Xueqing Xia

Universal Marker and Detection Tool for Human Sarcoma Circulating Tumor Cells

To date, no specific marker exists for the detection of circulating tumor cells (CTC) from different types of sarcomas, though tools are available for detection of CTCs in peripheral blood of patients with cancer for epithelial cancers. Here, we report cell-surface vimentin (CSV) as an exclusive marker on sarcoma CTC regardless of the tissue origin of the sarcoma as detected by a novel monoclonal antibody. Utilizing CSV as a probe, we isolated and enumerated sarcoma CTCs with high sensitivity and specificity from the blood of patients bearing different types of sarcoma, validating their phenotype by single cell genomic amplification, mutation detection, and FISH. Our results establish the first universal and specific CTC marker described for enumerating CTCs from different types of sarcoma, thereby providing a key prognosis tool to monitor cancer metastasis and relapse.

Epithelial–Mesenchymal Transitioned Circulating Tumor Cells Capture for Detecting Tumor Progression

Purpose: This study aimed to detect cell-surface vimentin (CSV) on the surface of epithelial–mesenchymal transitioned (EMT) circulating tumor cells (CTC) from blood of patients with epithelial cancers. Experimental Design: In this study, 101 patients undergoing postsurgery adjuvant chemotherapy for metastatic colon cancer were recruited. EMT CTCs were detected from blood of patients using the 84-1 monoclonal antibody against CSV as a marker. EMT CTCs isolated were characterized further using EMT-specific markers, fluorescent in situ hybridization, and single-cell mutation analysis. Results: Using the 84-1 antibody, we detected CSV exclusively on EMT CTCs from a variety of tumor types but not in the surrounding normal cells in the blood. The antibody exhibited very high specificity and sensitivity toward different epithelial cancer cells. With this antibody, we detected and enumerated EMT CTCs from patients. From our observations, we defined a cutoff of <5 or ≥5 EMT CTCs as the optimal threshold with respect to therapeutic response using ROC curves. Using this defined threshold, the presence of ≥5 EMT CTCs was associated with progressive disease, whereas patients with <5 EMT CTCs showed therapeutic response. Conclusion: Taken together, the number of EMT CTCs detected correlated with the therapeutic outcome of the disease. These results establish CSV as a universal marker for EMT CTCs from a wide variety of tumor types and thus provide the foundation for emerging CTC detection technologies and for studying the molecular regulation of these EMT CTCs. Clin Cancer Res; 21(4); 899–906. ©2014 AACR.

Interleukin-30: A novel antiinflammatory cytokine candidate for prevention and treatment of inflammatory cytokine-induced liver injury

The liver is the major metabolic organ and is subjected to constant attacks from chronic viral infection, uptake of therapeutic drugs, life behavior (alcoholic), and environmental contaminants, all of which result in chronic inflammation, fibrosis, and, ultimately, cancer. Therefore, there is an urgent need to discover effective therapeutic agents for the prevention and treatment of liver injury, the ideal drug being a naturally occurring biological inhibitor. Here we establish the role of IL30 as a potent antiinflammatory cytokine that can inhibit inflammation‐induced liver injury. In contrast, interleukin (IL)27, which contains IL30 as a subunit, is not hepatoprotective. Interestingly, IL30 is induced by the proinflammatory signal such as IL12 through interferon‐gamma (IFN‐γ) / signal transducer and activator of transcription 1 signaling. In animal models, administration of IL30 by way of a gene therapy approach prevents and treats both IL12‐, IFN‐γ‐, and concanavalin A‐induced liver toxicity. Likewise, immunohistochemistry analysis of human tissue samples revealed that IL30 is highly expressed in hepatocytes, yet barely expressed in inflammation‐induced tissue such as fibrous/connective tissue. Conclusion: These novel observations reveal a novel role of IL30 as a therapeutic cytokine that suppresses proinflammatory cytokine‐associated liver toxicity. (Hepatology 2012)

Discovery of a Linear Peptide for Improving Tumor Targeting of Gene Products and Treatment of Distal Tumors by IL-12 Gene Therapy

