine whether KCNMB1 knockdown differentially regulates migration and proliferation. Scale bars: 800 µM. Results are representative of 3 independent experiments. (F) Relative viability of control and KCNMB1 knockdown cells by using cell titer glow. Graph shows all three independent experiments performed in triplicate. (G) Schematic representing natural killer and cancer cell co-culture experiments and quantitation of time to cell death following 1:1 contact and immune synapse formation with a natural killer cell. Co-cultures were imaged for 18 hours, and cancer cell death was determined by propidium iodide (PI) accumulation and cellular rupture. Graphs represent data compiled from 2 independent experiments. Control (pLKO) = 30 cells, shKCNMB1 = 46 cells, shJPH2 = 21 cells; Mann-Whitney test. All error bars are mean±s.e.m.

Article Snippet: Thus, we queried which cells of the tumor microenvironment expressed the molecular target of BMS-204352, KCNMA1 and its associated proteins KNCMB1 and JPH2 in cancer patients’ primary tumors and in healthy lung cells annotated in the Human Protein Atlas.

Techniques: Expressing, Microarray, Knockdown, Migration, Control, Co-Culture Assay, Quantitation Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: Ionic Regulation of Mechanosurveillance and Metastasis via the MRTFA/KCNMB1 Axis

doi: 10.64898/2026.01.13.699089

Figure Lengend Snippet: (A) Orthotopic mammary fat pad tumor-take rates in mice injected with E0771 cells and treated with the indicated pharmacological agents. n= 9-10 female mice per treatment group (B) Orthotopic mammary fat pad tumor growth in mice listed in (A). Error bars, s.e.m. (C) Longitudinal analyses of metastatic burden in lungs of C57BL/6 mice over the course of 3 weeks by using bioluminescent imaging. N = 10 mice per treatment group. p/s: photons per second. P-values were derived from Mann-Whitney rank-sum test of two groups at a time. ns, not significant; * P <0.05; ** P <0.01; *** P <0.001. (D) Longitudinal analyses of metastatic burden in lungs of C57BL/6 mice pretreated with anti-GM1 antibodies to deplete NK cells. n = 10 female mice per group. p-value was derived from Mann-Whitney rank-sum test. (E) Longitudinal analyses of metastatic burden in lungs of athymic nude mice that lacks functional T-cells. n = 10 female mice per group. p-value was derived from Mann-Whitney rank-sum test. (F) UMAP representation of KCNMA1 expression in cells of the human lung accessed through the Human Protein Atlas database. Darker colors indicate higher degree of expression. Clusters c1-c26 correspond to the clusters c1-c26 in (G). (G) KCNMA1 expression in the clusters of cells represented in the UMAP as in (F). nTPM: transcripts per million. (H) Macrophage (F4/80) labeling of lungs from metastasis bearing mice that treated as in (C). Scale bar: 300µm. Images are representative of 8 mice in each group. (I) Macrophage polarization as judged by the surface expression of anti-tumor M1 marker iNOS and pro-tumor M2 marker CD206 in RAW 264 cell line. Representative of 2 independent experiments.

Article Snippet: Thus, we queried which cells of the tumor microenvironment expressed the molecular target of BMS-204352, KCNMA1 and its associated proteins KNCMB1 and JPH2 in cancer patients’ primary tumors and in healthy lung cells annotated in the Human Protein Atlas.

Techniques: Injection, Imaging, Derivative Assay, MANN-WHITNEY, Functional Assay, Expressing, Labeling, Marker

Journal: bioRxiv

Article Title: Ionic Regulation of Mechanosurveillance and Metastasis via the MRTFA/KCNMB1 Axis

doi: 10.64898/2026.01.13.699089

Figure Lengend Snippet: (A-J) Assessment of Left ventricular systolic function and mass 3 days and 21 days after tail-vein injection of mammary tumor cells in vehicle/IgG control treated and antiPD1 and BMS-204352 treated female mice. n = 5 mice per group M-mode images of parasternal long axis (LSAX) and short axis (SAX) were analyzed for stroke volume (A, F), ejection fraction (B, G), fractional shortening (C, H), cardiac output (D, I) and left ventricular mass (E, J). Data presented as mean ± standard error of the mean with two-way ANOVA with posthoc TukeyHSD analyses; ** - P value <0.001 *** - P value = 0.0001. (K) KCNMA1, KCNMB1 and JPH2 expression in the cells of the breast tumor microenvironment accessed from Broad Institute’s Single Cell Portal.

Article Snippet: Thus, we queried which cells of the tumor microenvironment expressed the molecular target of BMS-204352, KCNMA1 and its associated proteins KNCMB1 and JPH2 in cancer patients’ primary tumors and in healthy lung cells annotated in the Human Protein Atlas.

Techniques: Injection, Control, Expressing