Supplementary Figures S1-S6 from A Molecularly Annotated Model of Patient-Derived Colon Cancer Stem–Like Cells to Assess Genetic and Nongenetic Mechanisms of Resistance to Anti-EGFR Therapy

Abstract

<p>Supplementary Figure 1. A, Cell viability assay of KRASmut (n=18), KRASwt Ctx-R (n=12) and Ctx-S (n=18) xenospheres kept for 4 days in basal stem-cell medium without any growth factor (No GF) or with 0,4 ng/ml of EGF (Basal Medium). Dots represent the ratio of raw viability data between EGF and NoGF. Red line: mean value for the group. Statistical analysis was performed with one-way ANOVA (p<0,0001). Bonferroni Multiple Comparison Test was applied to compare each group (n.s.=not significative; **p<0,01; ***p<0,001). B, Cell viability assay of CRC0264 xenospheres kept for 4 days in basal stem-cell medium containing 0,4 ng/ml of EGF (basal medium) or 20 ng/ml of the indicated growth factors, and treated with cetuximab (10 μg/ml) or vehicle. Columns represent raw viability data. Supplementary Figure 2. A, Western Blot analysis of total protein extracts from the indicated KRASwt and KRASmut xenospheres cultured in standard stem-cell medium (i.e. containing 20 ng/ml of EGF and 10 ng/ml bFGF). B, Cell viability assay of CRC0729 xenospheres kept for 4 days in basal stem-cell medium containing 0,4 ng/ml of EGF (Basal Medium) or 20 ng/ml of the indicated growth factors, and treated with cetuximab (10 μg/ml) or vehicle. Columns represent raw RLU data. Supplementary Figure 3. A, Schematic representation of binding properties of the EGF family ligands in xenospheres. In orange, EGF-like ligands that bind EGFR and induce formation of three different receptor heterodimers, all containing EGFR (red: EGFR, yellow: ERBB2, blue: ERBB3). Cetuximab is likely to inhibit dimer formation by outcompeting these ligands. In blue, NRG1 binds ERBB3 and induces two different heterodimers, which are not inhibited by cetuximab as this does not compete with NRG1 for ERBB3 binding. B, Western Blot analysis of total protein extracts from CRC0078 xenospheres kept for 4 days in basal stem-cell medium containing 0.2, 2 or 20 ng/ml of EGF. C, Flow cytometric analysis of EGFR on CRC0078 xenospheres cultured as described in B. D, Western Blot analysis of total protein extracts from CRC0059 and CRC0078 xenospheres longtermed cultured in stem-cell medium containing either EGF or NRG1 (20 ng/ml). Supplementary Figure 4. Scatter plots of the Log2 expression values of gene expression profiles of the indicated xenospheres long-term cultured either in standard stem-cell medium containing EGF-bFGF or in a modified medium containing NRG1-bFGF (GEO accession number GSE101792). R2 values are indicated in the graphs. Supplementary Figure 5. A, Schematic representation of the induction of an NRG1 autocrine loop in KRASwt xenospheres. B, qPCR showing the expression of NRG1 in parental (CRC0078) and transduced (CRC0078- NRG1 and CRC0059-NRG1) xenospheres. A549 cells were used as a positive control for NRG1 expression. Actin B (ACTB) and ubiquitin (UBQ) were used as housekeeper genes. C, Micrographs of phase contrast images of parental and NRG1-expressing CRC0078 xenospheres kept in standard cell culture conditions (i.e. with 10% serum and adhesive plastic) for 1 week, and treated either with vehicle, cetuximab (10 μg/ml), lapatinib (0,5 μM), or their combination (Combo). 20X magnification. D, qPCR showing the expression of NRG1 in parental (mFIBR) and NRG1- expressing mouse fibroblasts. mHPRT was used as housekeeper gene. E, Cell viability assay of CRC0078 xenospheres kept for 4 days in stem-cell medium without any growth factor (No GF) or with 0,4 ng/ml EGF (Basal Medium), or conditioned medium of parental and NRG1-expressing murine fibroblast (mFIBR-CM and mFIBR-NRG1-CM, respectively), and treated with vehicle, cetuximab (10 μg/ml), or lapatinib (0,5 μM). Columns represent raw RLU data {plus minus} s.e.m (n = 3). Supplementary Figure 6. A, Tumor growth curves of CRC0078 spheropatients treated as indicated. Graphs represent tumor volume increase vs. day 0 {plus minus} s.e.m. (n = 6/group). B, Western Blot analysis of total protein extracts from CRC0078 and CRC0078-NRG1 spheropatients treated for 3 weeks (as in Figure 6F).</p>