Dong-Ming Kuang

Activated monocytes in peritumoral stroma of hepatocellular carcinoma foster immune privilege and disease progression through PD-L1

Macrophages (Mφ) are prominent components of solid tumors and exhibit distinct phenotypes in different microenvironments. We have recently found that tumors can alter the normal developmental process of Mφ to trigger transient activation of monocytes in peritumoral stroma. We showed that a fraction of monocytes/Mφ in peritumoral stroma, but not in cancer nests, expresses surface PD-L1 (also termed B7-H1) molecules in tumors from patients with hepatocellular carcinoma (HCC). Monocytes activated by tumors strongly express PD-L1 proteins with kinetics similar to their activation status, and significant correlations were found between the levels of PD-L1+ and HLA-DRhigh on tumor-infiltrating monocytes. Autocrine tumor necrosis factor α and interleukin 10 released from activated monocytes stimulated monocyte expression of PD-L1. The PD-L1+ monocytes effectively suppressed tumor-specific T cell immunity and contributed to the growth of human tumors in vivo; the effect could be reversed by blocking PD-L1 on those monocytes. Moreover, we found that PD-L1 expression on tumor-infiltrating monocytes increased with disease progression, and the intensity of the protein was associated with high mortality and reduced survival in the HCC patients. Thus, expression of PD-L1 on activated monocytes/Mφ may represent a novel mechanism that links the proinflammatory response to immune tolerance in the tumor milieu.

Peritumoral neutrophils link inflammatory response to disease progression by fostering angiogenesis in hepatocellular carcinoma

BACKGROUND & AIMS Substantial evidence indicates that inflammation is a critical component of tumor progression. Hepatocellular carcinoma (HCC) is usually derived from inflamed cirrhotic liver with extensive leukocyte infiltration. Neutrophils are the common inflammatory infiltrate in tumors, but their nature and regulation in human cancers remain elusive. METHODS A total of 238 HCC patients were enrolled randomly. Immunohistochemistry and SuperArray Real-Time PCR were used to analyze the distribution and clinical relevance of neutrophils in different microanatomical areas. The regulation and function of neutrophils were assessed by both in vitro and in vivo studies. RESULTS Neutrophils were enriched predominantly in peritumoral stroma of HCC tissues and their levels could serve as a powerful predictor for poor survival in HCC patients. Proinflammatory IL-17 is a critical mediator of the recruitment of neutrophils into peritumoral stroma of HCC tissues by epithelial cell-derived CXC chemokines. The accumulated peritumoral neutrophils were the major source of matrix metalloproteinase-9 in HCC tissues; this secreted protein stimulated proangiogenic activity in hepatoma cells. Accordingly, high infiltration of peritumoral neutrophils was positively correlated with angiogenesis progression at tumor-invading edge of HCC patients. Furthermore, we found that selective depletion of neutrophils effectively inhibited tumor angiogenesis and growth, in vivo. CONCLUSIONS These data provide direct evidence supporting the critical role of neutrophils in human tumor progression and reveal a fine-tuned collaborative action between cancer cells and immune cells in distinct tumor milieu, which reroutes the inflammatory response into a tumor-promoting direction.

Tumor-Activated Monocytes Promote Expansion of IL-17–Producing CD8+ T Cells in Hepatocellular Carcinoma Patients

