Abstract
Since a significant portion (30%) of individuals diagnosed with epithelial neoplasms have highly chemotherapy resistant CD44+CD24−/low cancer stem cells in the peripheral blood as well as in solid tumor nodules at diagnosis, we have been developing methods of cancer treatment which depend on methods of killing the cancer stem cells which depend on mechanisms other than chemotherapy for treatment. Vaccination has been one of these approaches. Unfortunately, it has been known for a long time that the immune response is diminished in older individuals due to decreased numbers of CD4 and CD8 T cells and due to acquisition of functional defects in CD4 cells. An example is the diminished expression of the CD40L in activated CD4 T cells in older individuals which limits the cellular and humoral response to vaccines in the older age groups. The presence of the CD40L on the CD4 helper T cells is important for vaccine induced expansion of antigen specific T cells and B cells. In order to overcome these problems, we have developed an Ad-sig-TAA/ecdCD40L vector prime-TAA/ecdCD40L protein boost vaccine strategy. The Ad-sig-TAA/ecdCD40L carries a transcription unit encoding the extracellular domain (ecd) of the CD40 ligand (CD40L) linked to tumor associated antigen (TAA). The TAA are in turn linked to a secretory signal peptide (sig) so that the TAA/ecdCD40L protein will be secreted from the vector infected cells at the injection site of the vector continuously over a 10–14 day period. The CD40L targets the TAA to the DCs, activates the DCs, and carries the TAA into the DC so that the TAA fragments are presented on Class I MHC. The sc injection of the TAA/ecdCD40L protein booster following the sc administration of the Ad-sig-TAA/ecdCD40L (we call this the TAA/ecdCD40L VPP vaccine) induces complete regressions of pre-existing tumor even in aged mice (18 months old). We then chose human MUC-1 as the TAA for testing the efficacy of our vaccine platform for cancer stem cells. Over 90% of the late stage patients with cancers of the breast, ovary, prostate, and lung have been reported to overexpress a hypoglycosylated form of MUC-1 which correlates with decreased time to progression. The hypoglycosylation on MUC-1 in cancer cells creates a tumor specific target for vaccines. Kufe’s lab has shown that overexpression of MUC-1 in the cancer cell promotes resistance to therapy by reducing p53 (Cancer Cell7: 167, 2005) and induces proliferation by activation of the NFkappaB (Nature Cell Biology9: 1419, 2007). Finn’s lab has shown that MUC-1 is a marker present on >90% of the “tumor stem cell” population as well as the more mature cells in breast cancer (Ca Res68: 2419, 2008). In order to test the efficacy of the hMUC-1/ecdCD40L VPP vaccine on tumor stem cells, we first isolated hMUC-1 positive Lewis Lung carcinoma cells by stably transfecting the hMUC-1 cDNA into these cells. We showed that the Ad-sig-hMUC-1/ecdCD40L vector prime-hMUC-1/ecdCD40L protein boost vaccine overcomes anergy to hMUC-1 antigen (hMUC-1) in hMUC-1.Tg mice. We then injected 100,000 human MUC- 1 positive Lewis Lung mouse carcinoma cells (LL2/LL1hMUC-1) mouse cancer cells into the syngeneic C57BL/6J mice. ELISPOT assay showed that the hMUC-1/ecdCD40L VPP vaccine increased the level of antigen specific CD8 effector cells in the lymph node draining the site of injection of the tumor to 900 hMUC-1 specific CD8 T cells/100,000 total cells and to 325 hMUC-1 specific CD8 T cells/100,000 cells in the spleen. The level of cytotoxicity of spleen cells increased 6 fold following vaccination (spleen cells from control and vaccinated mice were exposed in vitro to mitomycin C treated hMUC positive LL2/LL1hMUC-1 cells for 5 days before testing). Measurement of the growth of the sc tumor nodule at the sc injection site showed a 20 fold decrease in the total size of the tumor in the vaccinated vs the control mice and the size of the tumor was decreasing in the vaccinated mice vs the control group at the time of sacrifice at 24 days. Finally, the tumor nodule from the vaccinated and unvaccinated mice were excised post mortem, minced, treated with collagenase and DNAse I and strained through gauze. FACS analysis showed that in the vaccinated mice, the number of the MUC-1 positive tumor stem cells with the CD44+CD24− immunophenotype decreased over 20 fold during the treatment period. These results suggest that the hMUC-1/ecdCD40L VPP vaccine could suppress the levels of hMUC-1 cancer stem cells in pre-existing tumor nodules. This vaccine platform has entered clinical phase I testing.
Immunomodulatory Molecules On Lung Cancer Stem Cells From Lymph Nodes Aspirates