FIGURE 4.

Flk-1 vaccines can protect mice from experimental metastases and induce weak Her-2/neu epitope spreading in a more aggressive tumor model for breast cancer. A, Mice were immunized thrice with each vaccine, and then injected with 50,000 4T1 cells i.v.; tumors were allowed to grow for 25 days, and then mice were sacrificed. H&E-stained sections were performed on lung tissues, and tumor nodes were counted by hand. Graph shows the number of lung metastases per lobe per animal, mean ± SEM; *, p < 0.05; Mann-Whitney U test, experiment repeated once, n = 5 mice shown. B, Spleens from these animals were processed and rechallenged ex vivo in IFN-γ ELISPOT assays for Her-2/neu epitope spreading. The 4T1 cell line does express low levels of mouse Her-2/neu (48). Spreading is seen only in the Flk-1-E1-immunized mice. Graph shows mean ± SEM for spot number/well as compared with control Lm group; *, p < 0.05; Mann-Whitney U test, experiment repeated once, n = 5 per group. C, A similar experiment in which mice were protected via immunization with each vaccine for a series of 3 wk, and then injected with 50,000 4T1-Luc cells i.v.; mice were imaged longitudinally over the course of 4 wk, looking for the incidence of lung seeding and rate of metastasis (top panel). Average radiance in photons (p) captured per second per cm² for the surface area (sr) gated in the region of interest is shown in the bottom panel. Graph shows mean ± SEM; *, p < 0.05; Mann-Whitney U test. Significance for mice is as follows: day 18, only Flk-E1 significant; day 25, both Flk-E1 and Flk-I1 significantly different when compared with control Lm. D, Primary s.c. 4T1 tumors grow more slowly in Lm-LLO-Flk-1-protected animals. Mice were immunized thrice with each vaccine, and then injected with s.c. and i.v. with 50,000 4T1 cells. Graph shows mean ± SEM for tumor volume.