Like many effective therapeutics, interleukin-12 (IL-12) therapy often causes side effects. Tumor targeted delivery may improve the efficacy and decrease the toxicity of systemic IL-12 treatments. In this study, a novel targeting approach was investigated. A secreted alkaline phosphatase (SEAP) reporter gene-based screening process was used to identify a mini-peptide which can be produced in vivo to target gene products to tumors. The coding region for the best peptide was inserted into an IL-12 gene to determine the antitumor efficacy. Affinity chromatography, mass spectrometry analysis, and binding studies were used to identify a receptor for this peptide. We discovered that the linear peptide VNTANST increased the tumor accumulation of the reporter gene products in five independent tumor models including one human xenogeneic model. The product from VNTANST-IL-12 fusion gene therapy increased accumulation of IL-12 in the tumor environment, and in three tumor models, VNTANST-IL-12 gene therapy inhibited distal tumor growth. In a spontaneous lung metastasis model, inhibition of metastatic tumor growth was improved compared to wild-type IL-12 gene therapy, and in a squamous cell carcinoma model, toxic liver lesions were reduced. The receptor for VNTANST was identified as vimentin. These results show the promise of using VNTANST to improve IL-12 treatments.

Cell-surface Vimentin: A mislocalized protein for isolating csVimentin+CD133- novel stem-like hepatocellular carcinoma cells expressing EMT markers

Recent advances in cancer stem cell biology have shown that cancer stem‐like cells with epithelial–mesenchymal transition (EMT) phenotypes are more aggressive and cause relapse; however, absence of a specific marker to isolate these EMT stem‐like cells hampers research in this direction. Cell surface markers have been identified for isolating cancer stem‐like cells, but none has been identified for isolating cancer stem‐like cells with EMT phenotype. Recently, we discovered that Vimentin, an intracellular EMT tumor cell marker, is present on the surface of colon metastatic tumor nodules in the liver. In our study, we examined the potential of targeting cell surface Vimentin (CSV) to isolate stem‐like cancer cells with EMT phenotype, by using a specific CSV‐binding antibody, 84‐1. Using this antibody, we purified the CSV‐positive, CD133‐negative (csVim+CD133−) cell population from primary liver tumor cell suspensions and characterized for stem cell properties. The results of sphere assays and staining for the stem cell markers Sox2 and Oct4A demonstrated that csVim+CD133− cells have stem‐like properties similar to csVim−CD133+ population. Our investigation further revealed that the csVim+CD133− cells had EMT phenotypes, as evidenced by the presence of Twist and Slug in the nucleus, the absence of EpCAM on the cell surface and basal level of expression of epithelial marker E‐cadherin. The csVimentin‐negative CD133‐positive stem cells do not have any EMT phenotypes. csVim+CD133− cells exhibited more aggressively metastatic in livers than csVim−CD133+ cells. Our findings indicate that csVim+CD133− cells are promising targets for treatment and prevention of metastatic hepatocellular carcinoma.

FGL2 as a Multimodality Regulator of Tumor-Mediated Immune Suppression and Therapeutic Target in Gliomas

