Experimental Study On Egfr-Mediated C-erbB2 Antisense Imaging For The Detection Of Breast Cancer

OBJECTIVE:To explore the possibility of receptor-mediated antisense imaging as a novel method for the earlier detection of breast cancer, we carry through this experiment to make the breast cancer patients to be diagnosed earlier and treated in time, and decrease mortality. METHODS: Antisense oligonucleotide for mRNA of c-erbB2 oncogene was labeled with ^ï½›99m｝-technetium, ^ï½›99m｝Tc, a common isotope whose physical performance is excellent, through a self-synthetic biofunctional chelator N-hydroxysuccinimide ester of mercaptoacetylglycylglycylglycine. Its labeling efficiency, radiochemical purity, specific activity, stability, biodistribution, rate of combination with plasma protein, pharmacokinetics, uptake rate, refluent rate, growth and oncoprotein expression of SKBR-3 cells were investigated. Then ^ï½›99m｝Tc-ON will be injected into tumor-bearing nude mice through their tail vein for imaging application.Three man-made synthetic single stranded oligonucleotides are antisense, sense and nonsense ones. Each contains 15 bases. Their sequences are antisense 5'-NH₂-FTCCATGGTGCTCAC-3', sense 5'-NH₂-QTGAGCACCATGGAG-3' and nonsense 5'-NH2-FGCCTTATCCGTAGC-3' ( F and Q represent phosphorothioate bases C and G, respectively). Antisense oligonucleotides were targeted to the initial code of mRNA of c-erbB2, sense and nonsense ones for this mRNA acting as control. These oligonucleotiedes labeled with ^ï½›99m｝Tc were separated and purified through Sephadex G25 column chromatography by using 0.25M/L ammonium acetate (pH=5.2) as eluent. Then the leaching curve will be made, labeling efficiency calculated, the first radiation peak collected. The radiochemical purity of these labeled oligonucleotides will be measured by paper chromatography, and their stability after being put 4 hours at room temperature or incubation with serum analyzed. Ninety culture flasks with volume of 50ml were inoculated with 2.0×10⁵ cells for each one. Having been cultured in CO₂ incubator for 2 days, confluent cells being to 50-60 percent, these cells were transfected with 2μg EGF-^ï½›99m｝Tc-ASON or ^ï½›99m｝Tc-ASON respectively. The uptake rate of cells are tested at 20, 40, 60, 120, 240min, and refluent rate detected in 18h. Culture plate with 96 wells was inoculated with 5.0×10³ SKBR-3 cells for each one. Having been cultured for 48 hours with 50-60 percent of confluent cells, the plates were transfected with three types of EGF-^ï½›99m｝Tc-ON in different concentration 0.5, 1, 2, 5μCi and done with EGF-PL compound in different concentration 0.2, 0.4, 0.8, 1.6μg respectively. Each concentration was added to three wells. Then these plates were transferred to CO₂ incubator, after 24 hours MTT were added to these wells. The absorbency was tested by enzyme-linked immunoassay. Eighteen culture flasks with volume of 50ml were inoculated with 2.0×10⁵ SKBR-3 cells for each one. Having been cultured for 48 hours, each was transfected with 2μg EGF-^ï½›99m｝Tc-ASON or three types of ^ï½›99m｝Tc-ON or 6μg EGF-PL compound. Each group contains threeflasks. The oncoprotein of c-erbB2 was detected at 24h after transfection. The rate of combination with plasma protein will be tested through trichloroacetic acid precipitation. EGF-^ï½›99m｝Tc-ASON and ^ï½›99m｝Tc-ASON are injected into rabbits through auricular marginal vein. Then the blood will be gotten at 1, 3, 5, 10, 20, 40, 60, 120, 240min, the radioactivity counts obtained through calibrator. The pharmacokinetics curves will be made according to the radioactivity counts of each gram blood, equations obtained through 3P97 software. Sixty female BALB/c mice classified into two groups were used to test the biodistribution in vivo. About 3μCi EGF-^ï½›99m｝Tc-ASON or ^ï½›99m｝Tc-ASON was injected into each mouse through its tail vein. Then the radioactivity counts of such organs or tissues as liver, spleen, kidney, heart, blood, lung, stomach, intestine, bone, muscle and brain were measured at 0.5, 1, 2, 4, 8 and 24h after injection. The cell line of breast cance...