Background A study of the anti-tumor T-cell response and immunological memory following successful 4T1 breast cancer immunotherapy with the combination of surgical resection of the primary tumor and subsequent macrophage/dendritic cell reprogramming using injections of the pharmaceutical TLR4-agonist.

Materials and Methods 15,000 cells of the 4T1 mouse breast carcinoma inoculated subcutaneously into BALB/c mice generated solid tumors and metastatic disease ended by the death of all the tumor-bearing animals during 30-40 days. Surgical resection of the primary tumor was performed on day 11. Pharmaceutical TLR4-agonist (Immunomax®) administered intraperitoneally in dose of 14 µg every 2-3 days, in total seven injections per course. Sorted macrophage/dendritic cells reprogramming was examined by RT-PCR. Tumor-reactive IFNγ-secretory T cells were counted using ELISPOT in ex vivo co-cultures of sorted CD4 T cells or CD8 T cells with the tumor lysate-loaded syngeneic dendritic cells or alive 4T1 tumor cells. Sorted CD8 effector T cell cytotoxicity was measured in their co-culture with different numbers of 4T1 target cells.

Results Using a combination of surgical resection of the primary 4T1 tumor and immunotherapy with the pharmaceutical TLR4-agonist for the treatment of metastatic disease in BALB/c mice a complete recovery of 20-30% mice was achieved. The complete responder mice effectively generated CD4 T cells and CD8 T cells, which specifically respond to 4T1 tumor antigens by IFN-production and kill 4T1 tumor cells in ex vivo co-cultures. The T-cell response is systemic, as tumor-specific T cells accumulate in the spleen. The second or third inoculation of the 4T1 tumor is accompanied by a complete absence of tumor growth in 50% and inhibition of tumor growth in the rest of the immune mice. An accumulation of significant numbers of T cells that respond to 4T1 tumor antigens by IFNγ-secretion, as well as of CD8 T cells that kill 4T1 tumor cells in a cytotoxic test was found in the secondary (tertiary) tumors, as well as in the draining lymph nodes. Immunological memory in complete responder mice that recovered due to the treatment with resection of the primary tumor and immunotherapy with a 4T1-agonist persisted for a long time (maximum observation period of 260 days).

Conclusions Macrophage/dendritic cell reprogramming with the TLR4-agonist for the post-resectional immunotherapy of 4T1 breast cancer metastatic disease induce tumor-specific CD4 and CD8 T cell responses and T-cell mediated long-living immune memory.

Funding This study was supported by the Russian Science Foundation (project no. 20-15-00391).

Disclosure Information E. Ushakova: None. E. Lebedeva: None. A. Pichugin: None. R. Ataullakhanov: None.