BACKGROUND Fibrinogen-like protein 2 (FGL2) may promote glioblastoma multiforme (GBM) cancer development by inducing multiple immune-suppression mechanisms. METHODS The biological significance of FGL2 expression was assessed using the The Cancer Genome Atlast (TCGA) glioma database and tumor lysates analysis. The therapeutic effects of an anti-Fgl2 antibody and the role of immune suppression regulation by Fgl2 were determined in immune-competent, NOD-scid IL2Rgammanull (NSG), and FcɣRIIB-/- mice (n = 3-18 per group). Data were analyzed with two-way analysis of variance, log-rank survival analysis, and Pearson correlation. All statistical tests were two-sided. RESULTS In low-grade gliomas, 72.5% of patients maintained two copies of the FGL2 gene, whereas 83.8% of GBM patients had gene amplification or copy gain. Patients with high levels of FGL2 mRNA in glioma tissues had a lower overall survival (P = .009). Protein levels of FGL2 in GBM lysates were higher relative to low-grade glioma lysates (11.48±5.75ng/mg vs 3.96±1.01ng/mg, P = .003). In GL261 mice treated with an anti-FGL2 antibody, median survival was 27 days compared with only 17 days for mice treated with an isotype control antibody (P = .01). The anti-FGL2 antibody treatment reduced CD39(+) Tregs, M2 macrophages, programmed cell death protein 1 (PD-1), and myeloid-derived suppressor cells (MDSCs). FGL2-induced increases in M2, CD39, and PD-1 were ablated in FcɣRIIB-/- mice. CONCLUSIONS FGL2 augments glioma immunosuppression by increasing the expression levels of PD-1 and CD39, expanding the frequency of tumor-supportive M2 macrophages via the FcγRIIB pathway, and enhancing the number of MDSCs and CD39(+) regulatory T cells. Collectively, these results show that FGL2 functions as a key immune-suppressive modulator and has potential as an immunotherapeutic target for treating GBM.

Cell-surface Vimentin

Recent advances in cancer stem cell biology have shown that cancer stem‐like cells with epithelial–mesenchymal transition (EMT) phenotypes are more aggressive and cause relapse; however, absence of a specific marker to isolate these EMT stem‐like cells hampers research in this direction. Cell surface markers have been identified for isolating cancer stem‐like cells, but none has been identified for isolating cancer stem‐like cells with EMT phenotype. Recently, we discovered that Vimentin, an intracellular EMT tumor cell marker, is present on the surface of colon metastatic tumor nodules in the liver. In our study, we examined the potential of targeting cell surface Vimentin (CSV) to isolate stem‐like cancer cells with EMT phenotype, by using a specific CSV‐binding antibody, 84‐1. Using this antibody, we purified the CSV‐positive, CD133‐negative (csVim+CD133−) cell population from primary liver tumor cell suspensions and characterized for stem cell properties. The results of sphere assays and staining for the stem cell markers Sox2 and Oct4A demonstrated that csVim+CD133− cells have stem‐like properties similar to csVim−CD133+ population. Our investigation further revealed that the csVim+CD133− cells had EMT phenotypes, as evidenced by the presence of Twist and Slug in the nucleus, the absence of EpCAM on the cell surface and basal level of expression of epithelial marker E‐cadherin. The csVimentin‐negative CD133‐positive stem cells do not have any EMT phenotypes. csVim+CD133− cells exhibited more aggressively metastatic in livers than csVim−CD133+ cells. Our findings indicate that csVim+CD133− cells are promising targets for treatment and prevention of metastatic hepatocellular carcinoma.

CD8+T cell–specific induction of NKG2D receptor by doxorubicin plus interleukin-12 and its contribution to CD8+T cell accumulation in tumors

BackgroundIncreased infiltration of CD8+T cells into tumors has a positive impact on survival. Our previous study showed that doxorubicin (Dox) plus interleukin-12 (IL-12) boosted the accumulation of CD8+T cells in tumors and had a greater antitumor effect than did either agent alone. The purpose of this study was to determine the impact of NKG2D expression on CD8+T cell infiltration and antitumor efficacy.MethodsTumor-bearing mice were administered Dox, IL-12 plasmid DNA, or both via intraperitoneal injection or intramuscular electroporation. The induction of NKG2D on CD8+T cells and other lymphocytes was analyzed via flow cytometry, and NKG2D-positive CD8+T cell–specific localization in tumors was determined by using immunofluorescence staining in various types of immune cell–depleted mice.ResultsThe combination of Dox plus IL-12 specifically increased expression of NKG2D in CD8+T cells but not in other types of immune cells, including NK cells, which naturally express NKG2D. This induced NKG2D expression in CD8+T cells was associated with increased accumulation of CD8+T cells in murine tumors. Administration of NKG2D-blocking antibody or CD8+T cell–depletion antibody abrogated the NKG2D+CD8+T cell detection in tumors, whereas administration of NK cell–depletion antibody had no effect. Increased NKG2D expression in CD8+T cells was associated with increased antitumor efficacy in vivo.ConclusionWe conclude that Dox plus IL-12 induces NKG2D in CD8+T cells in vivo and boosts NKG2D+CD8+T-dependent antitumor immune surveillance. This discovery reveals a novel mechanism for how chemoimmunotherapy synergistically promotes T cell–mediated antitumor immune surveillance.

