Fig 1.
Cell line models of acquired cetuximab resistance exhibit increased IL-6 secretion.
A) PE/CA-PJ49 parental and CtxR cells were treated for 96 h with vehicle (PBS) or cetuximab (0.1 nM– 1 μM), then stained with crystal violet. Student’s two-tailed t-test was used to determine whether differences in absorbance at 590 nm were statistically significant (compared to vehicle-treated cells). n = 4. B) Cells were plated at low density and treated the next day with vehicle (PBS) or 100 nM cetuximab. Cells were stained with crystal violet after 12 days of cetuximab treatment. Media containing vehicle or cetuximab was changed every 4 days. C) RNA was extracted from PE/CA-PJ49 parental and CtxR cells and qPCR was conducted using the IL6 primers listed in S1 Table (normalized to TBP). n = 3. D) PE/CA-PJ49 parental cells and CtxR cells were plated in serum-free medium. Conditioned medium was collected after 72h and concentration of IL-6 was measured using ELISA. Student’s two-tailed t-test was used to determine whether differences in IL6 expression and secreted IL-6 were statistically significant (compared to parental cells). n = 4. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; n.s., not significant.
Fig 2.
Addition of recombinant IL-6 does not promote cetuximab resistance in PE/CA-PJ49 parental cells.
A) PE/CA-PJ49 parental cells were serum starved for 4 hours (no FBS) or remained in media containing 10% FBS (10% FBS), then treated with 50 ng/mL rhIL6 for 15 min or 4 hours. Cells were lysed in RIPA buffer and immunoblot was performed as described in Materials and Methods. β-tubulin image shown is from the STAT3 blot. B) PE/CA-PJ49 parental cells were treated for 96 h with vehicle (PBS), 50 ng/mL rhIL6, 100 nM Ctx, or the combination of Ctx and rhIL6, then stained with crystal violet. Images shown are representative of three biological replicates. C) Quantification of crystal violet staining in Fig 2B. Student’s two-tailed t-test was used to determine whether differences in absorbance at 590 nm were statistically significant (compared to vehicle-treated cells). n = 3. **p<0.01; n.s., not significant.
Fig 3.
Inhibition of IL-6 signaling does not impact cetuximab response in PE/CA-PJ49 parental and CtxR cells.
A) PE/CA-PJ49 parental cells were transfected with 10 nM nontargeting (nt) siRNA or one of two siRNAs targeting IL6 (siIL6 A and B). RNA was extracted 96 hours post-transfection and qPCR was conducted using the IL6 primers listed in S1 Table (normalized to TBP). n = 3. *p<0.05. B) PE/CA-PJ49 parental and CtxR cells were plated at a low density and transfected with 10 nM siRNA the next day. On the following day, and every four days thereafter, the cells were treated with vehicle (PBS) or 100 nM Ctx. The cells were stained with crystal violet 13 days post-transfection.
Fig 4.
Expression of components of the IL-6 pathway are altered in HNSCC cells that have acquired resistance to cetuximab.
A,B) RNA was extracted from PE/CA-PJ49 parental cells and cetuximab-resistant clones and qPCR was conducted using the IL6ST (A) or IL6R (B) primers listed in S1 Table (normalized to TBP). n = 3. C) Cells were lysed in RIPA buffer and immunoblot was performed. Images depicted are representative of three biological replicates. D) Densitometry was performed on the blots depicted in (C) using ImageJ as described in Materials and Methods. Densitometry values for gp130 were normalized to those for the loading control (β-tubulin). Student’s t-test was used to determine whether differences in the gp130/β-tubulin ratios in CtxR cells were statistically significant compared to parental cells. n = 3. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.
Fig 5.
IL-6 signaling is impaired in PE/CA-PJ49 CtxR cells.
A) PE/CA-PJ49 parental and CtxR cells were lysed in RIPA buffer and immunoblot was performed. Images shown are representative of three biological replicates. β-tubulin image shown is from the STAT3 blot. B) Densitometry was performed using ImageJ as described in Materials and Methods. Densitometry values for P-STAT3Y705 were normalized to those for total STAT3. Student’s t-test was used to determine whether differences in the P-STAT3Y705:STAT3 ratios in CtxR cells were statistically significant compared to parental cells. n = 3. *p<0.05; **p<0.01. C) PE/CA-PJ49 parental and CtxR cells were serum starved for 4 hours, then treated for 15 minutes with 50 ng/mL rhIL6 or rhLIF. Cells were lysed in RIPA buffer and immunoblot was performed. β-tubulin image shown is from the P-STAT3Y705 blot.
Table 1.
SNVs and indels identified in all PE/CA-PJ49 parental and CtxR cell lines.
Table 2.
SNVs and indels unique to individual cell lines.