The proinflammatory IL-17–producing CD8+ T cells (Tc17 cells) have recently been detected in tumors, but the nature and regulation of these cells in human tumors are presently unknown. We have recently found that IL-17+ cells are accumulated in human hepatocellular carcinomas (HCC), where they promote disease progression by fostering angiogenesis. In this study, we showed that Tc17 cells constitute a remarkable portion of IL-17–producing cells in human HCC. Although most circulating Tc17 cells were negative for IFN-γ, >80% of Tc17 cells in HCC tissues were positive for IFN-γ, and they were enriched predominantly in invading tumor edge. Most CD68+ cells located in invading tumor edge exhibited an activated phenotype and, accordingly, the activated monocytes isolated from HCC tissues were significantly superior to those isolated from nontumor tissues in inducing expansion of Tc17 cells in vitro with phenotypic features similar to those isolated from tumors. Compared with IL-17−IFN-γ+CD8+ cells, these IFN-γ+Tc17 cells have significantly higher expression of proinflammatory cytokines (IL-2, IL-22, and TNF-α), but reduced expression of granzyme B and perforin. Moreover, we found that tumor-activated monocytes secreted a set of key cytokines (IL-1β, IL-6, and IL-23) to trigger the proliferation of Tc17 cells. These data reveal an intriguing mechanism in which human Tc17 cells are generated by a fine-tuned collaborative action between different types of immune cells in distinct tumor microenvironments.

Neutrophils promote motility of cancer cells via a hyaluronan-mediated TLR4/PI3K activation loop.

Inflammation is a component of tumour progression mechanisms. Neutrophils are a common inflammatory infiltrate in many tumours, but their regulation and functions in neoplasia are not understood. We recently demonstrated that pro‐inflammatory IL‐17‐producing cells recruited blood neutrophils into the peritumoural stroma of hepatocellular carcinoma by epithelium‐derived CXC chemokines. Here we show that a substantial population of neutrophils accumulates in the peritumoural stroma of hepatocellular, cervical, colorectal, and gastric carcinomas, and that this correlates with metastases in hepatocellular and gastric carcinomas. Exposure of neutrophils to culture supernatants from several types of solid tumour cells (TSN) resulted in sustained survival and pro‐tumourigenic effects of cells. Kinetic experiments reveal that, shortly after exposure to TSN, neutrophils began to provoke activation and then produced significant inflammatory cytokines and expressed more anti‐apoptotic Mcl‐1 but less pro‐apoptotic Bax. These long‐lived neutrophils effectively enhanced the cancer cell motility via a contact‐dependent mechanism; this effect, together with early activation and subsequent longevity of TSN‐exposed neutrophils, could be reversed by blocking the activation of PI3K/Akt signalling in neutrophils. Moreover, we found that hyaluronan (HA) fragments constitute a common factor produced by various tumours that mimics the effect of TSN to induce long‐lived neutrophils and subsequent malignant cell migration. The effects of TSN were inhibited by function blocking interactions between HA and its receptor TLR4 on neutrophils, suggesting that this is a key signalling pathway involved. These results indicate that HA derived from malignant cells educates neutrophils to adopt an activated phenotype, and in that way stimulates the metastasis of malignant cells, which represents a positive regulatory loop between tumours and their stroma during neoplastic progression. Copyright

Activated CD69+ T Cells Foster Immune Privilege by Regulating IDO Expression in Tumor-Associated Macrophages

Substantial evidence indicates that immune activation at stroma can be rerouted in a tumor-promoting direction. CD69 is an immunoregulatory molecule expressed by early-activated leukocytes at sites of chronic inflammation, and CD69+ T cells have been found to promote human tumor progression. In this study, we showed that, upon encountering autologous CD69+ T cells, tumor macrophages (MΦs) acquired the ability to produce much greater amounts of IDO protein in cancer nests. The T cells isolated from the hepatocellular carcinoma tissues expressed significantly more CD69 molecules than did those on paired circulating and nontumor-infiltrating T cells; these tumor-derived CD69+ T cells could induce considerable IDO in monocytes. Interestingly, the tumor-associated monocytes/MΦs isolated from hepatocellular carcinoma tissues or generated by in vitro culture effectively activated circulating T cells to express CD69. IL-12 derived from tumor MΦs was required for early T cell activation and subsequent IDO expression. Moreover, we found that conditioned medium from IDO+ MΦs effectively suppressed T cell responses in vitro, an effect that could be reversed by adding extrinsic IDO substrate tryptophan or by pretreating MΦs with an IDO inhibitor 1-methyl-DL-tryptophan. These data revealed a fine-tuned collaborative action between different types of immune cells to counteract T cell responses in tumor microenvironment. Such an active induction of immune tolerance should be considered for the rational design of effective immune-based anticancer therapies.

