Trailblazer cells in C3-TAg basal-like tumors are distinct from trailblazer cells in PyMT luminal B tumors.

A. There is intra- and inter-tumor invasive heterogeneity in C3-TAg organoids. Graphic shows the process for the derivation and analysis of tumor organoids. Representative images show the invasive heterogeneity of C3-TAg organoids derived from a single tumor. The area of invasion is determined using an image mask of the nuclei of invading cells. Violin plot shows the quantification of organoid invasion within 48 h of tumor isolation. (Mann Whitney test, n= organoids). Scale bars, 50 µm. B. C3-TAg tumors contain trailblazer cells that can lead the collective invasion of intrinsically less invasive cells. Graphic shows the process for generating homogeneous and heterogeneous clusters. Images show clusters immunostained with anti-SV40 antibody (detects TAg in all tumor cells), counterstained with Hoechst (nuclei) and H2B:mCherry fluorescence (B6 cells only). C3-TAg tumor cells (unlabeled) promoted the invasion of B6 cells (H2B:mCherry). Solid arrow indicates a C3-TAg tumor cell leading invasion. Dashed arrow indicates a B6 cell invading behind the C3-TAg cell. Graph shows the relative area of B6 cell invasion from 3 independent experiments (mean±SEM, Mann Whitney test, n=clusters). Scale bars, 20 µm. C. Krt14 expression does not correlate with C3-TAg tumor invasion. Krt14, SV40 (C3-TAg) and E-Cad (PyMT) expression in the tumor core and tumor-stromal interface of C3-TAg and PyMT tumors. Solid and dashed arrows indicate Krt14pos and Krt14neg cells respectively leading collective invasion. Violin plot shows the percentage of tumor cell area expressing Krt14. (Mann Whitney Test, n=ROIs from 5 tumors). Scale bars, 100 µm. D. Krt14 expression positively correlates with PyMT organoid invasion. Violin plot shows mean Krt14 expression in invasive and non-invasive PyMT organoids. (Mann Whitney Test, n=organoids from 3 tumors). Scale bars, 100 µm. E. Krt14 expression does not correlate with C3-TAg organoid invasion. Violin plot shows mean Krt14 expression in invasive and non-invasive C3-TAg organoids. (Mann Whitney Test, n=organoids from 3 tumors). Scale bars, 100 µm. F. Krt14 expression does not correlate with trailblazer phenotype in C3-TAg organoids. Solid and dashed arrows indicate Krt14neg and Krt14pos trailblazer cells respectively. Bar graph shows Krt14 expression status in trailblazer cells. (mean±SEM, unpaired Student’s t test, n=organoids from 3 tumors) Scale bars, 50 µm. G. C3-TAg organoids are more invasive than PyMT organoids. Bar graph shows the area of invasion (mean±SEM, Mann Whitney test, n=clusters, r=2) Scale bars, 50 µm. H. Model depicting the relationship between Krt14 expression and trailblazer phenotype in PyMT and C3-TAg tumors.

Trailblazer cells are distinguished by a subset of EMT traits in C3-TAg basal-like mammary tumors.

A. Graphic shows preparation of C3-TAg organoids and non-invasive clonal cell line spheroids for RNA-seq analysis. Heatmap shows unsupervised clustering and differentially expressed genes in non-invasive C3-TAg clones (B6, C6, E8) and invasive organoids from 4 C3-TAg tumors. B. Boxplots showing the expression of the basal genes Krt14 and Trp63. C. There was increased expression of Hallmark EMT genes in C3-TAg tumor organoids compared to non-invasive C3-TAg clones. Plot shows the details of the Hallmark EMT generated by GSEA. D-F. Boxplots showing the expression of canonical mesenchymal (Vimentin, Axl), epithelial (E-cadherin, EpCAM) and EMT-TF (Snai1, Slug, Twist1, Zeb1, Zeb2, Fra1) genes in C3-TAg tumor organoids compared to non-invasive clonal spheroids from RNA-seq data (Mann Whitney test for E-cad, Student’s t-test for all others, n=6 for clones, (3 biological replicates); n=8 for tumor (4 biological replicates). G. Vimentin is more highly expressed in invasive C3-TAg primary tumors compared to noninvasive C3-TAg tumors and PyMT tumors. Solid arrows indicate Vimpos tumor cells leading collective invasion. Dashed arrows indicate Vimneg tumor cells. Violin plot shows percentage of tumor cell area expressing Vimentin (Mann Whitney Test, n=ROIs from 5 tumors). Scale bars, 100 µm. H. Model showing the distinct features of trailblazer cells in PyMT and C3-TAg tumors.

