presumably due to the poor chemical stability of carbonates and carbamates.

Several studies reported that the response to EGFR TKI is associated with specific mutation in the tyrosine domain of EGFR or with a high EGFR gene copy number. Later studies, however, indicated that mutations in the tyrosine domain of EGFR were also found in nonresponding tumors, suggesting that the response to therapy may be due to other mechanisms. Bortezomib We have recently reported that in multiple carcinomas, EGFR was phosphorylated not only on tumor cells but also on tumor associated endothelial cells. The phosphorylation of EGFR on tumor associated endothelial cells, however, was only found in the vasculature of tumors that produced TGF /EGF. In nude mice implanted with human carcinoma cells into the relevant orthotopic organs, treatment with specific EGFR TKI produced apoptosis of tumor cells and tumor associated endothelial cells. On the basis of these findings, we hypothesized that a major determinant for neoplastic sensitivity to EGFR TKI is the production of TGF /EGF by tumor cells and activation of EGFR on tumorassociated Cellular differentiation endothelial cells. To test this hypothesis, we used the SW620CE2 human colon cancer cells. These cells do not express EGFR or human epidermal growth factor receptor 2 but do express TGF. The cells were transduced with lentivirus small hairpin RNA or lentivirus TGF shRNA. The three different SW620CE lines were implanted into the cecal wall of nude mice, and 2 weeks later, treatment with a specific EGFR TKI began. Only tumors producing TGF were sensitive to the therapy. Because none of the tumor cells expressed EGFR, the data identified the EGFR expressed by tumor associated endothelial cells as the primary target. Colon Cancer Cell Line and Culture Conditions SW620 human colon cancer cells obtained from Cyclopamine Dr. Gary Gallick, M. D. Anderson Cancer Center were maintained in minimal essential medium supplemented with 10% fetal bovine serum, sodium pyruvate, nonessential amino acids, L glutamine, a two fold vitamin solution, and a penicillin/ streptomycin mixture. Adherent monolayer cultures were maintained on plastic and incubated at 37 C in a mixture of 5% CO2 and 95% air. The cultures were free of Mycoplasma and pathogenic murine viruses and were maintained for no longer than 12 weeks after recovery from frozen stocks. In Vivo Selection of Highly Tumorigenic Variants from the SW620 Human Colon Cancer Cell Line SW620 cells were injected into the cecal wall of nude mice. Three months after the injection, cecal tumors were harvested and treated with DNase and collagenase as described previously. Cells were established in culture. Primary cultures were passaged in vitro two or three times, and then cells were harvested by trypsinization and were injected into the cecum of another set of nude mice. The selection cycle was repeated twice to yield the cell line designated as SW620CE2.