Isolating Circulating Cancer Stem Cells (CCSCs) from Human Whole Blood.

Measuring circulating tumor cells (CTCs) or circulating cancer stem cells (CCSCs) in blood, which shed from primary tumors, is a noninvasive method to screen and/or diagnose patients with a high risk of developing metastatic cancers or relapse. Here, we describe an optimized and standardized laboratory method for isolating CCSCs from human blood samples, using cancer-specific stem cell biomarkers (Kantara et al., Lab Invest 95:100-112, 2015). When performing this technique, 0-1 circulating epithelial tumor cells/mL blood should be expected in patients free of malignant adenocarcinomas compared to over 3 circulating cancer stem cells/mL blood in patients positive for malignant adenocarcinomas (Kantara et al., Lab Invest 95:100-112, 2015).

Nuclear Countermeasure Activity of TP508 Linked to Restoration of Endothelial Function and Acceleration of DNA Repair.

There is increasing evidence that radiation-induced damage to endothelial cells and loss of endothelial function may contribute to both acute radiation syndromes and long-term effects of whole-body nuclear irradiation. Therefore, several drugs are being developed to mitigate the effects of nuclear radiation, most of these drugs will target and protect or regenerate leukocytes and platelets. Our laboratory has demonstrated that TP508, a 23-amino acid thrombin peptide, activates endothelial cells and stem cells to revascularize and regenerate tissues. We now show that TP508 can mitigate radiation-induced damage to endothelial cells in vitro and in vivo. Our in vitro results demonstrate that human endothelial cells irradiation attenuates nitric oxide (NO) signaling, disrupts tube formation and induces DNA double-strand breaks (DSB). TP508 treatment reverses radiation effects on NO signaling, restores tube formation and accelerates the repair of radiation-induced DSB. The radiation-mitigating effects of TP508 on endothelial cells were also seen in CD-1 mice where systemic injection of TP508 stimulated endothelial cell sprouting from aortic explants after 8 Gy irradiation. Systemic doses of TP508 that mitigated radiation-induced endothelial cell damage, also significantly increased survival of CD-1 mice when injected 24 h after 8.5 Gy exposure. These data suggest that increased survival observed with TP508 treatment may be due to its effects on vascular and microvascular endothelial cells. Our study supports the usage of a regenerative drug such as TP508 to activate endothelial cells as a countermeasure for mitigating the effects of nuclear radiation.

Novel regenerative peptide TP508 mitigates radiation-induced gastrointestinal damage by activating stem cells and preserving crypt integrity.

In recent years, increasing threats of radiation exposure and nuclear disasters have become a significant concern for the United States and countries worldwide. Exposure to high doses of radiation triggers a number of potentially lethal effects. Among the most severe is the gastrointestinal (GI) toxicity syndrome caused by the destruction of the intestinal barrier, resulting in bacterial translocation, systemic bacteremia, sepsis, and death. The lack of effective radioprotective agents capable of mitigating radiation-induced damage has prompted a search for novel countermeasures that can mitigate the effects of radiation post exposure, accelerate tissue repair in radiation-exposed individuals, and prevent mortality. We report that a single injection of regenerative peptide TP508 (rusalatide acetate, Chrysalin) 24 h after lethal radiation exposure (9 Gy, LD100/15) appears to significantly increase survival and delay mortality by mitigating radiation-induced intestinal and colonic toxicity. TP508 treatment post exposure prevents the disintegration of GI crypts, stimulates the expression of adherens junction protein E-cadherin, activates crypt cell proliferation, and decreases apoptosis. TP508 post-exposure treatment also upregulates the expression of DCLK1 and LGR5 markers of stem cells that have been shown to be responsible for maintaining and regenerating intestinal crypts. Thus, TP508 appears to mitigate the effects of GI toxicity by activating radioresistant stem cells and increasing the stemness potential of crypts to maintain and restore intestinal integrity. These results suggest that TP508 may be an effective emergency nuclear countermeasure that could be delivered within 24 h post exposure to increase survival and delay mortality, giving victims time to reach clinical sites for advanced medical treatment.