Trailblazer cells are distinguished by a subset of EMT traits in basal-like subtype of breast cancer.

A. KRT14 mRNA is more highly expressed in the basal-like breast cancer patient tumors compared to other breast cancer subtypes. Z-scores relative to all samples are shown (Kruskal Wallis test with Dunn’s multiple comparisons test) From the TCGA. P-values are in comparison to the basal-like patient tumors. B. Krt14 expression in TNBC patient tumors does not correlate with invasion. Violin plot shows percentage of Krt14 expressing cells in regions of low (dashed arrows) and high invasion (solid arrows) (Mann Whitney test, n=tumor sections). Scale bars, 50 µm. C. VIM mRNA is more highly expressed in the basal-like breast cancer patient tumors compared to other breast cancer subtypes. Z-scores relative to all samples are shown (Kruskal Wallis test with Dunn’s multiple comparisons test) From the TCGA. P-values are in comparison to basal-like tumors. D. Invasive regions in TNBC patient tumors trend towards higher Vimentin expression (solid arrows) compared to regions of low invasion (dashed arrows). Violin plot shows percentage of Vimentin expressing tumor cells (Mann Whitney test, n=tumor sections). Scale bars, 50 µm. E. Vimentin is expressed in trailblazer cells in HCI-001 basal-like TNBC patient derived xenografts. Solid arrows indicate high Vimentin expression. Dashed arrows indicate low Vimentin expression. Violin plot shows percentage of tumor cell area expressing Vimentin (Mann Whitney Test, n=ROIs from 10 HCI-001 tumors). Scale bars, 100 µm. F. metastasis data. G. Model showing that Vimentin expression specifies trailblazer cells in basal-like C3-TAg tumors and TNBC patient tumors.

TGFβ induces a trailblazer state in C3-TAg tumors.

A. GSEA was performed on RNA-seq data from C3-TAg organoids and non-invasive C3-TAg clonal cell line spheroids to identify pathway enrichment in C3-TAg organoids. Plot shows the GSEA for the KEGG: TGFβ signaling pathway. B. The Tgfbr1 inhibitor A83-01 suppresses C3-TAg organoid trailblazer phenotype within 48 h of tumor isolation. Graph shows the quantification of invasion (mean±SEM, Mann Whitney test, n=organoids from 4 tumors). Scale bars, 50µm. C. 1339-orgs grown in A83-01 were less invasive than organoids tested immediately after isolation from primary tumors. Graph shows organoid circularity. Greater circularity indicates a reduced trailblazer phenotype (mean±SEM, Mann Whitney test, n=organoids). Scale bars, 50 µm. D. Tgfbr1 inhibitors suppress the autocrine and exogenous TGFβ1 induced trailblazer phenotypes in 1339-orgs. Organoids were treated with the Tgfbr1 inhibitors A83-01 (500 nM) or SB431542 (1 µM) and exogenous TGFβ1 (2 ng/ml) as indicated. Violin plots show the quantification of organoid circularity (Mann Whitney test, n=organoids, r=3). Scale bars, 50 µm. E. Persistent exposure to exogenous TGFβ1 progressively enhanced the invasive phenotype in C3-TAg organoid lines. Graphs show the quantification of organoid circularity and the number of invading single cells (Mann Whitney test, n=organoids, r=3) Scale bars, 50 µm. F. Graphic shows the process for generating new primary tumors from control 1339-org line cells (Control 1339-org tumors) and 1339-org cells treated with TGFβ1 for 21 days (+TGFβ1 1339-org tumors). The expression of Vimentin was similar in Control 1339-org tumors and +TGFβ1 1339-org tumors. Violin plot shows quantification of Vimentin expression (Mann Whitney Test, 4 tumors from each group). Scale bars, 100 µm. G. The trailblazer phenotype of control 1339-org line cells was enhanced after tumor growth and similar to +TGFβ1 1339-org tumor cells. Violin plot shows quantification of organoid circularity (Mann Whitney Test, n=organoids representative of 4 tumors from each group).

