Figure 5. PI3Ki suppresses lactate production from treated PTEN/p53-deficient PC cells and secondary histone lactylation within activated TAM, resulting in enhanced TAM phagocytosis.

(A) Schema illustrating lactate add-back phagocytosis experiment on ex vivo CM. Single cell suspensions of PTEN/p53-deficient prostate GEMM tumors were treated with copanlisib (C, 100 nM) and CM was collected 24 hours following treatment. FACS-sorted TAM from PTEN/p53-deficient prostate tumors were cultured ex vivo with CM for 24 hours in presence or absence of lactate (100 nmol/μL). After PBS wash, TAM were co-cultured with CTV dye stained-AC1/SC1 cells for 2 hours. Bar graphs demonstrate fold change (FC) in phagocytic activity (B) and histone lactylation status (C) of activated MHC-II^hi/PD-1^lo and MHC-II^hi/PD-1^hi TAM, relative to untreated group (U). (D-E) Single cell suspensions were prepared from human bone/lymph node metastatic PC patient samples (BMET-1/−2 and LMET-1/−2/−3) and underwent single-cell RNA using Chromium controller 10x Genomics platform. Annotated tumor cells, TAM and myeloid cells were characterized for aerobic glycolysis (D, E), phagocytosis suppression (D) and M1-polarization status (E), respectively, using a normalized gene score, as described in the methods section. For ex vivo studies, n=2 independent experiments and for bioinformatics analysis, n = 355 tumor cells and 389 TAM in bone metastatic PC patients; n = 367 tumor cells and 101 myeloid cells in lymph node metastatic PC patients. Significances/p-values were calculated by one-way ANOVA (panel B-C), Wilcoxon rank-sum text (panel D-E) and indicates as follows, *p<0.05, **p<0.01 and ***p<0.001; ns = not statistically significant.