Comparison of immunity in mice cured of primary/metastatic growth of EMT6 or 4THM breast cancer by chemotherapy or immunotherapy.

PURPOSE: We have compared cure from local/metastatic tumor growth in BALB/c mice receiving EMT6 or the poorly immunogenic, highly metastatic 4THM, breast cancer cells following manipulation of immunosuppressive CD200:CD200R interactions or conventional chemotherapy. METHODS: We reported previously that EMT6 tumors are cured in CD200R1KO mice following surgical resection and immunization with irradiated EMT6 cells and CpG oligodeoxynucleotide (CpG), while wild-type (WT) animals developed pulmonary and liver metastases within 30 days of surgery. We report growth and metastasis of both EMT6 and a highly metastatic 4THM tumor in WT mice receiving iv infusions of Fab anti-CD200R1 along with CpG/tumor cell immunization. Metastasis was followed both macroscopically (lung/liver nodules) and microscopically by cloning tumor cells at limiting dilution in vitro from draining lymph nodes (DLN) harvested at surgery. We compared these results with local/metastatic tumor growth in mice receiving 4 courses of combination treatment with anti-VEGF and paclitaxel. RESULTS: In WT mice receiving Fab anti-CD200R, no tumor cells are detectable following immunotherapy, and CD4+ cells produced increased TNFα/IL-2/IFNγ on stimulation with EMT6 in vitro. No long-term cure was seen following surgery/immunotherapy of 4THM, with both microscopic (tumors in DLN at limiting dilution) and macroscopic metastases present within 14 d of surgery. Chemotherapy attenuated growth/metastases in 4THM tumor-bearers and produced a decline in lung/liver metastases, with no detectable DLN metastases in EMT6 tumor-bearing mice-these latter mice nevertheless showed no significantly increased cytokine production after restimulation with EMT6 in vitro. EMT6 mice receiving immunotherapy were resistant to subsequent re-challenge with EMT6 tumor cells, but not those receiving curative chemotherapy. Anti-CD4 treatment caused tumor recurrence after immunotherapy, but produced no apparent effect in either EMT6 or 4THM tumor bearers after chemotherapy treatment. CONCLUSION: Immunotherapy, but not chemotherapy, enhances CD4+ immunity and affords long-term control of breast cancer growth and resistance to new tumor foci.

Cure of metastatic growth of EMT6 tumor cells in mice following manipulation of CD200:CD200R signaling.

In previous studies, we observed that regulation of expression of CD200, both on cells of a transplantable breast cancer, EMT6, and of the host, as well as of the receptor, CD200R in host mice, regulated local tumor growth and metastasis in immunocompetent animals. This in turn led to an improved ability to document immunity to EMT6 in CD200R1KO mice. In the current study, we have explored the ability to cure BALB/c CD200KO or CD200R1KO mice of tumors ≤1 cm(3) in size by surgical resection of localized tumor, followed by immunization with irradiated EMT6 cells along with CpG as adjuvant. While control animals treated in this fashion developed significant pulmonary and liver metastases within 30 days of surgery, significant protection was seen in both CD200KO or CD200R1KO mice, with no macroscopic lung/liver metastases observed in CD200R1KO mice on sacrifice at day 300. Following surgical resection and immunization, draining lymph nodes from control mice contained tumor cells cloned at limiting dilution in vitro even before pulmonary and hepatic metastasis was seen. In contrast, within the limits of detection of the assay used (sensitivity ~1 in 10(7) cells), no tumor cells were detected at limiting dilution in similarly treated CD200R1KO mice, and significant reductions were seen in CD200KO mice. Infusion of anti-CD4, but less so anti-CD8, mAb into surgically treated and immunized CD200R1KO mice attenuated protection from both macroscopic (liver/lung) and microscopic (assayed by limiting dilution of DLN) metastasis. Adoptive transfer of lymphocytes from treated CD200R1KO mice to surgically treated control mice also attenuated metastatic growth of tumor, which was abolished by pretreatment of transferred cells with anti-CD4 mAb. Our data suggest that CD200:CD200R attenuates a potentially tumor-protective CD4 host response to breast cancer.

Further evidence for a role of tumor CD200 expression in breast cancer metastasis: decreased metastasis in CD200R1KO mice or using CD200-silenced EMT6.

Previous studies reported that CD200 expression on cells of the transplantable EMT6 mouse breast cancer line was increased during growth in immunocompetent mice. Low levels of expression persisted in NOD-SCID.IL-2(γr-/-) mice or mice with generalized over-expression of a CD200 transgene (CD200(tg) mice), despite the faster tumor growth in both of these latter strains. We also showed that CD200 expression (by the host and/or tumor cells) led to increased seeding of tumor cells to DLN in immunocompromised (CD200(tg) or NOD-SCID.IL-2(γr-/-)) vs immunocompetent mice, using limiting dilution cloning of tumor cells from DLN (vs contralateral lymph nodes, CLN). Evidence for an important role for CD200 expression in this increased metastasis came from the observation that neutralization of CD200 by anti-CD200mAbs decreased tumor metastasis and increased levels of cytotoxic anti-tumor immune cells in DLN. In the current studies, we have extended these observations by exploring tumor growth/metastasis in CD200R1 KO mice in which we have previously shown, in a transplant model, that expression of CD200 fails to deliver an immunosuppressive signal. In addition, we have studied local and metastatic growth in healthy control mice of EMT6 tumor cells stably transduced with shRNA able to silence CD200 expression. In both scenarios, decreased metastasis was observed, with increased immunity to EMT6 detected by cytotoxicity assays. In addition, adoptive transfer of DLN to control mice attenuated EMT6 metastases implying a potential therapeutic benefit from neutralizing CD200 expression in breast cancer.