Shanshan Wan

Tumor-associated macrophages produce interleukin 6 and signal via STAT3 to promote expansion of human hepatocellular carcinoma stem cells.

BACKGROUND & AIMS Cancer stem cells (CSCs) can contribute to hepatocellular carcinoma (HCC) progression and recurrence after 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 signal transducer and activator of transcription 3 was knocked down with small hairpin RNAs in HepG2 cells. Xenograft tumors were grown in NOD-SCID/Il2Rg(null) 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 of xenograft tumors in mice. In human HCC samples, the numbers of TAMs correlated with the 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 signal transducer and activator of transcription 3 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.

Cancer mediates effector T cell dysfunction by targeting microRNAs and EZH2 via glycolysis restriction

Aerobic glycolysis regulates T cell function. However, whether and how primary cancer alters T cell glycolytic metabolism and affects tumor immunity in cancer patients remains a question. Here we found that ovarian cancers imposed glucose restriction on T cells and dampened their function via maintaining high expression of microRNAs miR-101 and miR-26a, which constrained expression of the methyltransferase EZH2. EZH2 activated the Notch pathway by suppressing Notch repressors Numb and Fbxw7 via trimethylation of histone H3 at Lys27 and, consequently, stimulated T cell polyfunctional cytokine expression and promoted their survival via Bcl-2 signaling. Moreover, small hairpin RNA–mediated knockdown of human EZH2 in T cells elicited poor antitumor immunity. EZH2+CD8+ T cells were associated with improved survival in patients. Together, these data unveil a metabolic target and mechanism of cancer immune evasion.

Myeloid-Derived Suppressor Cells Endow Stem-like Qualities to Breast Cancer Cells through IL6/STAT3 and NO/NOTCH Cross-talk Signaling

Myeloid-derived suppressor cells (MDSC) contribute to immune suppression in cancer, but the mechanisms through which they drive metastatic progression are not fully understood. In this study, we show how MDSC convey stem-like qualities to breast cancer cells that coordinately help enable immune suppression and escape. We found that MDSC promoted tumor formation by enhancing breast cancer cell stem-like properties as well as by suppressing T-cell activation. Mechanistic investigations indicated that these effects relied upon cross-talk between the STAT3 and NOTCH pathways in cancer cells, with MDSC inducing IL6-dependent phosphorylation of STAT3 and activating NOTCH through nitric oxide leading to prolonged STAT3 activation. In clinical specimens of breast cancer, the presence of MDSC correlated with the presence of cancer stem-like cells (CSC) and independently predicted poor survival outcomes. Collectively, our work revealed an immune-associated mechanism that extrinsically confers cancer cell stemness properties and affects patient outcome. We suggest that targeting STAT3-NOTCH cross-talk between MDSC and CSC could offer a unique locus to improve cancer treatment, by coordinately targeting a coupled mechanism that enables cancer stemness and immune escape. Cancer Res; 76(11); 3156-65. ©2016 AACR.

Myeloid cells in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is highly associated with inflammation. Myeloid cells, including tumor‐associated macrophages and myeloid‐derived suppressor cells, are abundant in the HCC microenvironment and are often associated with poor prognosis. Myeloid cells in HCC play a vital role in supporting tumor initiation, progression, angiogenesis, metastasis, and therapeutic resistance. Here, we summarize our current knowledge about myeloid cells in HCC and focus on their immune‐suppressive activities and tumor‐promoting functions, as well as the relevance to potential new therapies in HCC. (Hepatology 2015;62:1304‐1312)

CD4+ T lymphocyte ablation prevents pancreatic carcinogenesis in mice.