Immune checkpoint regulator PD-L1 expression on tumor cells by contacting CD11b positive bone marrow derived stromal cells.

BackgroundExpression of programmed cell death ligand 1 (PD-L1) is an important process by which tumor cells suppress antitumor immunity in the tumor microenvironment. Bone marrow (BM)–derived immune cells are an important component of the tumor microenvironment. However, the link between PD-L1 induction on tumor cells and communication with BM cells is unknown.ResultsThis study demonstrates that BM cells have a direct effect in inducing PD-L1 expression on tumor cells, which contributes to the tumor cells’ drug resistance. This novel discovery was revealed using a co-incubation system with BM cells and tumor cells. BM cells from wild-type C57BL6 mice and the immune-deficient mouse strains B-cell−/−, CD28−/−, perforin−/−, and Rag2−/− but not CD11b−/− dramatically increased the expression of tumor cell surface PD-L1. This PD-L1 induction was dependent on CD11b-positive BM cells through direct contact with tumor cells. Furthermore, p38 signaling was activated in tumor cells after co-incubation with BM cells, whereas the expression of PD-L1 was remarkably decreased after co-culture of cells treated with a p38 inhibitor. The increase in PD-L1 induced by BM cell co-culture protected tumor cells from drug-induced apoptosis.ConclusionsPD-L1 expression is increased on tumor cells by direct contact with BM-derived CD11b-positive cells through the p38 signaling pathway. PD-L1 may play an important role in drug resistance, which often causes failure of the antitumor response.

IL6 mediated inflammatory loop reprograms normal to EMT(+) metastatic CSCs in pre-neoplastic liver of TGFβ deficient β2SP(+/-) mice.

Hepatocellular carcinoma (HCC) is the second‐leading cause of cancer‐related deaths worldwide with a poor survival rate. As many as 40% of HCCs are clonal, with alteration of key tumor‐suppressor pathways in stem cells as the primary cause of HCC initiation. However, mechanisms that generate metastatic stem cells in preneoplastic liver tissue are not well understood. We hypothesized that chronic inflammation is a major driver of the transformation of genetically defective liver stem cells (LSCs) into highly metastatic liver cancer cells in premalignant liver tissue. We developed models of chronic inflammation in wild‐type (WT) and β2‐spectrin (β2SP)+/− (SPTBN1) mice. CD133+ LSCs derived from preneoplastic livers of β2SP+/− mice treated with interleukin‐6 (pIL6; IL6β2SP+/− LSCs) were highly tumorigenic and metastatic, whereas those derived from WT mice treated with pIL6 (IL6WT LSCs) had significantly less proliferation and no tumorigenic properties. IL6β2SP+/− LSCs not only exhibited nuclear localization of Twist and Slug, markers of epithelial‐mesenchymal transition (EMT), but also constitutive activation of nuclear factor kappa B (NFκB; RELA). Knockdown of NFκB decreased the EMT phenotypes and metastatic capacity of these cells. NFκB in IL6β2SP+/− LSCs was activated by transforming growth factor β (TGFβ)‐activated kinase 1 (TAK1; MAP3K7), which is associated with poor survival in HCC and interleukin‐6 (IL6) expression. The amount of constitutively activated NFκB increased dramatically from normal to cirrhotic to HCC tissues from human patients. Conclusion: IL6‐mediated inflammation programs constitutive activation of the TAK1‐NFκB signaling cascade in CD133+ LSCs, and this program interacts with deficient TGFβ signaling, thereby accelerating the transformation of normal LSCs to metastatic cancer stem cells (mCSCs). Indeed, this study delineates the development of EMT‐positive mCSCs in HCC‐free liver tissue upon chronic inflammation. (Hepatology 2017;65:1222‐1236).