Eric Sceusi

Endothelial Cells Promote the Colorectal Cancer Stem Cell Phenotype through a Soluble Form of Jagged-1

We report a paracrine effect whereby endothelial cells (ECs) promote the cancer stem cell (CSC) phenotype of human colorectal cancer (CRC) cells. We showed that, without direct cell-cell contact, ECs secrete factors that promoted the CSC phenotype in CRC cells via Notch activation. In human CRC specimens, CD133 and Notch intracellular domain-positive CRC cells colocalized in perivascular regions. An EC-derived, soluble form of Jagged-1, via ADAM17 proteolytic activity, led to Notch activation in CRC cells in a paracrine manner; these effects were blocked by immunodepletion of Jagged-1 in EC-conditioned medium or blockade of ADAM17 activity. Collectively, ECs play an active role in promoting Notch signaling and the CSC phenotype by secreting soluble Jagged-1.

Overexpression of Snail induces epithelial- mesenchymal transition and a cancer stem cell- like phenotype in human colorectal cancer cells

Epithelial–mesenchymal transition (EMT) is a critical process providing tumor cells with the ability to migrate and escape from the primary tumor and metastasize to distant sites. Recently, EMT was shown to be associated with the cancer stem cell (CSC) phenotype in breast cancer. Snail is a transcription factor that mediates EMT in a number of tumor types, including colorectal cancer (CRC). Our study was done to determine the role of Snail in mediating EMT and CSC function in CRC. Human CRC specimens were stained for Snail expression, and human CRC cell lines were transduced with a retroviral Snail construct or vector control. Cell proliferation and chemosensitivity to oxaliplatin of the infected cells were determined by the MTT (colorimetric 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide) assay. Migration and invasion were determined in vitro using modified Boyden chamber assays. EMT and putative CSC markers were analyzed using Western blotting. Intravenous injection of tumor cells was done to evaluate their metastatic potential in mice. Snail was overexpressed in human CRC surgical specimens. This overexpression induced EMT and a CSC‐like phenotype in human CRC cells and enhanced cell migration and invasion (P < 0.002 vs. control). Snail overexpression also led to an increase in metastasis formation in vivo (P < 0.002 vs. control). Furthermore, the Snail‐overexpressing CRC cells were more chemoresistant to oxaliplatin than control cells. Increased Snail expression induces EMT and the CSC‐like phenotype in CRC cells, which enhance cancer cell invasion and chemoresistance. Thus, Snail is a potential therapeutic target in metastatic CRC.

Identification of cancer stem cells in human gastrointestinal carcinoid and neuroendocrine tumors

BACKGROUND & AIMS Metastatic gastrointestinal neuroendocrine tumors (NETs) frequently are refractory to chemotherapy. Chemoresistance in various malignancies has been attributed to cancer stem cells (CSCs). We sought to identify gastrointestinal neuroendocrine CSCs (N-CSCs) in surgical specimens and a NET cell line and to characterize novel N-CSC therapeutic targets. METHODS Human gastrointestinal NETs were evaluated for CSCs using the Aldefluor (Stemcell Technologies, Vancouver, Canada) assay. An in vitro, sphere-forming assay was performed on primary NET cells. CNDT2.5, a human midgut carcinoid cell line, was used for in vitro (sphere-formation) and in vivo (tumorigenicity assays) CSC studies. N-CSC protein expression was characterized using Western blotting. In vivo, systemic short interfering RNA administration targeted Src. RESULTS By using the Aldefluor assay, aldehyde dehydrogenase-positive (ALDH+) cells comprised 5.8% ± 1.4% (mean ± standard error of the mean) of cells from 19 patient samples. Although many primary cell lines failed to grow, CNDT96 ALDH+ cells formed spheres in anchorage-independent conditions, whereas ALDH- cells did not. CNDT2.5 ALDH+ cells formed spheres, whereas ALDH- cells did not. In vivo, ALDH+ CNDT2.5 cells generated more tumors, with shorter latency than ALDH- or sham-sorted cells. Compared with non-CSCs, ALDH+ cells demonstrated increased expression of activated Src, Erk, Akt, and mammalian target of rapamycin (mTOR). In vivo, anti-Src short interfering RNA treatment of ALDH+ tumors reduced tumor mass by 91%. CONCLUSIONS CSCs are present in NETs, as shown by in vitro sphere formation and in vivo tumorigenicity assays. Src was activated in N-CSCs and represents a potential therapeutic target in gastrointestinal NETs.

