Article Figures & Data
Figures
- Figure 1.
Generation of a cetuximab resistance model in vivo. A, T24, CAL33, A431, OSC-19, SCC1, and SCC1c8 cells were used to generate xenografts in athymic nude mice (n = 6 for CAL33, A431, OSC-19, SCC1, and SCC1c8; n = 12 for T24) that were exposed to increasing concentrations of cetuximab by i.p. injection (0.8 mg 2×/wk increased to 1.0 mg 2×/wk and then increased to 0.8 mg 3×/wk; doses increased in nonresponsive xenografts only as indicated by arrows). Resistant tumor cells were then harvested and propagated in culture and (B) reinoculated to form xenografts that were treated with 0.8 mg 3×/wk immediately following tumor formation.
- Figure 2.
Validation of cetuximab resistance model in vitro and in vivo. A, xenografts were generated by subcutaneous inoculation of 1 million tumor cells in athymic nude mice (n = 7) from cetuximab-sensitive T24 or cetuximab-resistant T24PR3 cells and treated with cetuximab (2.0 mg 3×/wk by i.p. injection) immediately following tumor formation, generally at 7 to 10 days (**, P < 0.005). TUNEL staining was carried out on frozen, fixed tumors to detect apoptotic cells. Data shown are the average of cells counted in quadruplicate 20× fields of view for 2 tumors per cell type (*, P < 0.05). B, invasion studies were carried out with media containing either 1 μmol/L cetuximab or drug-free media. Invasion chambers were placed in the same media containing 10% FBS. After 24 hours, invading cells were stained and counted (**, P < 0.005). Data are the result of 2 independent experiments run in duplicate.
- Figure 3.
611-CTF is hyperphosphorylated in cetuximab-resistant cells. A, cell lysates were collected under basal conditions when cells reached 70% confluence. Whole lysates were analyzed by Western blotting and probed for EGFR, HER2, and pHER2 (185 kDa) and 611-CTF and p611-CTF (110-kDa HER2 fragment). Densitometry is the result of 3 individual experiments where intensity of the p611-CTF bands was compared with the intensity of 611-CTF bands on the same gel (*, P < 0.05). A full image of these gels is available (Supplementary Fig. S1). B, cell lysates were collected under basal conditions when cells reached 70% confluence. Whole lysates were analyzed by Western blotting and probed for p-serine and cortactin. Densitometry is the result of 6 individual experiments where intensity of the p-serine bands for each cell type was compared with their respective cortactin bands from the same gel. Phosphorylation ratios for each cell line were compared with the T24 cell line for reference (*, P < 0.05).
- Figure 4.
A, T24PR3 cells were transduced with HER2 shRNA–containing lentiviral particles, and a representative image of the Western blots for full-length HER2 and 611-CTF from 4 clones and scramble control lysates are included here. Invasion studies in this figure were conducted as described earlier (*, P < 0.05), and all data are the result of 2 independent experiments run in duplicate. B, invasion studies were conducted using T24PR3 cells with media containing vehicle, cetuximab, trastuzumab to inhibit full-length HER2, or both cetuximab and trastuzumab. C, invasion studies were conducted using T24PR3 cells with media containing vehicle, cetuximab, the EGFR-HER2 kinase inhibitor afatinib, or both cetuximab and afatinib.
- Figure 5.
Dual kinase inhibition of EGFR and HER2 in vivo. Xenografts were created using cetuximab-resistant T24PR3 cells (A) or cetuximab-sensitive T24 cells (B) in athymic nude mice (n = 40). Mice were randomized on the basis of tumor volumes and treated with vehicle control, afatinib (0.4 mg daily by oral gavage), cetuximab (1.0 mg 3×/wk by i.p. injection), or both drugs concurrently for 21 days. Tumor volumes were measured 3 times per week for a total of 3 weeks. P values were generated using a Mann–Whitney test (*, P < 0.05). C, cell lysates were created from snap-frozen tumor tissue. Whole lysates were analyzed by Western blotting and probed for 611-CTF. Densitometry is the result of 4 technical replicates from 1 individual tumor of each treatment group normalized to the β-tubulin loading control for each tumor (*, P < 0.05; **, P < 0.005).
Additional Files
Supplementary Data
Files in this Data Supplement:
- Supplementary Figure 1 - PDF file - 166K
Volume 17, Issue 18
