(A) (Top) Representative M1 macrophage staining for F4/80 (red) and MHCII (green) for 21% and 7% protein diet cohorts in the LuCaP23.1 model. (Bottom) Representative M2 macrophage staining for F4/80 (red) and CD206 (MMR) (green) for 21% and 7% protein diet cohorts in the LuCaP23.1 model. (B) (Left) Blinded quantification of M1 macrophage presence in the TME based on random selection of five fields per tissue. (Right) Blinded quantification of M2 macrophage presence in the TME. (Bottom right) Western blot analysis showing M2 marker CD206 presence in 21% versus 7% protein diet conditions. (C) Schematic of in vitro macrophage stimulation. (D) Flow cytometry analysis for M1 (CD45+ CD11b+ F4/80+ iNOS+) and M2 (CD45+ CD11b+ F4/80+ Arg1+) in AA-restricted BMDMs 48 hours post-stimulation. The experiment was repeated at least 3 times independently. Legend: 1. Control media, 2. 1/2 methionine media, 3. 1/2 methionine 1/3 cystine media (E) mRNA fold change expression levels of M1 and M2 macrophage markers, iNOS and Arg1, respectively. The experiment was repeated at least 3 times independently. n = 9. Results are expressed as the mean ± SE. Statistical significance was determined by student’s t-test with Welch’s correction. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.