Monocyte/macrophage-elicited natural killer cell dysfunction in hepatocellular carcinoma is mediated by CD48/2B4 interactions.

Defects in natural killer (NK) cell functions are necessary for tumor immune escape, but their underlying regulatory mechanisms in human cancers remain largely unknown. Here we show, in detailed studies of NK cells in 294 untreated patients with hepatocellular carcinoma (HCC), that accumulation of functional NK cells in HCC tissues could predict improved survival of patients. However, in patients with advanced‐stage HCC, NK cells were significantly decreased in number with impaired tumor necrosis factor alpha (TNF‐α) and interferon‐gamma (IFN‐γ) production. High infiltration of peritumoral stroma monocytes/macrophages was positively correlated with impaired functional activities of NK cells in intratumoral areas. Further kinetic experiments revealed that soon after exposure to tumor‐derived monocytes, NK cells underwent a rapid, transient activation, but then they became exhausted, and eventually died. The monocytes from HCC tissues, but not from nontumoral liver, strongly express CD48 proteins; and such monocyte‐induced NK cell dysfunction was markedly attenuated by blocking CD48 receptor 2B4 on NK cells, but not by blockade of NKG2D and NKp30. Conclusion: These data reveal that human NK cells are regulated by a fine‐tuned collaborative action between different types of immune cells, which may reflect a novel immune‐escape mechanism by which tumors dynamically regulate their functions at distinct tumor microenvironments. (HEPATOLOGY 2013)

Interleukin‐17‐educated monocytes suppress cytotoxic T‐cell function through B7‐H1 in hepatocellular carcinoma patients

Substantial evidence indicates that inflammation is a critical component of tumor progression. The proinflammatory IL‐17‐producing cells have recently been detected in tumors, but the effect of IL‐17 on antigen‐presenting cells in tumors is presently unknown. We recently found that B7‐H1+ macrophages (Mφs) were enriched predominantly in the peritumoral stroma of hepatocellular carcinomas (HCCs). Here, we found a positive correlation between IL‐17‐producing cells and B7‐H1‐expressing Mφs in the same area. The B7‐H1+ monocytes/Mφs from HCC tissues expressed significantly more HLA‐DR, CD80, and CD86 than B7‐H1– cells. Accordingly, IL‐17 could activate monocytes to express B7‐H1 in a dose‐dependent manner. Although culture supernatants derived from hepatoma cells also induced B7‐H1 expression on monocytes, IL‐17 additionally increased hepatoma‐mediated B7‐H1 expression. Autocrine inflammatory cytokines released from IL‐17‐activated monocytes stimulated B7‐H1 expression. Moreover, these IL‐17‐exposed monocytes effectively suppressed cytotoxic T‐cell immunity in vitro; the effect could be reversed by blocking B7‐H1 on those monocytes. Consistent with this, cytotoxic T cells from HCC tissues expressed significant B7‐H1 receptor programmed death 1 (PD‐1) and exhibited an exhausted phenotype. These data reveal a fine‐tuned collaborative action between different stromal cells to counteract T‐cell responses in tumors. Such IL‐17‐mediated immune tolerance should be considered for the rational design of effective immune‐based anti‐cancer therapies.

Human Macrophages Promote the Motility and Invasiveness of Osteopontin-Knockdown Tumor Cells

