Flt3L therapy induces a Treg-promoting cDC1 state in triple-negative breast cancer

Conventional dendritic cells (cDCs) are at the forefront of activating the immune system to mount an anti-tumor immune response. Flt3L is a cytokine required for DC development that can increase DC abundance in the tumor when administered therapeutically. However, the impact of Flt3L on the phenotype of distinct cDC subsets in the tumor microenvironment is still largely undetermined. Here, using multi-omic single-cell analysis, we show that Flt3L therapy increases all cDC subsets in orthotopic E0771 triple-negative breast cancer, but this did not result in a reduction of tumor growth. Interestingly, a CD81+migcDC1 population, likely developing from cDC1, was induced upon Flt3L treatment. This subset is characterized by the expression of both canonical cDC1 markers as well as migratory cDC activation and regulatory markers and displayed a higher Treg-inducing potential compared to other migcDCs. To shift the cDC phenotype towards a T-cell stimulatory phenotype, CD40 agonist therapy was administered in combination with Flt3L. However, while aCD40 reduced tumor growth, Flt3L failed to improve the therapeutic response to aCD40 therapy. Interestingly, Flt3L+aCD40 combination therapy increased the abundance of Treg promoting CD81+migcDC1. Nonetheless, while Treg-depletion and aCD40 therapy were synergistic, the addition of Flt3L to this combination did not result in any added benefit. Overall, these results indicate that merely increasing cDCs in the tumor by Flt3L treatment cannot improve anti-tumor responses and therefore might not be beneficial for the treatment of triple-negative breast cancer, though could still be of use to increase cDC numbers for autologous DC-therapy. Overall design: Tumor-bearing mice were treated at day 12 after E0771 inoculation and treated for 9 consecutive days with vehicle or Flt3L. Similarly sized tumors, collected at day 21 after tumor inoculation, were pooled from three mice/condition. Living CD45+ cells were sorted using FACS (Fluorescence-activated cell sorting) and subsequently analyzed using CITE-seq.