Fig 1.
Mesenchymal stem cells enhance metastasis of NSCLC cells.
PC9 and HCC827 cells were cultured with or without human bone-marrow derived mesenchymal stem cells (MSCs) (1:1 ratio) for 3 days. Equal numbers of cells were used for intracardiac injection (4x105 cells/mouse for PC9 and MSC-PC9; 1x105/mouse for HCC827 and MSC-HCC827). Both PC9 and HCC827 cells were labeled with luciferase for detection by bioluminescence imaging (BLI). (A) Mice harboring PC9 and MSC-PC9 cells were imaged at day 28 post injection. Representative images are shown. (B) BLI total flux photons/sec (p/s) counts were plotted (PC9 n = 6; MSC-PC9 n = 7). (C) HCC827 and MSC-HCC827 mice were imaged at day 31 post injection. Representative images are shown. (D) BLI total flux (p/s) counts were plotted (HCC827 n = 6; MSC-HCC827 n = 7). Statistical analysis was performed using Student’s unpaired two-tailed t test (* p<0.05).
Fig 2.
MSCs induce EMT in lung cancer cells.
PC9 cells were cultured with or without MSCs followed by FACS sorting to separate PC9 (tomato+) from MSC (violet+) cells. (A) RT-PCR was performed for expression of indicated epithelial-mesenchymal transition (EMT) mRNAs in PC9 cells from single cell culture or sorted following co-culture with MSCs. Statistical analysis was performed using unpaired two-tailed t test (***p<0.001). Error bars represent ± SEM (n = 3). (B) Total lysates of PC9 cells from single culture and FACS-sorted PC9 cells following co-culture with MSCs were analyzed for expression of EMT proteins using indicated antibodies. (C) HCC827, HCC4006 and H1650 cells were cultured with or without MSCs followed by sorting. Total cell lysates from single cell culture and sorted from co-culture with MSCs were analyzed for expression of EMT proteins as indicated. Each protein was normalized to tubulin and presented as fold change shown under the protein blots.
Fig 3.
MSCs enhance expression and secretion of MMP9 in lung cancer cells.
(A) PC9 cells were cultured with or without MSCs followed by FACS to separate sorted PC9 (S-PC9) and sorted MSC (S-MSC) cells from co-culture. RT-PCR analysis was performed for expression of indicated MMP mRNAs. Statistical analysis was performed using One-way ANOVA followed by Tukey’s multiple comparison post hoc testing (*p<0.05, **p<0.01, ***p<0.001, ns = not significant). Error bars represent ± SEM (n = 3). (B) PC9 or HCC827 lung cancer cells were cultured with or without MSCs for 3 days, after which media was changed to serum-free conditions for an additional 24h. Culture supernatant (SN) and total cell lysates were analyzed by western blotting with indicated antibodies. Supernatant MMP9 was normalized to total MMP9 in lysate and presented as fold change. (C) HCC4006 or H1650 lung cancer cells were cultured with or without MSCs and analyzed as in (B). (D) Gelatin-zymography assay measuring MMP9 activity of culture supernatants from MSC or PC9 single culture or MSC+PC9 co-culture conditions. MMP9 (93kd) and MMP2 (63kd) proteins are indicated. (E-F) Quantification of MMP9 (E) and MMP2 (F) activities by gelatin zymography assay. Statistical analysis was performed using One-way ANOVA followed by Tukey’s multiple comparison post hoc testing (***p<0.001; ns = not significant). Error bars represent ± SEM (n = 4).
Fig 4.
MSCs induce ABL activation in lung cancer cells which is required for MMP9 secretion and activity.