The requirement for freshly isolated human colorectal cancer (CRC) cells in isolating CRC stem cells

Background:Isolation of colorectal cancer (CRC) cell populations enriched for cancer stem cells (CSCs) may facilitate target identification. There is no consensus regarding the best methods for isolating CRC stem cells (CRC-SCs). We determined the suitability of various cellular models and various stem cell markers for the isolation of CRC-SCs.Methods:Established human CRC cell lines, established CRC cell lines passaged through mice, patient-derived xenograft (PDX)-derived cells, early passage/newly established cell lines, and cells directly from clinical specimens were studied. Cells were FAC-sorted for the CRC-SC markers CD44, CD133, and aldehyde dehydrogenase (ALDH). Sphere formation and in vivo tumorigenicity studies were used to validate CRC-SC enrichment.Results:None of the markers studied in established cell lines, grown either in vitro or in vivo, consistently enriched for CRC-SCs. In the three other cellular models, CD44 and CD133 did not reliably enrich for stemness. In contrast, freshly isolated PDX-derived cells or early passage/newly established CRC cell lines with high ALDH activity formed spheres in vitro and enhanced tumorigenicity in vivo, whereas cells with low ALDH activity did not.Conclusions:PDX-derived cells, early passages/newly established CRC cell lines and cells from clinical specimen with high ALDH activity can be used to identify CRC-SC-enriched populations. Established CRC cell lines should not be used to isolate CSCs.

Varying Opinions on the Authenticity of a Human Midgut Carcinoid Cell Line – Letter

In 2007, our laboratory published an article in Clinical Cancer Research reporting on the development of a human midgut carcinoid tumor cell line ([1][1]). This cell line has now been shared with over 20 laboratories around the world. Investigators in the field of neuroendocrine tumor research are

Abstract 5132: Hypo-glycosylation of vascular endothelial growth factor receptor-1 may contribute to intracrine signaling in human colorectal cancer cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Introduction: Vascular endothelial growth factor receptor-1 (VEGFR-1) is highly expressed in endothelial cells, macrophages and cancer cells in the tumor microenvironment. VEGFR-1 plays an important role in tumor angiogenesis, inflammation and metastasis. In colorectal cancer (CRC) cells with hyperactive Wnt signaling, its function has been linked to cell motility, anchorage-independent growth, and cell survival. To better understand the role of VEGFR-1 in CRC, we performed studies to define the subcellular location and post-translational modifications of VEGFR-1 in human CRC cells. Methods/Results: Utilizing IHC analysis, GFP-tagged imaging and cell surface receptor assays, we observed that VEGFR-1 locates mostly in intracellular compartments in CRC cells, which is distinct from its membrane receptor status in endothelial cells. Moreover, spontaneous apoptosis was increased with endogenous VEGF knockout whereas treatment with the VEGF neutralizing antibody bevacizumab had no effect on apoptosis (Samuel et al. Oncogene, in press); these studies suggest that CRC cells require VEGF/VEGFR-1 intracrine signaling for suppression of apoptosis. In order to understand the means by which VEGFR-1 cellular localization is determined, we analyzed the glycosylation of VEGFR-1 in CRC cells, endothelial cells and clinical specimens of metastatic CRC from the liver. We found that VEGFR-1 is partially N-glycosylated by high mannose oligosaccharides in the CRC cells and human tumors, in contrast to complex, mature N-glycosylation of VEGFR-1 in the endothelial cells and normal liver tissue. Conclusion: VEGFR-1 is differentially post-translationally modified and intracellularly localized in CRC cells and metastatic tumors. Because N-glycosylation is critical for the membrane receptors intracellular trafficking and ligand interaction on the cell surface, hypo-glycosylation of VEGFR-1 in CRC cells likely contributes to its cytoplasmic localization and intracrine activation through a non-RTK pathway. The implications of intracrine function of VEGFR-1 in CRC warrant further investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5132. doi:10.1158/1538-7445.AM2011-5132

Abstract 365: Autocrine vascular endothelial growth factor signaling mediates survival and chemoresistance of human colorectal cancer cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Introduction. Although the effects of VEGF on angiogenesis and vascular function are well known, the autocrine effects of VEGF on tumor cell function remain to be elucidated. We studied phenotypic changes in human colorectal cancer (CRC) cells with homologous deletion of VEGF alleles to determine any potential autocrine/intracrine role of VEGF on tumor cell function. Methods. The human CRC cell lines HCT116 and LS174T with homologous recombination-mediated deletion of VEGF alleles were generated previously (Dang et al, Can Res 2006). To exclude the effect of exogenous VEGF, the cells were grown in low serum condition for all assays. MTT assay was used to detect changes in cell growth. Migration and invasion were determined using modified Boyden chamber assays. Cell death and apoptosis were assessed by FACS analysis following PI and Annexin V/PI staining respectively. Changes in protein levels of VEGF signaling intermediates and mediators of apoptosis were evaluated by Western blotting. Chemosensitivity was determined by FACS analysis following 5-FU treatment and PI staining of cells. Results. Loss of VEGF expression decreased cell growth and increased spontaneous apoptosis in CRC cells. Cells null for VEGF demonstrated increases in migration and invasion. Loss of VEGF expression also increased the sensitivity of cells to the cytotoxic effects of the chemotherapeutic drug, 5-FU, as shown by increased cell death and apoptosis. These effects are mediated via upregulation of the pro-apoptotic mediators cleaved caspase-3 and PARP and, downregulation of the anti-apoptotic mediator survivin. In addition, loss of VEGF led to increased levels of PlGF, SDF-1 and phosphorylated VEGFR-1 in CRC cells. Conclusion. Loss of autocrine/intracrine VEGF signaling leads to an increase in spontaneous apoptosis and chemosensitivity of colon cancer cells. In addition, loss of VEGF signaling led to increases in PlGF and SDF-1, suggesting that loss of VEGF signaling can lead to induction of compensatory pathways that may be mediated by numerous cell types in the tumor microenvironment. These findings have implications for a better understanding of mechanisms of action for VEGF targeted therapies when combined with chemotherapy in patients with metastatic CRC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 365.

