Fig 2: 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 3: Autocrine RTK signaling in BCa cell lines.(A) Left: Axl kinase activation (phospho-Axl/total-Axl protein, pAxl/tAxl), as determined by 2-site immunoassay, in serum-deprived J82 cells treated with Gas6 (5.7 nM, 1 h at 37°C) in the presence or absence of cabozantinib (cabo; 300 nM), relative to untreated cells. Maximum possible Axl kinase activation was measured by adding ATP during the immunoassay (20 micromolar, 20 min at 25°C) of lysates prepared from untreated J82 cells. Right top: Axl kinase activation (pAxl/tAxl) in serum-deprived J82 cells that had been transfected with GAS6 siRNA or scrambled (control) siRNA. In both panels, values are the mean +/- SD from triplicate samples; asterisks indicate significant difference from untreated control (p < 0.05). Right bottom: ethidium bromide visualization of PCR products specific for GAS6 mRNA (upper panel) vs 18S rRNA (lower panel) resolved by agarose gel electrophoresis. (B) Left: Akt kinase activation (pAkt/tAkt), as determined by 2-site immunoassay, in serum-deprived J82 cells treated with Gas6 (5.7 nM, 1 h at 37°C) in the presence or absence of cabozantinib (cabo; 300 nM), relative to untreated cells. Right: Akt kinase activation (pAkt/tAkt) in serum-deprived J82 cells that had been transfected with Gas6 siRNA or scrambled (control) siRNA. In both panels, values are the mean +/- SD from triplicate samples; asterisks indicate significant difference from untreated control (p < 0.05). (C) Left: Erk kinase activation (pErk/tErk), as determined by 2-site immunoassay, in serum-deprived J82 cells treated with Gas6 (5.7 nM, 1 h at 37°C) in the presence or absence of cabozantinib (cabo; 300 nM), relative to untreated cells. Right: Erk kinase activation (pErk/tErk) in serum-deprived J82 cells that had been transfected with GAS6 siRNA or scrambled (control) siRNA. In both panels, values are the mean +/- SD from triplicate samples; asterisks indicate significant difference from untreated control (p < 0.05). (D) Upper panels: Immunoblot (IB) of phospho-MST1R (pMST1R) and MST1R (left) or pCSF1R and CSF1R (right) in lysates from serum-deprived FL3 cells after transfection with siRNA directed against MST1 (siMST1, left) or CSF1 (siCSF1, right) or control siRNA (siCont). Lower panels: Ethidium bromide visualization of PCR products (PCR) specific for MST1 (left) or CSF1 (right) mRNA or 18S RNA resolved by agarose gel electrophoresis. (E) Left: Akt kinase activation (pAkt/tAkt) in serum-deprived FL3 cells treated with MST1 (5 nM) or CSF1 (10 nM) in the presence or absence of cabozantinib (cabo; 300 nM), relative to untreated cells. Right: Akt kinase activation (pAkt/tAkt) in FL3 cells that had been transfected with siRNA directed against CSF1 or MST1, or with control siRNA. In both panels, values are the mean +/- SD from triplicate samples; asterisks indicate significant difference from control (p < 0.05). (F) Left: Erk kinase activation (pErk/tErk) in serum-deprived J82 cells treated with MST1 or CSF1 in the presence or absence of cabozantinib (cabo), relative to untreated cells. Right: Erk kinase activation (pErk/tErk) in FL3 cells that had been transfected with siRNA directed against CSF1 or MST1, or with control siRNA. In both panels, 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.

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).