Immunoblot analysis of PC9 (A) or HCC827 (B) cells cultured with or without MSCs followed by FACS sorting. ABL kinase activity was measured by tyrosine phosphorylation of ABL (Y245), and phosphorylation of ABL downstream targets, STAT3 (Y705) and CrkL (Y207). Phosphorylated protein was normalized to its corresponding total protein and presented as fold change under phospho-protein blots. (C-D) PC9 (C) or HCC827 (D) cells were cultured with or without MSCs and in the presence or absence of ABL kinase inhibitor GNF5 (5 μM) or ABL001 (10 μM) for 48 or 72h. Culture supernatant (SN) was analyzed for MMP9 and Amphiregulin (AREG) proteins. Total cell lysates were analyzed for p-CrkL and p-STAT3 to confirm Abl kinase activity inhibition by GNF5 or ABL001. Supernatant MMP9 was normalized to total MMP9 in lysates and presented as fold change. Phosphorylated protein was normalized to its corresponding total protein and presented as fold change under phospho-protein blots. (E-F) Culture supernatants collected from PC9 cells cultured with or without MSCs in the presence or absence of the ABL kinase inhibitor GNF5 were analyzed for MMP9 and MMP2 activity on gelatin zymography assay. A representative zymographic band is shown (top), and quantification of corresponding proteins (bottom) was performed with Fiji software. Statistical analysis was performed using One-way ANOVA followed by Tukey’s multiple comparison post hoc testing. (***p<0.001; ns = not significant). Error bars represent ± SEM (n = 3).
Fig 5.
Depletion of ABL kinases reduces expression and secretion of MMP9.
PC9 cells were transduced with lentiviruses encoding either scramble control shRNA (SCR) or shRNAs specific for ABL1 and ABL2 (AA). Cells were then co-cultured with or without MSCs followed by FACS sorting to separate PC9-SCR or PC9-AA cells from MSCs. (A) RT-PCR analysis of PC9-SCR, PC9-AA, sorted PC9-SCR and sorted PC9-AA cells for MMP9 mRNA expression. Statistical analysis was performed using One-way ANOVA followed by Tukey’s multiple comparison post hoc testing (***p<0.001; *p<0.05). Error bars represent ± SEM (n = 4). (B) Culture supernatants (SN) from PC9-SCR or PC9-AA cells grown alone or with MSCs under co-culture conditions were analyzed by Western blotting for MMP9 protein and total cell lysates were blotted with indicated antibodies. MMP9 protein in supernatants was normalized to total MMP9 in lysates and presented as fold change. (C-D) Culture supernatants of PC9-SCR or PC9-AA with or without MSC co-culture were analyzed for MMP9 (C) and MMP2 (D) using gelatin-zymography. A representative zymographic band is shown (top) with corresponding band quantification (bottom). Statistical analysis was performed using One-way ANOVA followed by Tukey’s multiple comparison post hoc testing (***p<0.001, **p<0.01, ns = not significant). Error bars represent ± SEM (n = 3).
Fig 6.
High MMP9 expression correlates with poor survival in lung adenocarcinoma patients.
Analysis of lung adenocarcinoma patient microarray data for MMP9 expression was performed using the KM plot analysis tool (kmplot.com). Patient groups were divided into tertiles for high, medium and low MMP9 expression. (A) Overall survival (OS) (n = 719 total patients across all cohorts for OS) and (B) Progression-free survival (PFS) (n = 461 total patients across all cohorts for PFS) for lung adenocarcinoma patients. Statistical analysis was performed by Log-Rank Mantel-Cox testing.
Fig 7.
Depletion of ABL kinases in lung cancer cells abrogates MSC-induced increase of metastatic activity.
PC9 or HCC827 cells labeled with luciferase were transduced with viruses carrying either scramble control shRNA (SCR) or shRNAs specific for ABL1 and ABL2 (AA). Cells were then cultured with MSCs (1:1 ratio) for 3 days. Equal number of cells were intracardially-injected into nude mice (4x105/mouse for PC9 or 1x105 /mouse for HCC827). (A-B) Representative BLI images at day 21 post injection are shown (A) and BLI total flux (p/s) counts were plotted (B) (n = 9 for each group) for mice injected with MSC+PC9-SCR or MSC+PC9-AA cells. (C) Percent survival for mice injected with MSC+PC9-SCR vs. MSC+PC9-AA cells over 100 days. Survival statistical analysis was calculated using Log rank (Mantel Cox) test (n = 9 each group). (D-E) Representative BLI images at day 24 post injection are shown (D) and BLI total flux (p/s) counts were plotted (E) (n = 7 for each group) for mice injected with MSC+HCC827-SCR or MSC+HCC827-AA cells. Total flux p values were calculated with unpaired two-tailed T test (* p<0.05). (F-G) Total lysates were probed with indicated antibodies to assess knockdown of ABL1 and ABL2 in PC9 (F) and HCC827 (G) cells transduced with lentiviruses carrying either control (SCR) or shRNAs specific for ABL1 and ABL2 (AA).