Fig 2: Autocrine RTK driven proliferation and colony formation in BCa cell lines.(A) PCR products showing siRNA suppression (upper and middle panels in each group) vs. 18S rRNA loading controls (lower panel in each group) resolved by agarose gel electrophoresis and visualized using ethidium bromide staining, from cell lines used for results shown in panels B–F. Upper left: J82 cells transfected with siRNA directed against GAS6 or AXL used for results shown in panel B. Upper middle: FL3 cells transfected with siRNA directed against MST1 or MST1R used for results shown in panel C. Upper right: FL3 cells transfected with siRNA directed against CSF1 or CSF1R used for results shown in panel D. Lower left: J82 cells transfected with siRNA directed against AXL used for results shown in panel E. Lower right: FL3 cells transfected with siRNA directed against MST1R or CSF1R used for results shown in panel F. (B) Proliferation (abs at 570 nm, 3 d, 37°C) of J82 cells in 1% FBS previously transfected with siRNA directed against GAS6 (siGAS6), AXL (siAXL) or control siRNA (siControl) that were left untreated or treated with Gas6 (5.7 nM) in the absence or presence of cabozantinib (cabo, 300 nM). (C) Proliferation of FL3 cells in 1% FBS previously transfected with siRNA directed against MST1 (siMST1), MST1R (siMST1R) or control siRNA (siControl) that were left untreated or treated with MST1 (5 nM) in the absence or presence of cabozantinib (cabo, 300 nM). (D) Proliferation of FL3 cells in 1% FBS previously transfected with siRNA directed against CSF1 (siCSF1), CSF1R (siCSFR) or control siRNA (siControl) that were left untreated or treated with CSF1 (10 nM) in the absence or presence of cabozantinib (cabo, 300 nM). (E) Anchorage independent growth (abs at 570 nm) of J82 cells in 1% FBS previously transfected with siRNA directed against AXL (siAXL) or control siRNA (siControl) that were left untreated or treated with Gas6 (5.7 nM) in the absence or presence of cabozantinib (cabo, 300 nM). (F) Anchorage independent growth of FL3 cells in 1% FBS previously transfected with siRNA directed against MST1R (siMST1R), CSF1R (siCSFR) or control siRNA (siControl) that were left untreated or treated with MST1 (5 nM) or CSF1 (10 nM) in the absence or presence of cabozantinib (cabo, 300 nM). In panels C—F, values are the mean +/- SD from triplicate samples; asterisks indicate significant difference from untreated control (p < 0.05, ns, not significant). In all panels, data are representative of 3 or more independent experiments.

Fig 3: Potential autocrine RTK signaling in MIBC patient samples and BCa cell lines.(A) Heat map showing relative mRNA expression levels of ctRTK-cognate ligand pairs (RNA Seq V2) among 408 tumor samples from patients with MIBC [22] (left), and among 15 BCa-derived cell lines (absolute mRNA copy number, right). (B) Immunoblot detection of ctRTKs and their cognate ligands, CSF1/CSF1R, GAS6/AXL, MST1/MST1R, and evidence of DDR1, DDR2 and PTK7 proteins in 9 BCa-derived cell lines. GAPDH (bottom panel) serves as a loading control. (C) Left: heat map showing relative mRNA expression levels of 9 EMT-associated transcriptional activators (listed at right) among 408 tumor samples from patients with MIBC [22]. Samples are grouped on the horizontal axis by mRNA subtype classifications defined previously [22] indicated at the top (from left to right): neuronal (N, green); basal squamous (BS, red); luminal (L, gray); luminal infiltrating (LI, gold); luminal papillary (LP, purple), and clustered hierarchically on the vertical axis. Right: heat map showing absolute mRNA copy levels of 9 EMT-associated transcriptional activators among 15 BCa-derived cell lines (identified at bottom).

Fig 4: Serial passage of J82 cell xenografts in mice.(A) Tumor volume (mm3) of individual J82 (left) or MDXC1 (middle) cell xenografts in mice, or mean (+/- SD) tumor growth of both cell line xenografts (right), vs. days post implantation. (B) mRNA expression levels of AXL, TYRO3, MERTK, CSF1R, RET, MET, KDR, GAS6, KIT and MST1R in J82 cells and 8 J82 tumor-derived cell lines (MDXC1, MDXC2, MDXC31R, MDXC31L, MDXC33R, MDXC33L, MDXC34R and MDXC34L) as determined by qRT-PCR. (C) Immunoblot of lysates from J82 and MDXC1 lysates for phospho-MERTK (pMERTK, upper panels), MERTK (middle panels) or GAPDH (lower panels); intact cells had been treated prior to lysis with Gas6 (5.7 nM) or TP0903 (300 nM) as indicated. Results are representative of 3 or more independent experiments. (D) Light micrographs (10x objective magnification) of stained J82 (upper panels) of MDXC1 (lower panels) 24 h cell migration in the absence (control, left) or presence of added Gas6 (Gas6, 5.7 nM, right). (E) Heat map showing relative mRNA expression levels of 321 genes of the 770 gene Nanostring PanCancer Progression Panel (clustered hierarchically) that were significantly different among untreated J82 (left) and MDXC1 (right) cells (two-group comparison p = 0.021, q = 0.05; S5 Table in S1 File).

Fig 5: Autocrine RTK driven migration and proliferation in BCa cell lines.(A) Migration (16 h, 37°C) of serum deprived J82 cells previously transfected with siRNA directed against AXL (siAXL) or control siRNA (siControl), treated with Gas6 (5.7 nM) in the presence or absence of cabozantinib (cabo; 300 nM), relative to untreated cells. Values are the mean +/- SD from triplicate samples; asterisks indicate significant difference from control (p < 0.05; ns, not significant). All results are representative of at least 3 experiments. Inset: ethidium bromide visualization of PCR products specific for AXL mRNA (upper panel) or 18S rRNA (lower panel) resolved by agarose gel electrophoresis. (B) Migration (16 h, 37°C) of serum deprived FL3 cells previously transfected with siRNA directed against MST1R (siMST1R), CSF1R (siCSF1R) or control siRNA (siControl), treated with MST1 (5 nM) or CSF1 (10 nM) in the presence or absence of cabozantinib (cabo; 300 nM), relative to untreated cells. Values are the mean +/- SD from triplicate samples; asterisks indicate significant difference from untreated control (p < 0.05). All results are representative of at least 3 experiments. Inset: ethidium bromide visualization of PCR products specific for MST1R, (upper panel), CSF1R (middle panel), or 18S RNA (lower panel) resolved by agarose gel electrophoresis. (C) Proliferation (cell no. x 10-5, 6 d, 37°C) of 5637 (left) or J82 (right) cells in 1% FBS left untreated (control; circles) or treated with Gas6 alone (5.7 nM; squares), cabozantinib alone (300 nM, triangles) or Gas6 and cabozantinib (inverted triangles). Media with additives was replaced on days 1, 2, and 4; cells were counted on days 0, 3 and 6. Values are the mean +/- SD from triplicate samples; asterisks indicate significant difference from control (p < 0.05). (D) Proliferation (cell no. x 10-5, 6 d, 37°C) of FL3 cells in 1% FBS left untreated (control; circles) or treated with MST1 alone (5 nM; squares), cabozantinib alone (300 nM, triangles) or MST1 and cabozantinib (inverted triangles). Media replacement and cell counting as per panel C. Values are the mean +/- SD from triplicate samples; asterisks indicate significant difference from control (p < 0.05).