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

Abstract

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.

Figure 1. Comparison of lung and liver metastases of tumor cells in WT BALB/c mice receiving EMT6 or 4THM tumor cells and subsequently treated with surgical resection and chemotherapy/immunotherapy (see )Methods.

4 mice were used per group, with mice sacrificed at the times show post surgery (number above histogram bars) to measure macroscopic tumor metastases in the lung/liver. All data represent arithmetic means (±SD) for each group. nc indicates no metastatic colonies detected; *, p<0.05 relative to similar group receiving either immunotherapy or chemotherapy.

Figure 2. Attenuation of outgrowth of tumor from DLN of mice shown in Figure 1 as assessed by limiting dilution frequency (see Methods).

DLN cells from separate mice were also cloned alone at the time of surgery (data to far left of each panel-control*). All frequencies were calculated based on the input numbers of cells from DLN of control mice only. *, p<0.05 compared with control* mice

Figure 3. Effect of anti-CD4 mAb on lung/liver (panel a) or DLN (panel b) metastases in mice receiving EMT6 or 4THM tumor cells and treatment as in Figure 1 .

5 mice were used per group for sacrifice at the time post surgery points shown (numbers above histogram bars). Data show means for macroscopic tumor colonies/group; nc =  no visible tumor colonies. * indicates p<0.05, compared with control treated with surgery alone;

Figure 4. Specific protection from re-challenge with EMT6, but not 4THM, assaying either local tumor growth (panel a) or DLN metastases (panel c) in mice treated 90 d earlier by surgical tumor resection and immunotherapy.

Naïve mice had had no previous EMT6 or 4THM tumor implants. All mice were sacrificed at 20 d post re-challenge. Data represent means for group. No protection was seen in mice initially treated with 4THM tumors before treatment/re-challenge (panel b). *, p<0.05 compared with equivalent fresh control mice.

Figure 5. Adoptive transfer of splenocytes from immune- but not chemo-therapy treated mice receiving EMT6 tumors can decrease lung (panel a) and DLN (panel b) metastases in mice which had previously received EMT6 but not 4THM tumors.

The tumors in the latter mice were surgically removed 1 d before spleen transfer, and all mice sacrificed 14 d after spleen cell transfer. Data show means (±SD). *, p<0.01 relative to control (no cell transfer).