Development and characterization of HBOW cell lines.

A, Schematic depicting isolation and purification process of HBOW cell lines. B, Confocal imaging of HCR cell lines in 2D culture (image shows overlay of fluorescence and DIC). IHC staining for N-terminus and C-terminus domains of HER2 receptor on HCR cell pellets. C, Cell proliferation in vitro as quantified by daily imaging and cell segmentation. Three independent experiments were averaged and fit with a logistic growth model. D, Population doubling time and E, Carrying capacity calculated from “C”. F, Schematic depicting orthotopic transplant of cells into NOD/SCID mice. G, Tumor-free survival of transplants by genotype (n=10 for each genotype). H, Fluorescent microscopy of isolated transplanted tumors. Whole mount tumor imaging (epifluorescent dissecting microscope) and tissue section imaging (confocal microscopy) are shown. IHC staining of N-terminus and C-terminus domains of HER2 on FFPE sections of transplant tumors. I, Linear curve fit of log2 transformed overall tumor volume. J, Tumor volume doubling time (TVDT) from “I”. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparisons test. ns, not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Error bars are represented as mean±s.e.m. Scale bars: 5000 μm (A, H fluorescence-whole tissue), 100μm (B, H fluorescence-section, N-terminus, C-terminus), (mTFP1:cyan:WT HER2, EYFP:yellow:d16 HER2, and mKO:magenta:p95 HER2)

Timelapse live imaging and single cell tracking for motility analysis of HER2 Crainbow cell lines.

A, Schematic of experimental conditions and cell tracking approach. B, Snapshots of a representative well seeded on tissue culture plastic at 10,000 initial cell density with single cell tracks overlaid. C, Region of interest from “B”. D, Diagram of spatial correlation analysis using a 50µm neighborhood. E, Principal component analysis (PCA) of motility and spatial crowding metrics across all conditions and colored by genotype. F&G, PCA of motility and spatial crowding metrics across all experiments colored by initial cell seeding density (F) and surface coating (G).

Comparing cellular morphology and invasive behaviors in HCR cell lines.

A, Representative images of HCR-WT, HCR-16, and HCR-95 cells plated for 24 hours on gelatin-coated coverslips, stained with phalloidin (grey) and DAPI (pseudocolor: HCR-WT– cyan, HCR-16 – yellow, HCR-95 – magenta). B, Quantification of cell area measurements on phalloidin-stained images (n= 81-82cells/condition). C, Quantification of total F-actin fiber length per cell on phalloidin-stained images (n=75 cells/condition). D, Example of invadopodia structures overlying areas of fluorescent matrix degradation in HCR-95 cells (matrix – grey, degradation puncta - black, cortactin – green, DAPI – magenta pseudocolor). Arrowheads indicate active invadopodia. E, Representative images of each cell line stained with cortactin (grey), F-actin (not shown to enable better visualization of puncta only) and DAPI (pseudocolor: HCR-WT– cyan, HCR-16 – yellow, HCR-95 – magenta) to identify F-actin+/ cortactin+ puncta indicating individual invadopodia structures (arrowheads) and rosette invadopodia superstructure in HCR-95 (arrow with tail). F, Quantification of F-actin+/cortactin+ invadopodia structures in HCR cells (n=73 40x fields of view (FOV) across 3 biological replicates, points are average invadopodia/nuclei/FOV). G, Representative image of invadopodia rosettes (arrow with tail) seen in HCR-95 cells by staining with phalloidin (grey), Cortactin (green), and DAPI (magenta). H, Quantification of rosette number per nuclei, from live imaging of LifeAct transfected HCR cell lines (n=19-20 20x FOV across 2 biological replicates) I, Representative images of matrix degradation by each HCR cell line, seen as black puncta in regions where fluorescent gelatin plate coating was enzymatically degraded by invadopodia of overlying cells, J, Areas of mechanical matrix disruption below stress fiber adhesion points in HCR-95 (matrix – grey, degradation puncta - black, cortactin – green, DAPI – magenta pseudocolor). K, Quantification of matrix disruption area per nuclei (including both enzymatic degradation and mechanical disruption), displayed as log2 fold change. Statistical significance was determined by one-way ANOVA with Kruskal-Wallis multiple comparisons test (B, C, H),Tukey’s multiple comparisons test (F), or Holm-Sidak multiple comparisons test (K). ns, not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Error bars are represented as mean±s.e.m. Scale bars: 100 μm (A), 50 μm (I), 10 μm (D, E, G, J).

Single cell analysis of HER2 Crainbow cell lines.

A, Single cell sequencing of HCR cell lines integrated and clustered by UMAP projection. B, Expression of epithelial and mesenchymal genes within UMAP clusters. C, Dot plot of genotype-specific markers in each cell line. D, Volcano plot of myogenic genes enriched in HCR-95 cells. E, Protein level expression of MRTFA and its gene targets in HCR cell lysates by western blot. F, Fractionation western blot distinguishing expression of MRTFA and fibrillarin in the nucleus and cytoplasm or HCR cell lines and G, quantification. Statistical significance was determined by one-way ANOVA with Holm-Sidak’s multiple comparisons test. ns, not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Error bars are represented as mean±s.e.m.

Short hairpin RNA knock-down of MRTFA leads to loss of invasive characteristics in HCR-95 cells.