Increasing evidence indicates that macrophages in tumor stroma can significantly modify the malignant phenotypes of tumors. Osteopontin (OPN) is frequently overexpressed in cancers with high metastatic capacity and, thus, has been considered as a potential therapeutic target. To find out whether macrophages can affect the outcome of OPN-knockdown tumor cells, we used RNA interference (RNAi) to stably silence the OPN expression in the highly invasive human hepatoma cell line SK-Hep-1. Silencing of OPN markedly decreased the motility and invasiveness of the SK-Hep-1 cells. Further studies using this cell model revealed that coculture with human macrophages or macrophage-conditioned medium largely restored the migration and invasion potential of OPN-knockdown tumor cells. Moreover, such macrophage-promoted motility can be effectively blocked either by the addition of OPN-neutralizing antibody to the cocultured medium or by silencing OPN expression in macrophages. These results indicate that macrophage-derived OPN can compensate for the decrease of OPN and thereby restore the metastatic potential of OPN-knockdown tumor cells. Further characterization of the underlying mechanisms disclosed that macrophage-derived OPN exerted its function independently of the actin cytoskeleton rearrangement or the activation of matrix metalloproteinase and Rho families. Our results suggest that there are fine-tuned complex interactions between cancer cells and stroma cells, which may modify the outcome of cancer therapy, and therefore should be considered for the rational design of anticancer strategy.

Increased autophagy sustains the survival and pro-tumourigenic effects of neutrophils in human hepatocellular carcinoma

BACKGROUND & AIMS Neutrophils are common cells of the inflammatory infiltrate and are predominantly enriched in many cancers. We recently found that neutrophils are accumulated in human hepatocellular carcinoma (HCC), where they promote disease progression by releasing matrix metalloproteinase-9 (MMP9). The underlying mechanisms, however, that allow tumour microenvironments to educate neutrophils are largely unknown. METHODS Neutrophils were purified from HCC patients and healthy donors. Immunohistochemistry and immunoblotting were used for the evaluation of autophagy in neutrophils. The regulation and function of increased neutrophil autophagy were assessed by both in vitro and ex vivo studies. RESULTS Most neutrophils in HCC intratumoural regions, in contrast to those located in the paired non-tumoural areas and within tumour vessels, substantially expressed autophagy-specific protein LC3. Soluble factors derived from hepatoma, including hyaluronan fragments, triggered a considerable increase of functional LC3 and autophagosomes in neutrophils, but this was unrelated to the deactivation of mTOR signalling. Inhibiting the activation of Erk1/2, p38, and NF-κB signals could significantly attenuate such tumour-elicited autophagy. These neutrophils, undergoing autophagy, exhibited long-lived phenotypes with retained Mcl-1 and significantly more intact mitochondria as well as low cleaved caspase-3, which could be abolished by inhibiting the initiation of autophagy. Moreover, increased neutrophil autophagy also correlated with sustained production of pro-metastatic oncostatin M and MMP9 and advanced migration of cancer cells. CONCLUSIONS Increased autophagy in neutrophils may represent a novel mechanism that links the innate response to neoplastic progression in humans. Studying the mechanisms that selectively modulate neutrophil autophagy will provide a novel strategy for anti-cancer therapy.

B7-H1–expressing antigen-presenting cells mediate polarization of protumorigenic Th22 subsets

Classical IL-22-producing T helper cells (Th22 cells) mediate inflammatory responses independently of IFN-γ and IL-17; however, nonclassical Th22 cells have been recently identified and coexpress IFN-γ and/or IL-17 along with IL-22. Little is known about how classical and nonclassical Th22 subsets in human diseases are regulated. Here, we used samples of human blood, normal and peritumoral liver, and hepatocellular carcinoma (HCC) to delineate the phenotype, distribution, generation, and functional relevance of various Th22 subsets. Three nonclassical Th22 subsets constituted the majority of all Th22 cells in human liver and HCC tissues, although the classical Th22 subset was predominant in blood. Monocytes activated by TLR2 and TLR4 agonists served as the antigen-presenting cells (APCs) that most efficiently triggered the expansion of nonclassical Th22 subsets from memory T cells and classical Th22 subsets from naive T cells. Moreover, B7-H1-expressing monocytes skewed Th22 polarization away from IFN-γ and toward IL-17 through interaction with programmed death 1 (PD-1), an effect that can create favorable conditions for in vivo aggressive cancer growth and angiogenesis. Our results provide insight into the selective modulation of Th22 subsets and suggest that strategies to influence functional activities of inflammatory cells may benefit anticancer therapy.