In vitro and in vivo Evaluation of 111 In-labeled E. coli Heat-Stable Enterotoxin Analogs for Specific Targeting of Human Breast Cancers

Summary Research into the interaction between the E. coli heat-stable enterotoxin (ST h ) and the guanylin receptor guanylate cyclase C (GC-C) has generated >100 synthetic analogs of the peptide, several of which have been investigated as imaging or therapeutic agents for colorectal cancers. The evidence presented here suggests that in addition to ST h binding to GC-C expressing cell lines derived from human colon, ST h also specifically binds to an as yet unidentified receptor expressed in high densities on the surface of cell lines derived from human breast cancers. In vitro whole-cell crosslinking studies using 125 I-labeled F 19 -ST h (1–19) demonstrate that the putative ST h binding protein migrates as an approximately 120–125 kDa species by SDS-PAGE, significantly smaller than the glycosylated GC-C molecule found in the T84 human colon cancer cell line. RT-PCR using total RNA isolated from breast and colon cancer cell lines indicates that GC-C transcripts are undetectable in human breast cancer cell lines and abundant in human colon cancer cell lines. In vitro competitive binding studies using ST h analogs and the estrogen receptor positive (ER + ) T-47D cell line demonstrated IC 50 values between 2.6 and 8.5 nM. Similar studies on the estrogen receptor negative (ER − ) cell line MDA-MB-231 showed IC 50 ’s between 5.6 and 9.9 nM. Saturation binding analysis revealed receptor expression to fall between 40,000 and 120,000 sites per cell in these cell lines, receptor abundances equal to or greater than the abundance of GC-C in colorectal cancer cell lines. ST h binding to these cells, although of similar affinity to ST h binding to GC-C, is distinguishable from it on the basis of its ligand specificity. The characteristics of ST h analogs as radiopharmaceutical agents were tested in an in vivo model utilizing T-47D human breast cancer cell xenografts in SCID mice. Clearance of ST h analogs was rapid, primarily via renal excretion into the urine, with >85% ID excreted into the urine at 1 h p.i. Tumor uptake at 1 h p.i. in T-47D tumor cell xenografts was 0.67±0.23% ID/g, and was significantly decreased ( p <0.05) upon co-administration of 4 mg/kg unlabeled ST h . These results suggest that ST h may find application for the imaging and treatment of breast cancer.