Breast cancer (BCa) is the leading cause of cancer-related death among women worldwide. Unlike other cancers, immune checkpoint inhibitors do not improve patient survival, and new immunotherapies need to be tested. Using a syngeneic BCa model with the 4T1 cell line, we assessed the efficacy of autologous DC vaccines in eliciting an immune response against cancer cells.

Mouse bone marrow (BM) cells were differentiated into cDC2 using GM-CSF and IL-4. These GM-DCs were pulsed with 4T1 tumor lysates and activated with LPS and PAMPs from a prophylactic vaccine (Vanguard-Plus). Although plasma anti-4T1 antibody levels increased by 69% in mice treated with GM-DCs, there was no increase in tumor-infiltrating lymphocytes, and the 16% decrease in tumor volume could not be reproduced.

To improve DC preparation, we cultured BM cells with FLT3L and Delta-like 1 (DLL1), yielding a mix of cDC1 and cDC2 (~1:4 ratio). These DLL1-DCs were loaded with tumor antigen and activated with TLR3, TLR4, TLR7, and TLR9 agonists, increasing MHC-II, CD80, and CD86, as assessed by flow cytometry, and elevating mRNA levels of Il12b, Ifng, and Tnfa. In a preventive setting, DL1-DC-Vax initially caused a 38-47% decrease in tumor volume. However, the effect was lost, possibly due to CD8⁺ T cell exhaustion (31.9±4.0% PD-1⁺Tim3^- and 43.2±11.1% PD-1⁺Tim3⁺) and the presence of PMN-MDSCs (16.8%) and M-MDSCs (8.9%).

To eliminate MDSCs, we tested 5-fluorouracil. Three 5-FU injections (50mg/kg) significantly reduced the numbers of PMN- and M-MDSCs (-60% and -87%, respectively) in 4T1 tumors. Although the number of infiltrating CD8⁺ T cells did not change, 5-FU significantly decreased PD-1⁺Tim3^- CD8⁺ T cells (-26%) and increased PD-1^-Tim3^- CD8⁺ T cells (+35%).

Overall, these results indicate that DLL1-DC vaccination alone is not effective against 4T1 BCa cells and suggest that combining it with 5-FU and anti-PD-1 could improve efficacy and ultimately benefit BCa patients.