Fig 1.
Stress induces CXCR3 expression on BOWES RGP melanoma cells.
(A) Flow cytometry analysis of CXCR3 protein expression on a VGP melanoma cell line, C9. Cells were incubated with serum at 5% CO2. (B) Graph of CXCR3 protein expression on BOWES cells from 8 separate flow cytometry experiments. Cells were cultured either in normal culture conditions (far left bar), in serum-free media at 8% CO2 for 24, 48, or 72 hrs, or in serum-free media at 8% CO2 for 48 hrs followed by 48 hrs in normal culture conditions (far right bar). T-tests were performed on the averages for each time point, comparing treatment cells with the 0hr time point (NS = P>0.05, *** = P≤ 0.001). (C-D) Flow cytometry analysis of CXCR3 protein expression on an RGP cell line, BOWES. Cells were incubated (C) with serum at 5% CO2 or (D) without serum at 8% CO2 for 72 hrs. All flow cytometry plots and graphs are representative data from at least 3 separate experiments. Red lines = unstained cells, blue lines = cells stained with CXCR3.
Table 1.
Gene expression of stressed BOWES cells (cultured in serum-free media at 8% CO2) relative to those cultured under normal serum-containing conditions (10% FBS media at 5% CO2).
Fig 2.
IL-8 expression in stressed BOWES cells is associated with CXCR3 signaling and increased in the CXCR3High subpopulation of cells.
(A) BOWES cells were cultured for 48 hrs under stressful conditions (serum-free media at 8% CO2) in the presence of ligand (100ng/ml CXCL9 and CXCL10), with the addition of DMSO, 0.2μM, or 1μM AMG487. IL-8 expression was measured with RT-PCR, fold change was calculated relative to cells treated 1μM AMG487. (B) Flow cytometry analysis of representative data from 3 separate sorts. CXCR3Low and CXCR3High cells were sorted based on the gates shown. Red line = unstained cells, blue line = cells stained with CXCR3. (C) CXCR3 and (D) IL-8 RT-PCR results from unsorted cells grown with serum in 5% CO2 or without serum in 8% CO2 (stressed), and sorted stressed cells (CXCR3Low and CXCR3High). Fold change was calculated relative to unsorted stressed cells. T-tests were performed on the relative fold changes, pooled data from 3 separate experiments (* = P≤ 0.05, ** = P≤0.01, ***P≤ 0.001).
Fig 3.
Ectopic overexpression of CXCR3 in BOWES cells increases ligand mediated phosphorylation of ERK, cellular migration, and IL-8 expression.
BOWES cells stably transfected with CXCR3 or an empty vector (PCMV6) were grown in serum-containing media. (A) Flow cytometry and (B) RT-PCR were used to measure CXCR3 expression. (A) BOWES WT (red line), BOWES PCMV6 clone #1(blue line), and BOWES CXCR3 clones #1 (orange line), #2 (light green), and #3 (dark green line). (B) RT-PCR was normalized to CXCR3 clone #2. (C) BOWES PCMV6 and CXCR3 cells were grown in serum-free media for 2 hrs, then in the presence of CXCL9 and CXCL10 for 2, 5, 10, 20, or 30 minutes. Total protein was isolated and probed for pERK and total ERK via immunoblot analysis. (D) BOWES PCMV6 and CXCR3 cells were plated on a membrane above serum-free media with CXCL9/10 (+ligand) or without (-ligand), and total migrated cells per field (40X objective) were quantified after 6hrs. Cells were also plated on the membrane in the presence or absence of AMG487. T-tests were performed on the BOWES CXCR3 migrated cells ± ligand in the presence and absence of AMG487 (NS = P>0.05, **** = P≤ 0.0001). (E) BOWES PCMV6 and CXCR3 cells were grown in serum-containing media for 48 hrs in the presence of ligand, with 0, 0.2, or 1 μM AMG487. Expression of IL-8 was measured via RT-PCR, fold change was calculated relative to BOWES PCMV6 0 μM. T-tests were performed on the relative fold changes, representative data from 3 separate experiments (**** = P≤ 0.0001).
Fig 4.
IL-8 expression in BOWES cells is linked with MAPK signaling cascades in BRAFWT BOWES cells.
WM35 IL-8 signaling is CXCR3-signaling independent. (A) BOWES WT cells were grown in serum-free media for 48 hrs in the presence of ligand, with 0, 0.2, or 1 μM AMG487 and with DMSO (black bars) or 3 μM U0126 (gray bars). Expression of IL-8 was measured via RT-PCR, fold change was calculated relative to cells treated with 0 μM U0126 and 1 μM AMG487. (B-C) Flow cytometry analysis of CXCR3 protein expression on the BRAFV600E RGP cell line, WM35. Cells were incubated (B) with 5% serum at 5% CO2 or (C) without serum at 8% CO2 for 72 hrs. Red lines = unstained cells, blue lines = cells stained with CXCR3. (D) BOWES WT (black bars) and WM35 cells (gray bars) were grown in serum-free media for 48 hrs in the presence of ligand, with 0, 0.2, or 1 μM AMG487. Expression of IL-8 was measured via RT-PCR, fold change was calculated relative to BOWES cells treated with 0 μM AMG487. (E) WM35 cells were grown in serum-free media for 48 hrs in the presence of ligand, with 0, 0.2, or 1 μM AMG487 and either DMSO (black bars), 3 μM U0126 (striped bars), or 3 μM PLX4032 (gray bars). Expression of IL-8 was measured via RT-PCR, fold change was calculated relative to WM35 cells treated with 0 μM AMG487 and 3 μM U0126. All T-tests were performed on the relative fold changes, all data is representative from at least 3 separate experiments (NS = P>0.5, * = P≤ 0.05, ** = P≤0.01, **** = P≤ 0.0001).
Fig 5.
Ectopic overexpression of CXCR3 on BOWES RGP cells increases tumor frequency and metastases and IL-8 expression in vivo.
BOWES PCMV6 and CXCR3 cells were injected interdermally into NSG mice (5 x 105 cells per injection). Tumors were measured weekly with calipers and tumor volume (mm3) was calculated. Mean tumor volume and SEM were calculated from mice that had measurable tumors. (A) The graph shows mean tumor volume (with SEM) over the 6 week period. Linear regression analysis was performed on the two slopes (P = 0.006). (B) Tumor tissue from injected mice was resected at the time of harvest and RNA was isolated. IL-8 expression was measured in BOWES PCMV6 and CXCR3 tumor tissue via RT-PCR (**** = P≤ 0.0001). Representative individual tumors are presented in graph.
Table 2.
Tumor growth in vivo.