Figure 2. Loss of Kindlin-1 reduces tumor associated macrophages and increases cDC1 dendritic cells.

(A) Met-1 Kin1-WT or Kin1-NULL tumors were established via subcutaneous injection in FVB mice, and harvested at day 10 for immunophenotyping by flow cytometry. Major myeloid populations were quantified as a percentage of live (total) cells. Gating demonstrated in Figure 2—figure supplement 1. (B) Raw FACS plots demonstrating gating of cDC1 and cDC2 cells, and quantified (C) as a percentage of total DCs (CD11c⁺ MHC II⁺). (D) Quantification of CD80 expression on total DCs by flow cytometry. (E) Quantification of PD-L1 expression on cDC1 cells. (F) As in A but bulk tumors were harvested for RNA expression analysis using Nanostring PanCancer Immune panel. Differentially expressed genes related to the gene sets ‘MHC’ and ‘Antigen (Ag) presentation’ are shown. (G) Expression of Ifng using Nanostring PanCancer Immune panel comparing Met-1 Kin1-WT and Kin1-NULL cells. (H) Expression of Cd274 (PD-L1) on isolated CD45⁺ cells using Nanostring Immune Exhaustion panel comparing draining lymph nodes (dLN) and tumors from Met-1 Kin1-WT and Kin1-NULL tumor bearing mice. Example of two independent experiments (A–E), n=3–5 per group, error bars = SD. For F, fold change cut off = 1.2, FDR =< 0.05. Unpaired t-test with * =< 0.05, ** =< 0.01, *** =< 0.001. Further macrophage and dendritic cell profiling shown in Figure 2—figure supplement 2.

Figure 2—figure supplement 1. Flow cytometry gating examples of myeloid populations.

Example of tumor myeloid cell gating shown. First debris is removed by All cells gate, followed by singlets and live cells. CD45⁺ cells are selected for downstream identification of Macrophages (F4/80+), Neutrophils (CD11b⁺ F4/80^- Ly6Ghi Ly6cint) and Monocytes (CD11b⁺ F4/80^- Ly6Glo Ly6C⁺). Total dendritic cells (DCs) were defined as CD11c⁺ MHC II⁺ with further downstream gating of cDC1 (CLEC9a⁺ SIRPα^-) and cDC2 (CLEC9a^- SIRPα⁺). Expression of CD103 was then assessed on cDC1s and cDC2s.

Figure 2—figure supplement 2. Macrophage and dendritic cell profiling in Met-1 tumors and PD-L1- Kindlin-1 correlation in human breast cancer dataset.

(A) Quantification of MHC II and CD206 (left), MHC II and SIRPα (middle), and SIRPα and CD206 (right) as a percentage of total macrophages infiltrating MET-1 tumors. Data representative of two independent experiments. n=5 per group, error bars = SD. (B) Representative histograms of PD-L1 and CD80 expression on cDC1 cells in Met-1 Kin1-WT and NULL tumors. (C) Correlation analysis of FERMT1 (Kindlin-1) and CD274 (PD-L1) using METABRIC human breast cancer data set. n=1904,, r=0.1375 (95% confidence interval 0.09–0.18), p<0.0001.