TGFβ1 induces multiple trailblazer states through distinct phases of re-programming.

A.. RNA-seq was performed on 1339-org cells treated for 2, 7 and 22 days with TGFβ1. Heatmap shows unsupervised clustering of control and TGFβ1 treated cells along with 4 groups of DEGs. B. Graphs show the top 5 Gene Ontology terms associated with each group. C. Heatmap shows changes in expression of mesenchymal (Vim), EMT-TF (Zeb1 and 2, Twist1, Snail, Slug), epithelial (E-Cad), basal (p63, Krt14) and luminal (Krt8) markers after TGFβ1 exposure for 0, 2, 7 and 22 days. D. Heatmap shows that persistent activation of Tgfβ signaling increaseds the expression of previously defined trailblazer genes. E. Graph shows the enrichment of Group 3 and Group 4 induced genes in non-invasive clones and invasive C3-TAg tumor organoids. F-G. Trailblazer cells in multiple different EMT states lead collective invasion in freshly isolated C3-TAg tumor organoids and TGFβ1 treated 1339-org line organoids. Sections from paraffin-embedded organoids were immunostained with Vimentin and E-cadherin antibodies to define different EMT states. The phenotype of trailblazer cells leading invasion or invading single cells was quantified. Bar graphs indicate the mean number of trailblazer or single invading cells per organoid and their EMT phenotype. H. Model showing the heterogeneity in trailblazer states induced by TGFβ1.

The transcription factors Fra1, Zeb1 and Zeb2 confer a trailblazer state.

A. Invasion of 1863T cells transfected with siRNAs targeting TFs associated with EMT and invasion. Graph shows the quantification of cells vertically invading ≥40 µm into the ECM (mean±SEM, Mann Whitney test, n=4 wells from 2 independent experiments). Scale bars, 50 µm. Smad3 siRNA serves as a positive control for suppression of invasion. Blue arrows indicate genes prioritized for further analysis. B. Zeb1 siRNA transfection suppressed the invasion of TGFβ1 treated 1863 clusters. Dot plot shows the quantification of invasion (mean±SEM, Mann Whitney test, n=clusters, r=2). Scale bars, 50 µm. Bar graph shows Zeb1 and Zeb 2 expression (mean±SEM, n=2). C. Transfection with a miR200c-3p mimic decreased TGFβ1 treated 1863 cluster invasion and Zeb1 and Zeb2 expression. Dot plot shows the quantification of invasion (mean±SEM, Mann Whitney test, n=clusters, r=2). Scale bars, 50 µm. Bar graph shows Zeb1 and Zeb 2 expression, (mean±SEM, n=2). D. Fra1 siRNA pool 1 transfection suppressed GFβ1 treated 1863 cluster invasion (mean±SEM, Mann Whitney test n=clusters, r=5) Scale bars, 50 µm. E. Fra1 depletion did not suppress Zeb1 and Zeb2 expression (mean±SEM, n=2). F-G. E-cadherin, Vimentin, Fra1, Zeb1 and Zeb2 mRNA expression after 48 h treatment with (F) Tgfbr1 inhibitor A8301 and (G) TGFβ1 (mean±SEM, n=2). I. Model showing the parallel regulation of the trailblazer state by the Tgfβ signaling and a second pathway that induces Fra1.

Egfr signaling regulates Fra1 expression and C3-TAg trailblazer state.

A. Heatmap shows the expression of the Egfr ligands Epigen (Epgn), Amphiregulin (Areg) and Heparin-binding epidermal growth factor (Hbegf) in C3-TAg organoids and non-invasive clones. B. The Mek1/2 inhibitor trametinib (10 nM) and Egfr inhibitor erlotinib (1 µM) reduce Fra1 expression in the 1339-org line (mean±SEM, n=2). C. Immunoblot shows the response of 1863 and 1339 cells to trametinib, erlotinib and A83-01 treatment (n=2). D. Erlotinib (1 µM) suppresses the TGFβ1 induced invasion the 1339-org line. Violin plot shows the quantification of organoid circularity (Mann Whitney test, n=organoids, r=3) Scale bars, 50 µm. E. Trametinib suppresses the TGFβ1 induced invasion of the 1339-org line in a dose dependent manner. Violin plot shows the quantification of organoid circularity (Mann Whitney test, n=organoids, r=3). Scale bars, 50 µm. F. A83-01 suppresses the EGF induced invasion in the 1339-org line. Violin plot shows the quantification of organoid circularity (Mann Whitney test, n=organoids, r=4) Scale bars, 50 µm. G. Trametinib suppresses the invasion of freshly derived C3-TAg organoids within 24 h of original tumor processing. Graph shows the quantification of invasion from 4 different tumors (mean±SEM, Mann Whitney test, n=organoids) Scale bars, 50 µm. H. Images show dually phosphorylated Erk1/2 immunostaining in regions of high invasion (solid arrows) and low invasion (dotted arrow) in primary C3-TAg tumors. Scale bars, 50 µm. I. Model showing the parallel regulation of trailblazer cells by signaling pathways coordinated by Tgfβ and Egfr.