Abstract 2300: Overexpression of Snail induces epithelial-mesenchymal transition and cancer stem cell-like phenotypes in CRC cells

Background: Epithelial-to-mesenchymal transition (EMT) is a critical process providing tumor cells with the ability to migrate and escape from the primary tumor and metastasize to distant sites. Recently, EMT has been shown to be associated with the cancer stem cell (CSC) phenotype in breast cancer. Snail is a transcriptional factor that mediates EMT in a number of tumor types. Its aberrant expression has been found in various cancers including colorectal cancer (CRC). The role of Snail in mediating EMT and CSC function in CRC has not yet been fully elucidated. Methods: The human CRC cell lines HT29 and KM12L4 were transduced with a retrovirus containing the pBabe-puro-Snail expression construct or the empty vector. The Snail expressing cells and controls were selected with the medium containing 2µg/ml puromycin. Changes in cell proliferation and chemo-sensitivity were determined by MTT assay. Migration and invasion were determined using in vitro wound healing assay and modified Boyden Chamber assays, respectively. EMT makers (E-cadherin, vimentin) and the putative CSC marker CD133 was analyzed by Western blotting. ALDH1 enzyme activity was analyzed by the Aldefluor assay. Results: Overexpression of Snail in CRC cells induced an EMT phenotype with elevated expression of vimentin and reduced expression of E-cadherin. Snail-induced EMT did not alter cell proliferation but showed enhanced cell motility, in which Snail-expressing cells were 5- to 6-fold more migratory than the control cells. Snail also promoted the CSC-like phenotype demonstrated by increased expression of the CSC markers CD133. The aldefluor positive cell population in the Snail-expressing cells was increased ∼4 fold. Moreover, the Snail-expressing CRC cells showed increased chemoresistance to 5FU by 50%. Conclusion. High level of Snail expression confers the EMT and CSC phenotype in CRC cells, which enhanced cancer cell invasion and chemoresistance. Our data suggest that Snail may be a potential therapeutic target in metastatic colorectal cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2300.

Abstract 1281: Endothelial cells promote the cancer stem cell phenotype of human colorectal cancer cells

Background: Cancer stem cells (CSC), which possess the properties of self-renewal, multi-potential differentiation and tumor initiation, can arise either from normal stem cells or due to the influence of the tumor micro-environment. This study was done to determine the potential role of endothelial cell (EC) derived paracrine factors on promoting the CSC phenotype in human colorectal cancer (CRC) cells. Methods: RFP-labeled human CRC cells HCT116 were co-cultured with RF24 ECs (immortalized HUVECs) under low serum conditions. After FAC-sorting for the CRC cells, the CSC population was analyzed by 1) the Aldefluor assay, 2) sphere forming ability, and 3) Western blot analysis for the CSC markers CD133 and CD44. In addition, low serum condition media obtained from either ECs or parental CRC cells (control), was added to CRC cells to determine its effect on the CSC phenotype. In order to characterize the apoptotic effect of EC derived factors on CRC cells, we performed Annexin V staining by FACS, PARP cleavage, Caspase 3 cleavage, and Bcl2 levels by Western blotting. Using MTT assay, we determined the effect of EC derived factors on chemo-sensitivity of CRC cells exposed to 5-FU. Results: Co-culturing of CRC cells with ECs markedly increased the ALDH-positive population from 5% to 18%, and the sphere forming ability by more than 4-fold in CRC cells. In addition, CRC cells also displayed increased CD133 (6-fold) and CD44 (4-fold) protein levels. Furthermore, this effect could be mimicked simply by co-culturing CRC cells with conditioned media obtained from ECs. Similarly, treatment of CRC cells with conditioned medium from ECs significantly increased the ALDH-positive population from 4% to 8%, sphere forming ability >3-fold, and the expression of CD133 (7-fold) and CD44 (6-fold). Conditioned medium from ECs, concomitantly decreased spontaneous apoptosis in CRC cells as demonstrated by a 3-fold decrease in Annexin V-positive population (21% to 7%), down-regulation of cleaved PARP and Caspase 3, and up-regulation of Bcl2. CRC cells exposed to EC conditioned medium also displayed decreased sensitivity to 5-FU [>10-fold increase of the IC50 from 2.0 μg/ml to 22.3 μg/ml (p Conclusions: Exposure of CRCs to ECs increased CSC properties, and decreased spontaneous apoptosis. Since, this effect can also be mimicked by the conditioned media, additional experiments to identify and validate the soluble factors that mediate this effect are in progress. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1281.