Fig. 3.

CRT + CTX/L-NIL reprograms the myeloid tumor microenvironment. Established mEER tumors were treated with CRT and/or CTX/L-NIL and harvested after the first week of treatment, as shown in to Fig. 1e, and tumor myeloid cell infiltrate was analyzed using flow cytometry (a-e; see Additional file 1: Figure S4 for myeloid flow cytometry gating strategy) and ex vivo co-culture (f-h). a Myeloid-focused tSNE (among CD11b⁺/CD11c⁺ cells) from flow cytometry data for each treatment group. b Corresponding tSNE color-map (left) and radar plot (right) showing myeloid sub-type alterations between each treatment group as z-scores (myeloid sub-type color in radar plot corresponds with their color in tSNE map; N = 1 representative of 3; n = 8–9/group). c-e Percentage of myeloid sub-types among total myeloid cell tumor infiltrate (CD11b⁺/CD11c⁺), including inflammatory monocytes (c), M1-like macrophages (d), and granulocytic MDSCs (e) (N = 3; n = 23–25/group; Tukey’s multiple comparison test for inflammatory monocytes and Dunn’s multiple comparison test for M1-like macrophages and granulocytic MDSCs). f Experimental schematic used to test treatment-induced myeloid influence on CD8⁺ T cell cytotoxicity. Naïve splenic CD8⁺ T cells were stimulated with anti-CD3/CD28 antibodies and IL-2 in the absence or presence of myeloid cells (CD11b⁺ and CD11c⁺ cells) isolated from tumors that received different treatments for 3 days and then co-cocultured with mEER cells in presence of IL-2. After 24 h, apoptotic tumor cells were detected by Annexin V/ PI staining. g Representative images of mEER tumor cells stained for Annexin V (shown in green) following co-culture (scale bars show 200 μm). h mEER tumor cell apoptotic fold change (Annexin V⁺ / PI⁻) normalized to non-primed T cells (T cells not co-cultured with myeloid cells) (N = 4; n = 20–26/group; Dunn’s multiple comparison test). All bar graphs show mean +/− SD and each dot represents an individual mouse. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001