A compromise between the Tgbβ and Egfr signaling programs confers the trailblazer state.

A. Overview of the approach to test the interactions between the Egfr and Tgfβ signaling pathways. B. Polar plot organizing genes represented as individual dots based on their change in expression in response to treatment with TGFβ1 or co-treatment with TGFβ1 and erlotinib. Genes that underwent ≥2 fold-change with a p< 0.05 after Benjamini-Hochberg correction in at least one condition relative to one of the other conditions are shown (3 total comparisons). The distance from the center of the plot represents the Δlog2 value of each gene (increased difference in expression, further from the center). The bar plots on the surface of the circle show the number of genes at the degree point in the plot. Genes are grouped into 12 color-coded groups along 30 degree increments over the 360-degree plot. As an example, genes in cluster 5 are increased in expression by TGFβ1 and this increase in expression is inhibited by Erlotinib. In contrast, genes in cluster 1 are increased in expression by TGFβ1, and this induction is further enhanced by treatment with Erlotinib. C. Summary of the number of genes and represented biological processes associated with different modes of interaction between the Tgfbr1 and Egfr pathways in cells treated with TGFβ1 or TGFβ1+erlotinib. Also see Figure 8—figure supplement 1C-D and Supplementary file 5 for the assignment of polar plot clusters and detailed biological processes used to define the summary integration groups. D. Egfr suppressed the expression of a subset of genes induced by extrinsic TGFβ1. Line plots indicate the expression of genes in Cluster 1. Dashed line indicates the average scaled expression of all Cluster 1 genes. Lower bar graph shows the top 10 biological processes (GO-BP) associated with Cluster 1 genes (Fisher’s exact test). E. Egfr was necessary for the expression of a subset of genes induced by extrinsic TGFβ. Line plots show the expression of genes in Cluster 5. Dashed line indicates the average scaled expression of all Cluster 5 genes. Lower bar graph shows the top 10 biological processes (GO-BP) associated with Cluster 5 genes (Fisher’s exact test). F. Graph of Axl expression (mean±SEM, n=2). G. The Axl inhibitor BGB324 (1 µM) suppressed freshly isolated C3-TAg tumor organoid invasion. Graph shows the quantification of invasion (mean±SEM, Mann Whitney test, n=organoids, r=2). H. Model showing how the induction of the trailblazer state involves a compromise between the Tgfβ and Egfr regulatory programs. The Egfr dependent induction of genes, such as Axl, that are required for trailblazer cell motility has the cost of restricting the expression ECM remodeling genes known to promote invasion and metastasis.

Model of inter- and intra-tumor trailblazer cell heterogeneity.

The characteristic features and functional requirements of the trailblazer cells induced by Tgfβ and Egfr signaling programs in C3-TAg/basal-like tumors are fundamentally distinct from the trailblazer cells in PyMT/luminal B tumors.