Methods for detecting circulating cancer stem cells (CCSCs) as a novel approach for diagnosis of colon cancer relapse/metastasis.

Cancer stem cells (CSCs) are believed to be resistant to currently available therapies and may be responsible for relapse of cancer in patients. Measuring circulating tumor cells (CTCs) in the blood of patients has emerged as a non-invasive diagnostic procedure for screening patients who may be at high risk for developing metastatic cancers or relapse of the cancer disease. However, accurate detection of CTCs has remained a problem, as epithelial-cell markers used to date are not always reliable for detecting CTCs, especially during epithelial-mesenchymal transition. As CSCs are required to initiate metastatic tumors, our goal was to optimize and standardize a method for identifying circulating CSCs (CCSCs) in patients, using established CSC markers. Here, we report for the first time the detection of CCSCs in the blood of athymic nude mice, bearing metastatic tumors, and in the blood of patients positive for colonic adenocarcinomas. Using a simple and non-expensive method, we isolated a relatively pure population of CSCs (CD45-/CK19+), free of red blood cells and largely free of contaminating CD45+ white blood cells. Enriched CCSCs from patients with colon adenocarcinomas had a malignant phenotype and co-expressed CSC markers (DCLK1/LGR5) with CD44/Annexin A2. CSCs were not found in the blood of non-cancer patients, free of colonic growths. Enriched CCSCs from colon cancer patients grew primary spheroids, suggesting the presence of tumor-initiating cells in the blood of these patients. In conclusion, we have developed a novel diagnostic assay for detecting CSCs in circulation, which may more accurately predict the risk of relapse or metastatic disease in patients. As CSCs can potentially initiate metastatic growths, patients positive for CCSCs can be treated with inhibitory agents that selectively target CSCs, besides conventional treatments, to reduce the risk of relapse/metastatic disease for improving clinical outcomes.

Curcumin promotes autophagic survival of a subset of colon cancer stem cells, which are ablated by DCLK1-siRNA.

Curcumin is known to induce apoptosis of cancer cells by different mechanisms, but its effects on cancer stem cells (CSC) have been less investigated. Here, we report that curcumin promotes the survival of DCLK1-positive colon CSCs, potentially confounding application of its anticancer properties. At optimal concentrations, curcumin greatly reduced expression levels of stem cell markers (DCLK1/CD44/ALDHA1/Lgr5/Nanog) in three-dimensional spheroid cultures and tumor xenografts derived from colon cancer cells. However, curcumin unexpectedly induced proliferation and autophagic survival of a subset of DCLK1-positive CSCs. Spheroid cultures were disintegrated by curcumin in vitro but regrew within 30 to 40 days of treatment, suggesting a survival benefit from autophagy, permitting long-term persistence of colorectal cancer. Notably, RNA interference-mediated silencing of DCLK1 triggered apoptotic cell death of colon cancer cells in vitro and in vivo, and abolished colorectal cancer survival in response to curcumin; combination of DCLK1-siRNA and curcumin dramatically reversed CSC phenotype, contributing to attenuation of the growth of spheroid cultures and tumor xenografts. Taken together, our findings confirm a role of DCLK1 in colon CSCs and highlight DCLK1 as a target to enhance antitumor properties of curcumin.

Temporal characterization of lymphatic metastasis in an orthotopic mouse model of oral cancer.

BACKGROUND: The overall mortality rate in cases of head and neck squamous cell carcinoma (HNSCC) has not improved over the past 30 years, mostly because of the high treatment failure rate among patients with regionally metastatic disease. To better understand the pathobiologic processes leading to lymphatic metastasis development, there is an urgent need for relevant animal models. METHODS: HNSCC cell lines were implanted into the tongues of athymic nude mice. Histology, immunohistochemistry, and ex vivo 2-photon microscopy were used to evaluate tumor progress and spread. RESULTS: Orthotopic xenografts of different HNSCC cell lines produced distinct patterns of survival, tumor histology, disease progression rate, and lymph node metastasis development. Remarkably, all injected cell types reached the lymph nodes within 24 hours after injection, but not all developed metastasis. CONCLUSION: This orthotopic xenograft model closely mimics several characteristics of human cancer and could be extremely valuable for translational studies focusing on lymphatic metastasis development and pathobiology.

