Fig. 2. Combining local ablative IR with RA induces iNOS and TNF-α producing inflammatory macrophages.

MC38 tumors were treated as indicated and harvested 4 days post start of the IR/RA treatments for analysis in A-J.

(A) The gating strategy of iNOS⁺ myeloid cells.

(B) Frequency of CD11b⁺ myeloid cells in total cells of tumors.

(C) Percentage of major subsets of CD11b⁺ cells in tumors received IR/RA treatment.

(D) Gating strategy of iNOS⁺ inflammatory macrophages (Inf-MACs).

(E) Percentage of iNOS⁺ inflammatory macrophages in total cells of tumors.

(F) Percentage of subsets of iNOS⁺ Inf-MACs in terms of CD11c and Ly6C marker in total cells.

(G) Concentration of iNOS product nitrite in tumor homogenate. Treated tumors were harvested on day 4 post treatment and tumor fragments were cultured in vitro. Nitrite level in culture media was measured after 2 hours.

(H) TNF-α⁺ CD11b⁺ population in tumor receiving indicated treatment.

(I) Concentration of TNF-α in tumor homogenate at 4 days after start of the indicated treatments.

(J) Percentage of Inf-MACs in tumors grown in WT or CCR2^−/− mice, 4 days after start of IR and RA treatment.

(K) Tumor growth curve of MC38 tumors during treatments while CD11b⁺ cell recruitment was blocked.

(L) Tumor growth curve of MC38 tumors during treatments while iNOS inhibitor (1400W) was administered.

*, p<0.05; **, p<0.01; ***, p<0.001. Experiments were conducted 3 times with 3-5 mice in each group. Data in K, L are presented as mean ± SEM, the rest of the data are mean ± SD. Results from representative experiments are shown.