A. Representative masked images of C3-TAg organoids showing reduced organoid invasion which is represented by increased organoid circularity. Scale bars, 50 µm. B. C3-TAg organoids and dissociated cells from the same tumors were grown in ECM for 2 and 4 days respectively to evaluate invasion. Solid arrows indicate representative invasive cells. Dashed arrow indicates a representative non-invasive spheroid. Violin plot shows the quantification of circularity (Mann Whitney test, n=organoids, dissociated cell spheroids or individual dissociated cells, r=2). Scale bars, 50 µm. C. Graphic shows the methodology for deriving opportunist and trailblazer clonal cell lines. D. Representative images of non-invasive (G2) and invasive (C7) clonal cell line clusters. Scale bars, 100 µm (top), 20 µm (bottom, inset). E. Trailblazer D6-H2B:GFP cells promoted the collective invasion of non-invasive opportunist B6-H2B:mCherry cells. Top row, 2×2 tiled LED fluorescence images. Inset images are confocal sections of the ROIs indicated by the dashed line boxes. Graph shows the quantification of B6-H2B:mCherry cell invasion (mean±SEM, Mann Whitney test, n=clusters, r=2). Scale bars, top row 100 µm; inset images 20 µm. F. Immunoblots showing the expression of Krt14 and Krt8 in non-invasive (G2, B6) and invasive (D6, C7) C3-TAg clonal cell lines. Vinculin serves as a loading control. (n=2) G. Representative images from 2 independent experiments show that C3-TAg organoids are intrinsically more invasive than PyMT organoids in ECM composed of low Collagen I.

A. Non-invasive opportunist C3-TAg clonal cell lines were grown in ECM for at least 6 days. Violin plot shows the quantification of spheroid circularity (Mann Whitney test, n=spheroids, r=2). Scale bars, 50 µm. B. Immunoblots showing the expression of ΔNp63 in non-invasive (opportunist) (G2, B6) and invasive (trailblazer) (D6, C7) C3-TAg clonal cell lines (n=2). Lysates are from the same gel. C. Heatmap showing the expression of the indicated genes in human breast cancer cell lines. Trailblazer enriched populations are indicated in magenta. P=parental, T=trailblazer and O=opportunist when parental cell lines and their daughter trailblazer and opportunist subpopulations are shown (SUM159 and SUM149). D. GSEA was performed on RNA-seq data from C3-TAg organoids and non-invasive C3-TAg clones spheroids to identify pathway enrichment in C3-TAg organoids. Plots show the GSEA for the indicated KEGG pathways. E. Graph shows Hallmark pathways with NES ≥1. F. Overview images of noninvasive carcinoma in situ (CIS) and invasive carcinoma cells in C3-TAg tumors. Dashed boxes showing the ROIs from Figure 2G. Scale bars, 100 µm.

A. Full TNBC patient tumors images with dashed boxes showing the ROIs from Figure 3B. Scale bars, 200 µm. B. Full TNBC patient tumor images with dashed boxes showing the ROIs from Figure 3D. Scale bars, 200 µm.

A. 1863 cells derived in the presence of the Tgfbr1 inhibitor A83-01 are less invasive than 1863T cells, which were derived from the same tumor but without A83-01. Graph shows 1863 and 1863T cluster invasion (mean±SEM, Mann Whitney test, n=clusters, r=2). Scale bars, 50µm. B. Smad3 siRNA transfection suppresses TGFβ1 treated 1863 cluster invasion. Dot plot shows the quantification of invasion (mean±SEM, Mann Whitney test, n=clusters, r=2). Scale bars, 50 µm. Bar graph shows Smad3 expression (mean±SEM, n=2). C. Pooled and individual Smad3 siRNAs suppress 1863T vertical invasion (mean±SEM, unpaired Student’s t test, n=3). Scale bars, 50 µm.

A. Graphics show the process of deriving 1339-org, 1863-org and 1788-org lines and their relationship to 2D cell lines derived from the same tumors. B-C. Tgfbr1 inhibition suppresses autocrine and exogenous TGFβ1 induced invasion of C3-TAg (B) 1863-org and (C) 1788-org lines. Organoids were treated with the Tgfbr1 inhibitors A83-01 (500 nM), SB431542 (1 µM) or exogenous TGFβ1 (2 ng/ml) as indicated. Violin plot shows the quantification of organoid circularity (Mann Whitney test, n=organoids, r=2). Scale bars, 50 µm.

A. Graphic showing the process of CTC isolation from mice. Representative images show Krt8 expressing CTCs detected in mice with control 1339-org tumors and +TGFβ1 1339-org tumors. Violin plot shows percentage of total cells that were CTCs per field of view (Mann Whitney test, representative of blood collected from 7 mice per group, r=2). Scale bars, 100 µm; 50 µm (inset).