Zhang and colleagues show that Kras-expressing epithelial cells recruit CD4+ T cells that repress the activity of CD8+ T cells to establish the immunosuppressive microenvironment in the iKras* mouse model of pancreatic cancer; elimination of CD4+ T cells uncovers the antineoplastic function of CD8+ T cells. Pancreatic cancer, one of the deadliest human malignancies, is associated with oncogenic Kras and is most commonly preceded by precursor lesions known as pancreatic intraepithelial neoplasias (PanIN). PanIN formation is accompanied by the establishment of an immunotolerant microenvironment. However, the immune contribution to the initiation of pancreatic cancer is currently poorly understood. Here, we genetically eliminate CD4+ T cells in the iKras* mouse model of pancreatic cancer, in the context of pancreatitis, to determine the functional role of CD4+ T cells during mutant Kras-driven pancreatic carcinogenesis. We show that oncogenic Kras-expressing epithelial cells drive the establishment of an immunosuppressive microenvironment through the recruitment and activity of CD4+ T cells. Furthermore, we show that CD4+ T cells functionally repress the activity of CD8+ T cells. Elimination of CD4+ T cells uncovers the antineoplastic function of CD8+ T cells and blocks the onset of pancreatic carcinogenesis. Thus, our studies uncover essential and opposing roles of immune cells during PanIN formation and provide a rationale to explore immunomodulatory approaches in pancreatic cancer. Cancer Immunol Res; 2(5); 423–35. ©2014 AACR.

Neoadjuvant stereotactic body radiation therapy, capecitabine, and liver transplantation for unresectable hilar cholangiocarcinoma

Hilar cholangiocarcinoma (CCA) is a difficult malignancy to treat surgically because of its anatomical location and its frequent association with primary sclerosing cholangitis. Neoadjuvant chemoradiotherapy followed by liver transplantation in lymph node–negative patients has been advanced by select liver transplant centers for the treatment of patients with unresectable disease. This approach has most commonly used external‐beam radiotherapy in combination with biliary brachytherapy and 5‐fluorouracil–based chemotherapy. Our center recently embarked on a protocol using stereotactic body radiation therapy (SBRT) followed by capecitabine in lymph node–negative patients until liver transplantation. We, therefore, retrospectively determined the tolerability and pathological response in this pilot study. During a 3‐year period, 17 patients with unresectable hilar CCA were evaluated for treatment under this protocol. In all, 12 patients qualified for neoadjuvant therapy and were treated with SBRT (50‐60 Gy in 3‐5 fractions over the course of 2 weeks). After 1 week of rest, capecitabine was initiated at 1330 mg/m2/day, and it was continued until liver transplantation. During neoadjuvant therapy, there were 35 adverse events in all, with cholangitis and palmar‐plantar erythrodysesthesia being the most common. Capecitabine dose reductions were required on 5 occasions. Ultimately, 9 patients were listed for transplantation, and 6 patients received a liver transplant. The explant pathology of hilar tumors showed at least a partial treatment response in 5 patients, with extensive tumor necrosis and fibrosis noted. Additionally, high apoptotic indices and low proliferative indices were measured during histological examinations. Eleven transplant‐related complications occurred, and the 1‐year survival rate after transplantation was 83%. In this pilot study, neoadjuvant therapy with SBRT, capecitabine, and liver transplantation for unresectable CCA demonstrated acceptable tolerability. Further studies will determine the overall future efficacy of this therapy. Liver Transpl 20:81–88, 2014.

Alternative splicing of human NT5E in cirrhosis and hepatocellular carcinoma produces a negative regulator of ecto-5′-nucleotidase (CD73)

Alternative splicing of human NT5E generates CD73S, an endoplasmic reticulum–associated and dimerization-deficient glycoprotein that lacks enzymatic activity. CD73S functions as a negative regulator of canonical CD73 by promoting its proteasomal degradation, which may have significance in chronic liver disease and liver cancer.

