Abhisek Mitra

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.

Technologies for deriving primary tumor cells for use in personalized cancer therapy

For decades, immortal cancer cell lines have constituted an accessible, easily usable set of biological models to investigate cancer biology and explore the potential efficacy of anticancer drugs. However, numerous studies have suggested that these cell lines poorly represent the diversity, heterogeneity, and drug-resistant tumors occurring in patients. The derivation and short-term culture of primary cells from solid tumors have thus gained significant importance in personalized cancer therapy. This review focuses on our current understanding and the pros and cons of different methods for primary tumor cell culture. Furthermore, various culture matrices such as biomimetic scaffolds and chemically defined media supplemented with essential nutrients, have been prepared for different tissues. These well-characterized primary tumor cells redefine cancer therapies with high translational relevance.

IL‐30 (IL27p28) attenuates liver fibrosis through inducing NKG2D‐rae1 interaction between NKT and activated hepatic stellate cells in mice

Chronic hepatic diseases, such as cirrhosis, hepatocellular carcinoma, and virus‐mediated immunopathogenic infections, affect billions of people worldwide. These diseases commonly initiate with fibrosis. Owing to the various side effects of antifibrotic therapy and the difficulty of diagnosing asymptomatic patients, suitable medication remains a major concern. To overcome this drawback, the use of cytokine‐based sustained therapy might be a suitable alternative with minimal side effects. Here, we studied the therapeutic efficacy and potential mechanisms of interleukin (IL)−30 as antifibrosis therapy in murine liver fibrosis models. CCl4 or 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine (DDC) 0.1% (wt/wt) Purina 5015 Chow (LabDiet, St. Louis, MO) was fed for 3 weeks to induce liver fibrosis. Either control vector (pCtr) or pIL30 was injected hydrodynamically once per week. A significant decrease in collagen deposition and reduced expression of alpha‐smooth muscle actin (α‐SMA) protein indicated that IL‐30‐based gene therapy dramatically reduced bridging fibrosis that was induced by CCl4 or DDC. Immunophenotyping and knockout studies showed that IL‐30 recruits natural‐killer–like T (NKT) cells to the liver to remove activated hepatic stellate cells (HSCs) significantly and ameliorate liver fibrosis. Both flow cytometric and antibody‐mediated neutralization studies showed that liver NKT cells up‐regulate the natural killer group 2, member D (NKG2D) ligand and bind with the NKG2D ligand, retinoic acid early inducible 1 (Rae1), and positively activated HSCs to ameliorate liver fibrosis. Furthermore, adoptive transfer of liver NKT cells in T‐cell‐deficient mice showed reduction of fibrosis upon IL‐30 administration. Conclusions: Highly target‐specific liver NKT cells selectively remove activated HSCs through an NKG2D‐Rae1 interaction to ameliorate liver fibrosis after IL‐30 treatment. (Hepatology 2014;60:2026–2038)

An overview of effective therapies and recent advances in biomarkers for chronic liver diseases and associated liver cancer

Chronic liver diseases (CLDs) such as hepatitis, alcoholic liver disease, nonalcoholic fatty liver, and their downstream effect cancer affect more than a billion of people around the world both symptomatically and asymptomatically. The major limitation for early detection and suitable medical management of CLDs and liver cancer is either the absent of symptoms or their similar manifestations as other diseases. This detection impediment has led to a steady increase in the number of people suffering from CLDs with an ultimate outcome of liver failure and undergoing transplantation. A better understanding of CLD pathogenesis has helped us to develop novel therapies for patients who are at greatest risk for CLD progression to the most serious disease cancer. With the discovery of aberrant molecular pathways in CLDs, it is now possible to delineate a road map for selecting targeted therapies for CLDs. Technological advances in imaging as well as the availability of several stable, sensitive, early, noninvasive biomarkers for distinguishing different stages of CLDs and cancer have greatly facilitated both drug target identification and real-time monitoring of response to therapy. Biomarkers are the most useful in clinical practice for liver diseases like hepatocellular carcinoma (HCC), which is associated with secretion of various tumor-related proteins or nucleotides in peripheral circulation. The need for the identification of CLD biomarkers remains high. This article reviews the etiologies of CLDs, the results of recent clinical trials of treatments for CLDs, and development of noninvasive methodologies for detecting CLDs and monitoring their progression toward HCC.

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.

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.

IL6‐mediated inflammatory loop reprograms normal to epithelial‐mesenchymal transition+ metastatic cancer stem cells in preneoplastic liver of transforming growth factor beta–deficient β2‐spectrin+/− 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).

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).

TGF-β/β2-spectrin/CTCF-regulated tumor suppression in human stem cell disorder Beckwith-Wiedemann syndrome

Beckwith-Wiedemann syndrome (BWS) is a human stem cell disorder, and individuals with this disease have a substantially increased risk (~800-fold) of developing tumors. Epigenetic silencing of β2-spectrin (β2SP, encoded by SPTBN1), a SMAD adaptor for TGF-β signaling, is causally associated with BWS; however, a role of TGF-β deficiency in BWS-associated neoplastic transformation is unexplored. Here, we have reported that double-heterozygous Sptbn1+/- Smad3+/- mice, which have defective TGF-β signaling, develop multiple tumors that are phenotypically similar to those of BWS patients. Moreover, tumorigenesis-associated genes IGF2 and telomerase reverse transcriptase (TERT) were overexpressed in fibroblasts from BWS patients and TGF-β-defective mice. We further determined that chromatin insulator CCCTC-binding factor (CTCF) is TGF-β inducible and facilitates TGF-β-mediated repression of TERT transcription via interactions with β2SP and SMAD3. This regulation was abrogated in TGF-β-defective mice and BWS, resulting in TERT overexpression. Imprinting of the IGF2/H19 locus and the CDKN1C/KCNQ1 locus on chromosome 11p15.5 is mediated by CTCF, and this regulation is lost in BWS, leading to aberrant overexpression of growth-promoting genes. Therefore, we propose that loss of CTCF-dependent imprinting of tumor-promoting genes, such as IGF2 and TERT, results from a defective TGF-β pathway and is responsible at least in part for BWS-associated tumorigenesis as well as sporadic human cancers that are frequently associated with SPTBN1 and SMAD3 mutations.