Surrogate Tumor Antigen Vaccination Induces Tumor-Specific Immunity and the Rejection of Spontaneous Metastases

Abstract

The nonimmunogenic 4T1 murine mammary carcinoma model and a model surrogate tumor antigen (sTA) were employed to explore the possibility of inducing tumor-specific immunity through active immunization in the absence of defined tumor-associated antigens. Immunization of naive mice with protein-based sTA resulted in protection from s.c. challenge, with 4T1 modified to express the sTA (4T1.sTA), or from a sTA-expressing unrelated tumor cell line (mKSA). Immunization had no effect on parental 4T1 tumor growth or the formation of parental 4T1 spontaneous lung metastases. Mice that were sTA immunized and successfully rejected 4T1.sTA challenge also rejected a subsequent challenge in the contralateral flank with parental 4T1 and strikingly prevented the formation of spontaneous parental 4T1 lung metastases. The rejection of parental 4T1 seemed to be specific for and associated with unknown 4T1 tumor-associated antigens, because rejection of mKSA did not induce cross-protection against a challenge with parental 4T1. To evaluate the effect of this vaccine approach on established disease, mice were simultaneously challenged on day 0 with 4T1.sTA and parental 4T1 in contralateral flanks and then immunized on days 3, 10, 17, and 24 with sTA protein. Tumor growth and metastasis were delayed in four of five animals, and 20% (2 of 5) of the animals were tumor free at the completion of the experiment. Together, these data suggest that prior vaccination with a sTA followed by inoculation with poorly immunogenic tumor cells modified to express the sTA activates determinant spreading and the induction of systemic tumor immunity resulting in indigenous tumor rejection. (Cancer Res 2005; 65(7): 2938-46)

Induction of Tumorigenesis and Metastasis by the Murine Orthologue of Tumor Protein D52

“…Forty-eight hours after transfection, 3T3.V and 3T3.mD52 were selected with G418 at increasing concentrations over time until reaching a concentration of 1 mg/mL. Expression of mD52 mRNA was confirmed by reverse transcription-PCR (RT-PCR) using methods our laboratory previously published (25,26). Expression of mD52 was not detected in either the untransfected parental 3T3 cell line or the empty vector control, 3T3.V (Fig.…”

“…The 3T3.mD52 and 3T3.V (vector control) cell lines were generated by transfecting parent 3T3 fibroblasts (Swiss albino 3T3 cells that were not immortalized or virally transduced and known to be contact inhibited ATCC CCL 92; ref. 51) with pCDNA3.1 containing the full-length cDNA for mD52 (pcDNA.mD52) or empty pCDNA3.1(+), respectively, using LipofectAMINE Plus reagent (Invitrogen) and methods previously described (25). Forty-eight hours following transfection, 3T3.V and 3T3.mD52 were selected and subsequently maintained in 1 mg/mL G418.…”

“…Cells were harvested and counted, and whole cell protein lysates were prepared at a concentration of 1 Â 10 8 cell equivalents/mL using methods previously described (25). Briefly, 10 AL of lysate were loaded onto a standard SDS-PAGE (4% stacking, 12% resolving) and run at 200 V for 15 min and 150 V for 40 min, followed by electrolytic transfer onto nitrocellulose.…”

“…For single cell clones of 3T3.mD52 and 3T3.V, naïve female BALB/c mice were inoculated subcutaneously in the right flank with 5  10 6 viable cells. Tumor volume (mm 3 ) was calculated using the following formula: (a  b 2 )/2, where b is the smaller of the two measurements (25).…”

“…To analyze tumor metastasis to the lungs, mice were sacrificed, and their lungs removed and injected with India ink to visualize individual tumor nodules as described previously (25). Briefly, an India ink solution was injected through the trachea and allowed to fill the lungs.…”