A. FACS showing that TGFβ1 treated 1339-org cells undergo a shift in the distribution of EpCAM expression (representative of 2 independent experiments). B. Images showing a progressive increase of Vimentin expression in TGFβ1 treated 1339-org cells. Arrows indicate representative Vimentin expressing cells leading collective invasion. Graph shows Vimentin expression in 1339-org organoids (Student’s t-test, n=organoids). Scale bars, 100 µm. C. Immunoblots showing the expression of Zeb1, E-cadherin and Vimentin in D6, 1863T and 1863 cell lines. Vinculin serves as a loading control (n=2). D. Immunoblots showing the expression of E-cadherin and Vimentin in non-invasive B6 and trailblazer D6 cell lines. Vinculin serves as a loading control. (n=2). E. Representative images of D6 and 1339 clusters. Scale bars 50 µm.

A. Zeb1 and Zeb2 siRNA transfection suppresses 1863T cluster invasion (mean±SEM, Mann Whitney test, n=clusters, r=3). Scale bars, 50 µm. B. Transfection with a miR200c-3p mimic decreases 1863T cluster invasion (mean±SEM, Mann Whitney test, n=clusters, r=2). Scale bars, 50 µm.

A. Fra1 depletion by a second distinct Fra1 siRNA pool (pool 2) decreases TGFβ1 treated 1863 cluster invasion. Dot plot shows the quantification of invasion (mean±SEM, Mann Whitney test, n=clusters, r=2). Bar graph shows the depletion of Fra1 expression by two different pools of Fra1 siRNA (mean±SEM, n=3 for pool 1 and n=2 for pool 2). Scale bars, 50 µm. B. 1863T vertical invasion is suppressed by each of the four individual sequences from Fra1 siRNA pool 1 (mean±SEM, Mann Whitney test, n= 9 wells, r=3) Scale bars, 50 µm. C. Fra1 siRNA pool 2 reduces the vertical invasion of 1863T cells (mean±SEM, Mann Whitney test, n=12 wells, r=4). Scale bars, 50 µm. D. Fra1 depletion suppresses the invasion of 1863T clusters (mean±SEM, Mann Whitney test, n=clusters, r=2). Scale bars, 50 µm. E. Fra1 depletion reduces the vertical invasion of D6 cells (mean±SEM, Mann Whitney test, n= 9 wells, r=3). Scale bars, 50 µm. F. Fra1 depletion reduces the invasion of TGFβ1 treated 1339 clusters (mean±SEM, Mann Whitney test, n=clusters, r=2). Scale bars, 50 µm.

A. Jun depletion suppresses TGFβ1 induced 1863 cluster invasion (mean±SEM, Mann Whitney test, n=clusters, r=2) Scale bars, 50 µm. B. Depletion of Jun expression by Jun siRNA transfection (mean±SEM, n=2). C. Pooled and individual Jun siRNAs suppress 1863T vertical invasion (mean±SEM, unpaired Student’s t test, n=3). Scale bars, 50 µm.

A. FOSL1 (FRA1) mRNA is more highly expressed in the basal-like breast cancer patient tumors compared to other breast cancer subtypes. By comparison, ZEB1 and ZEB2 mRNA is more highly expressed in other breast cancer cancer subtypes compared to basal-like breast cancer. Z-scores relative to all samples are shown (Kruskal Wallis test with Dunn’s multiple comparisons test). P-values are in comparison to basal-like tumors B. Correlation of VIM and FOSL1 (FRA1) expression in breast cancer patient tumors.

A-B. GSEA was performed on RNA-seq data from C3-TAg organoids and non-invasive C3-TAg clone spheroids to identify pathway enrichment in C3-TAg organoids. Plots show the GSEA associated with Kras signaling. C. HBEGF mRNA is more highly expressed in the basal-like breast cancer patient tumors compared to other breast cancer subtypes. Z-scores relative to all samples are shown (Kruskal Wallis test with Dunn’s multiple comparisons test). P-values are in comparison to the basal-like tumors. D. Correlation of FOSL1 (FRA1) and HBEGF expression in breast cancer patient tumors.