Targeting ADAM17 inhibits human colorectal adenocarcinoma progression and tumor-initiating cell frequency

ADAM17 (a disintegrin and metalloproteinase 17)/TACE (TNFα converting enzyme) has emerged as a potential therapeutic target in colorectal cancer (CRC) and other cancers, due in part to its role in regulating various tumor cell surface proteins and growth factors and cytokines in the tumor microenvironment. The emergence of MEDI3622, a highly potent and specific antibody-based ADAM17 inhibitor, has allowed testing of the concept that targeting ADAM17 may be an important new therapeutic approach for CRC patients. We demonstrate that MEDI3622 is highly efficacious on tumor growth in multiple human CRC PDX models, resulting in improved survival of animals bearing tumor xenografts. MEDI3622 was further found to impact Notch pathway activity and tumor-initiating cells. The promising preclinical activity seen here supports further clinical investigation of this treatment approach to improve therapeutic outcome for patients diagnosed with metastatic CRC, including patients with KRAS-mutant tumors for whom other therapeutic options are currently limited.ADAM17 (a disintegrin and metalloproteinase 17)/TACE (TNFα converting enzyme) has emerged as a potential therapeutic target in colorectal cancer (CRC) and other cancers, due in part to its role in regulating various tumor cell surface proteins and growth factors and cytokines in the tumor microenvironment. The emergence of MEDI3622, a highly potent and specific antibody-based ADAM17 inhibitor, has allowed testing of the concept that targeting ADAM17 may be an important new therapeutic approach for CRC patients. We demonstrate that MEDI3622 is highly efficacious on tumor growth in multiple human CRC PDX models, resulting in improved survival of animals bearing tumor xenografts. MEDI3622 was further found to impact Notch pathway activity and tumor-initiating cells. The promising preclinical activity seen here supports further clinical investigation of this treatment approach to improve therapeutic outcome for patients diagnosed with metastatic CRC, including patients with KRAS-mutant tumors for whom other therapeutic options are currently limited.

KLRG1 restricts memory T cell antitumor immunity.

Killer cell lectin-like receptor subfamily G member 1 (KLRG1) has been found on human memory T lymphocytes. However, the roles of KLRG1 on human T cells especially in tumor microenvironment have not been fully understood. Our results showed KLRG1 expression on T cells significantly increased in tumor microenvironment. KLRG1+ T cells exhibited poor proliferative capacity with decreased effector cytokine production. Meanwhile, KLRG1+ T cells expressed abundant pro-inflammatory cytokines and demonstrated high level of Foxp3 expression. KLRG1+ T cells showed decreased expression of miRNA-101 and higher expression of CtBP2. Our results indicated KLRG1 might contribute to the impaired antitumor immunity of memory T cells in tumor microenvironment. Thus, repressing KLRG1 on human memory T cells might be a novel therapeutics against cancer.

Abstract 315: Immune cells promote hepatocellular carcinoma stemness phenotype

Hepatocellular carcinoma (HCC) is a common cause of cancer-related deaths and recurrence rates can be as high as 50% following resection. Evidence in several tumor types suggests that cancer stem cells (CSCs) contribute to tumorigenicity, therapeutic resistance and recurrence even in the presence of anti-tumor immunity. Furthermore, CSC phenotype may be influenced by tumor microenvironment. Therefore, we aimed to determine whether HCC stemness phenotype may be influenced by immune cell subsets. We have previously identified CD44 as a HCC-CSC marker as evidenced by enhanced tumorigenicity and sphere forming capacity in CD44 + HCC cells. To determine the effects of immune cells on HCC stemness, transwell co-culture experiments were performed using GFP-labeled HepG2 and T cells or monocytes/macrophages. The percentage of CD44 + HCC cells were increased during co-culture with anti-CD3/anti-CD28 activated T cells from donor PBMCs (n=9), suggesting that immune effector cells could enrich CD44 + HepG2 cells. In addition, CD44 + HCC cells were enriched during co-culture with HCC-associated macrophages (TAM) (n=3 HCC patients) or monocytes from healthy donor PBMCs (n=11). CD44 + HCC cells were positively correlated with TAM in primary HCC patients (R=0.69, n=12, p + HCC cells and promoted sphere formation capacity. In conclusion, HCC stemness phenotype is expanded by T cells, HCC associated macrophage and monocytes, as well as cytokines, IL-6 and IL-8. These findings suggest that immune cell derived factors in the tumor microenvironment may contribute to HCC recurrence or progression by promoting a stemness phenotype. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 315. doi:1538-7445.AM2012-315