Tumor-Associated Macrophages Produce Interleukin 6 and Signal via STAT3 to Promote Expansion of Human Hepatocellular Carcinoma Stem Cells

Abstract

Background & Aims Cancer stem cells (CSCs) can contribute to hepatocellular carcinoma (HCC) progression and recurrence following therapy. The presence of tumor-associated macrophages (TAMs) in patients with HCC is associated with poor outcomes. It is not clear whether TAMs interact with CSCs during HCC development. We investigated whether TAMs affect the activities of CSCs in the microenvironment of human HCCs. Methods HCCs were collected from 17 patients during surgical resection and single cell suspensions were analyzed by flow cytometry. CD14+ TAMs were isolated from the HCC cell suspensions and placed into co-culture with HepG2 or Hep3B cells, and CSC functions were measured. The interleukin 6 (IL6) receptor was blocked with a monoclonal antibody (tocilizumab), and STAT3 was knocked down with small hairpin RNAs in HepG2 cells. Xenograft tumors were grown in NOD-SCID/Il2Rgnull mice from human primary HCC cells or HepG2 cells. Results CD44+ cells from human HCCs and cell lines formed more spheres in culture and more xenograft tumors in mice than CD44− cells, indicating that CD44+ cells are CSCs. Incubation of the CD44+ cells with TAMs promoted expansion of CD44+ cells, and increased their sphere formation in culture and formation xenograft tumors in mice. In human HCC samples, numbers of TAMs correlated with numbers of CD44+ cells. Of all cytokines expressed by TAMs, IL6 was increased at the highest level in human HCC co-cultures, compared with TAMs not undergoing co-culture. IL6 was detected in the microenvironment of HCC samples and induced expansion of CD44+ cells in culture. Levels of IL6 correlated with stages of human HCCs and detection of CSC markers. Incubation of HCC cell lines with tocilizumab or knockdown of STAT3 in HCC cells reduced the ability of TAMs to promote sphere formation by CD44+ cells in culture and growth of xenograft tumors in mice. Conclusions CD44+ cells isolated from human HCC tissues and cell lines have CSC activities in vitro and form a larger number of xenograft tumors in mice than CD44− cells. TAMs produce IL6, which promotes expansion of these CSCs and tumorigenesis. Levels of IL6 in human HCC samples correlate with tumor stage and markers of CSCs. Blockade of IL6 signaling with tocilizumab, a drug approved by the Food and Drug Administration for treatment of rheumatoid arthritis, inhibits TAM-stimulated activity of CD44+ cells. This drug might be used to treat patients with HCC.

FAP Promotes Immunosuppression by Cancer-Associated Fibroblasts in the Tumor Microenvironment via STAT3–CCL2 Signaling

“…However, we still could not exclude other mechanisms by which MDSCs or macrophages contribute to tumor growth. For example, MDSCs and macrophages were recently reported to promote cancer stemness (35,36). We also confirmed it by showing that coculture of MSDCs enhanced the expression of stemness-related genes of Hepa1-6 cells and their proliferation.…”

Tumor-induced myeloid deviation: when myeloid-derived suppressor cells meet tumor-associated macrophages

“…In pancreatic tumors, TAM depletion arrests the proliferation of tumor-initiating cells (95). Indeed, TAMs can sustain CSC proliferation by releasing proinflammatory cytokines such as TNF-α and IL-6, which reinforce tumor cell proliferation through NF-κB and STAT3 signaling pathways (96,97). These same molecular pathways may be activated through a direct TAM-to-CSC contact via CD90 and ephrin A4 receptors (98).…”

STAT3 is a key transcriptional regulator of cancer stem cell marker CD133 in HCC

“…And in fact a recent study showed that tumor-associated macrophages (TAMs) promote the expansion of CSCs in HCC. Tocilizumab is a recently FDA approved drug that targets IL-6 receptor, and treatment of HCC cells with tocilizumab or STAT3 knockdown reduced the ability of TAMs to not only promote CSC expansion but also growth of xenograft tumors (8). Thus, STAT3 signaling appears to be a promising therapeutic target for HCC treatment.…”