Figure 3. TAMs promote HCC CSC expansion.

(A) Correlation of HCC TAM infiltration (Lin⁻CD11b⁺CD14⁺) and CD44⁺ HCC cells (Lin⁻CD44⁺) in HCC patients by FACS on fresh cell suspensions (n=17 patients, R=0.66, p<0.005, Spearman rank order correlation) and representative FACS shown.

(B) Correlation of CD14 and stemness-related genes in clinical HCC tumors (n=91, p<0.05) using a microarray database²⁰. Spearman rank order correlation co-efficient (R) and P-value (P) were determined.

(C) Enrichment of CD44⁺ HCC cells by TAMs after three day transwell co-culture followed by FACS of HepG2 cells (n=5 HCC patients), POU5F1 expression by qRT-PCR (n=5 HCC patients), and sphere formation capacity (n=10). Control is HepG2 cells alone. Representative FACS and spheres assays (10x mag) are shown.

(D) Increased tumor growth following co-culture with TAMs. 1000 HepG2 cells following co-culture without or with TAMs were injected subcutaneously into opposing flanks of NSG mice, respectively, and tumor volume determined, n=5 mice/group, one of four representative experiments is shown.

(E) CD44⁺ subset is expanded in vivo in response to TAMs. HCC tumors were established in NSG mice by HepG2 cell IP injection. Once established, TAMs were IP injected and HCC cells three days later underwent FACS for CD44 (n=5 mice/group).

*p < 0.05. Data presented as mean±SEM.