A. Trametinib (10 nM) and erlotinib (1 µM) reduce Fra1 expression in 1863 cells (mean±SEM, n=2). B. Immunoblot showing that Jun expression is not perturbed by treatment with trametinib (10 nM), erlotinib (1 µM) or A83-01 (500 nM) in 1863 and 1339 cells (n=2 for erlotinib, A8301; n=3 for trametinib). C. Immunoblot shows that trametinib (10 nM) does not reduce Zeb1 expression or Smad2 phosphorylation in C3-TAg cells (n=2). D. 1339-org organoids were persistently stimulated with exogenous TGFβ1 for 14 days to induce a trailblazer state (D14 TGFβ1 1339-org). The ability of trametinib and A83-01 to suppress then invasion of the D14 TGFβ1 1339-org cells was then determined. Violin plots show the quantification of invasion (Mann Whitney test, n=organoids, r=2). Scale bars, 100 µm. E. Trametinib and A83-01 suppress the invasion of D6 trailblazer cells (Mann Whitney test, n=spheroids, r=2). F-G. The Tgfbr1 inhibitor A83-01 suppresses the EGF induced invasion of (F) 1863-org and (G) 1788-org organoids. Violin plot shows the quantification of organoid circularity (Mann Whitney test, n=organoids, r=2). Scale bars, 50 µm.

A. Graphic and data shows how a MCF10A-trailblazer cell line was established be treating MCF10A cells with 10% FBS. MCF10A cells were supplemented with 10% FBS for 8 days in monolayer culture. The invasive ability of control and 10% FBS treated MCF10A cells was then determined. The 10% FBS induced a trailblazer state in the MCF10A cells, which we term “MCF10A-Trailblazer”. Violin plot shows quantification of circularity. (Mann Whitney test, n=spheroids, r=2). Scale bars, 100 µm. B. A83-01, trametinib and erlotinib suppresses MCF10A-trailblazer invasion (Mann Whitney test, n=cspheroids, r=2) Scale bars, 100 µm. C. A83-01, trametinib and erlotinib suppress the invasion of MCF10A-trailblazer cells treated with exogenous TGFβ1 (Mann Whitney test, n=spheroids, r=2) Scale bars, 100 µm.

A. A83-01, trametinib and erlotinib suppress MCFDCIS spheroid invasion (Mann Whitney test, n=spheroids, r=2) Scale bars, 100 µm. B. A83-01, trametinib and erlotinib suppress the TGFβ1 induced invasion of MCFDCIS spheroids (Mann Whitney test, n=spheroids, r=2) Scale bars, 100 µm.

A. Overview of the approach to test the interactions between the Egfr and Tgfβ signaling pathways when cells were treated with EGF or EGF+A83-01. B. Polar plot organizing genes represented as individual dots based on their change in expression in response to treatment with EGF or co-treatment with EGF and A83-01. Genes that underwent ≥2 fold-change with a p< 0.05 after Benjamini-Hochberg correction in at least one condition relative to one of the other conditions are shown (3 total comparisons). The distance from the center of the plot represents the Δlog2 value of each gene (increased difference in expression, further from the center). The bar plots on the surface of the circle show the number of genes at the degree point in the plot. Genes are grouped into 12 color-coded groups along 30 degree increments over the 360 degree plot. As an example, genes in group 5 were increased in expression by EGF and this increase in expression was inhibited by A83-01. In contrast, genes in cluster 1 were increased in expression by EGF, and this induction is further enhanced by treatment with A83-01. C. Plot shows the cluster locations along the 30 degree increments in the polar plots shown in Figure 8B and Figure 8—figure supplement 1B. D. Clusters assigned to different summary groups for TGFβ1 and TGFβ1+erlotinib treated organoids shown in Figure 8B. E. Clusters assigned to different summary groups for EGF and EGF+A83-01 treated organoids shown in Figure 8—figure supplement 1B. F. Summary of the number of genes and represented biological processes associated with different modes of interaction between the Tgfbr1 and Egfr pathways in cells treated with EGF and EGF+A83-01. Also see Supplementary file 5 for the assignment of polar plot clusters and detailed biological processes used to define the summary integration groups. G. Egfr restricta the expression of a subset of genes induced by autocrine Tgfβ. Line plots show the expression of genes in Cluster 1. Dashed line indicates the average scaled expression of all Cluster 1 genes. Graph shows the top 10 biological processes (GO-BP) associated with Cluster 1 genes (Fisher’s exact test). H. Tgfbr1 activity was necessary for the expression of a subset of genes induced by extrinsic EGF. Line plots show the expression of genes in Cluster 5. Dashed line indicates the average scaled expression of all Cluster 5 genes. Graph shows the top 10 biological processes (GO-BP) associated with Cluster 5 genes (Fisher’s exact test).