Fig. 5. ECM components promote resistance to HER2 and PI3K inhibition.
(A) Primary tumor cells were seeded on 24-well plates coated ± 10% growth factor-reduced Matrigel and treated with 10 μg/ml trastuzumab, 10 μg/ml pertuzumab and 2 μM BKM120 (TPB) for 6 days. Cell monolayers were stained with crystal violet. (B) Quantification of (A) using ImageJ software; % survival of TPB-treated cells compared to untreated controls is shown. Each bar represents the mean image intensity ± S.D. of at least 3 replicates. (C) Cells derived from parental tumors were seeded on plates coated ± Matrigel and treated as in (A). (D) #564 parental and (E) TPB-resistant cells were plated on uncoated or Collagen I- or II-coated 24-well plates and treated with trastuzumab, pertuzumab, and 1-2 μM BKM120 or 1 μM BYL719 for 4 days. Cells were trypsinized and counted using a Coulter counter. Each bar represents the mean cell number ± S.D. of at least 3 replicates. (*p<0.01, **p<0.01, ***p<0.001, ****p<0.0001, 2-way ANOVA followed by Bonferroni's multiple comparison test). All experiments were performed in media containing 1% FBS. (F) Parental HER2+/PIK3CAH1047R tumor cells were seeded on uncoated or Collagen I-coated plates and treated as in (D). Cell lysates were subjected to immunoblot analysis with the indicated antibodies. Bar graphs represent quantification of immunoblot bands using ImageJ software.