A, Representative images of HCR cell lines stained with phalloidin (green), anti-MRTFA (grey), and DAPI (pseudocolor: HCR-WT– cyan, HCR-16 – yellow, HCR-95 – magenta). B, Western blots of protein level expression of MRTFA and downstream targets aSMA and TGFB1I1 in HCR-95-shCTRL and HCR-95-shMRTFA cell lines. C, Representative images of HCR-95-shCTRL and HCR-95-shMRTFA cell lines stained with phalloidin (grey) and DAPI (pseudocolor: magenta) showing morphology and cytoskeletal organization. D, Quantification of cell area in HCR-95-shCTRL and HCR-95-shMRTFA cell lines on phalloidin stained images (n=128 cells/condition). E, Quantification of total F-actin fiber length/cell in HCR-95-shCTRL and HCR-95-shMRTFA cell lines (n=95-106 cells/condition). F, Representative images of matrix disruption by each HCR-95-shCTRL and HCR-95-shMRTFA cells, seen as black regions where fluorescent gelatin plate coating was degraded or ripped. G, Quantification of matrix disruption area per nuclei, displayed as log2 fold change. Statistical significance was determined by one-way ANOVA with Kruskal-Wallis multiple comparisons test (D, E) or Holm-Sidak’s multiple comparisons test (G). ns, not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Error bars are represented as mean±s.e.m. Scale bars: 100 μm (A), 50 μm (E), 10 μm (H).

Knock-down of TGFB1I1 leads to loss of invasive characteristics in HCR-95 cells.

A, Representative images of HCR cell lines stained with TGFB1I1 (grey) and DAPI (pseudocolor: HCR-WT– cyan, HCR-16 – yellow, HCR-95 – magenta). B, Representative images of HCR-95 stained with Phalloidin (green), anti-TGFB1I1 (grey), and DAPI (pseudocolor: magenta) to show TGFB1I1 co-expression with F-actin fibers (arrows). C, Representative images of HCR-95 stained with Phalloidin (green), TGFB1I1 (grey), and DAPI (pseudocolor: magenta) to show TGFB1I1 co-expression with rosette (arrows). D, Western blot of TGFB1I1 expression in two HCR-95-shTGFB1I1 knock down cell lines (shTGFB1I1 #1 and shTGFB1I1#2) and the vector control cell line (HCR-95-shCTRL). E, Timelapse live imaging of rosette formation using Lifeact in HCR-95-shCTRL and HCR-95-shTGFB1I1#2 showing formation of larger, more abnormally shaped and persistent F actin rings/rosettes upon TGFB1I1 knockdown (Supplemental Videos 4 and 6) . F, Quantification of rosette number in HCR-95-shCTRL and HCR-95-shTGFB1I1#2 from LifeAct imaging (n=18-20 20xFOV from 2 biological replicates; average rosette number/nuclei). G, Quantification of rosette area in HCR-95-shCTRL and HCR-95-shTGFB1I1#2 from LifeAct imaging (n=20 FOV from 2 biological replicates; average rosette area/nuclei). H, Representative images of matrix disruption by HCR-95-shCTRL and HCR-95-shTGFB1I1#2 cells, seen as black puncta regions where fluorescent gelatin plate coating was degraded or ripped. I, Quantification of matrix disruption area per nuclei, displayed as log2 fold change. Statistical significance was determined by Mann-Whitney test (F, G, I). ns, not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Error bars are represented as mean±s.e.m. Scale bars: 50 μm (A, B, C, E, H).

Functional selectivity of p95 receptor signaling through Y1139 promotes MRTFA activation and invasion.

A, HEK cells co-transfected with either a control EYFP, EYFP-2a-d16 HER2, EYFP-2a-p95 HER2, EYFP-2a-p95Y1139F HER2, or EYFP-2a-p95Y1221/2 HER2 (magenta) and an mTagBFP2-MRTFA fusion (green). B, Log2 fold change of MRTFA nuclear expression (n=3 biological replicates for a total range of 222 – 291 cells analyzed). C, Western blots of HCR-95 and HCR-1139 protein level expression of MRTFA targets ACTA2 and TGFB1I1 D, Representative images of 3D spheroid formation in Matrigel matrix at 96 hours. E, Quantification of sphere circularity distributions of each cell line. Experiment was performed 3 independent times, and all organoids were aggregated and compared (n=3 biological replicates for a total of 523-1144 spheres analyzed). F, Schematic of transwell invasion assay. G, Quantification by z-position of invaded cells as percentage of total population. H, Schematic of orthotopic transplants of HCR-95 and HCR-1139 into NOD/SCID mice. I, Tumor-free survival of orthotopic transplants of NOD/SCID mice with HCR-95 or the p95-Y1139F mutant (HCR-1139) cells. J, Linear regression of log2 transformed tumor volume measurements of HCR-95 (n=9) and HCR-1139 (n=11) transplants. K, Tumor volume doubling time of HCR-95 and HCR-1139 transplants. L, Clarified lungs of HCR-95 transplanted mouse with representative metastasis (inset zoom). M, Quantification of metastasis in HCR-95 and HCR-1139 transplanted mice by percentage of population with at least one metastatic lesion and N, number of metastases per mouse per condition. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparisons test (B, G) or Mann-Whitney test (J, N), or Anderson-Darling test with Dunn’s multiple comparisons test with Bonferroni correction (E). ns, not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Error bars are represented as mean±s.e.m. Scale bars: 10 μm (A), 100 μm (D), 1000 μm (L).

Summary of functionally selective behaviors engaged by HER2 isoforms.

Principal component model representing key cell line phenotypes. Average values from six different assays were collected and scaled for PCA analysis: Proliferation = inverse tumor volume doubling time from in vivo transplantation, Self-correlation and Zero Neighbors = self-correlation and nearest neighbors kinematic motility analyses, MRTFA Expression = whole cell lysate western blot analysis, Invasion = Boyden chamber transwell invasion quantification, Degradation = fluorescent degradation quantification.