Progastrin Peptides Increase the Risk of Developing Colonic Tumors: Impact on Colonic Stem Cells.

Pre-neoplastic lesions (ACF, aberrant-crypt-foci; Hp, hyperplastic/dysplastic polyps) are believed to be precursors of sporadic colorectal-tumors (Ad, adenomas; AdCA, adenocarcinomas). ACF/Hp likely originate due to abnormal growth of colonic-crypts in response to aberrant queues in the microenvironment of colonic-crypts. Thus identifying factors which regulate homeostatic vs aberrant proliferation/apoptosis of colonocytes, especially stem/progenitor cells, may lead to effective preventative/treatment strategies. Based on this philosophy, role of growth-factors/peptide-hormones, potentially available in the circulation/microenvironment of colonic-crypts is being examined extensively. Since the time gastrins were discovered as trophic (growth) factors for gastrointestinal-cells, the effect of gastrins on the growth of normal/cancer cells has been investigated, leading to many discoveries. Seminal discoveries made in the area of gastrins and colon-cancer, as it relates to molecular pathways associated with formation of colonic tumors will be reviewed, and possible impact on diagnostic/preventative/treatment strategies will be discussed.

Laminin interactions with head and neck cancer cells under low fluid shear conditions lead to integrin activation and binding.

Lymphatic metastasis of cancer cells involves movement from the primary tumor site to the lymph node, where the cells must be able to productively lodge and grow. It is there that tumor cells encounter cellular and non-cellular constituent elements that make up the lymph node parenchyma. Our work shows that head and neck squamous cell carcinoma (HNSCC) cell lines are able to bind to laminin, fibronectin, vitronectin, and hyaluronic acid, which are extracellular matrix elements within the lymph node parenchyma. HNSCC cell lines bound to laminin under lymphodynamic low shear stress (0.07 dynes/cm(2)), consistent with lymph flow via ß1 integrins, including α2ß1, α3ß1, and α6ß1. Binding occurred in the presence of shear stress and not in the absence of flow. Additionally, tumor cell binding to laminin under flow did result in calcium signaling. Our data indicate a novel role for ß1 integrin-mediated binding of HNSCC cells to laminin under conditions of lymphodynamic flow that results in intracellular calcium signaling within the cancer cell.

Progastrin overexpression imparts tumorigenic/metastatic potential to embryonic epithelial cells: phenotypic differences between transformed and nontransformed stem cells.

We recently reported that overexpression of progastrin (PG) in embryonic epithelial cells (HEKmGAS cells) increased proliferation of the cells compared to that of control HEKC cells. Here, we report the novel finding that tumorigenic and metastatic potential of HEKmGAS cells is also increased significantly compared to that of HEKC cells. Cell surface-associated annexinA2 (CS-ANXA2) binds PG and is overexpressed on cancer cells, allowing us to successfully use fluorescently labeled PG peptide for enumerating metastatic lesions of transformed/cancer cells in vivo. Next, we examined the hypothesis that increased tumorigenic/metastatic potential of isogenic HEKmGAS versus HEKC cells maybe due to transformed phenotype of stem cells. FACSorting/FACScanning of cells demonstrated significant increases in percent doublecortin-CAM-kinase-like1 (DCLK1)/Lgr5-positive stem cells, coexpressing cluster of differentiation44 (CD44)/CS-ANXA2, in HEKmGAS versus HEKC cells. Distinct differences were noted in the morphology of HEKC versus HEKmGAS spheroidal growths on nonadherent cultures (selective for stem cells). HEKC spheroids were rounded with distinct perimeters (e.g., basement membranes), whereas HEKmGAS spheroids were amorphous with no perimeters. Relative levels of DCLK1/Lgr5/CD44 and ANXA2/ß-catenin/pNFκBp65/metalloproteinases were significantly increased in HEKmGAS versus HEKC cells, growing as monolayer cultures, 3D spheroids (in vitro), or xenografts (in vivo). Interestingly, HEKC cells enriched for CS-ANXA2 developed amorphous spheroids, whereas downregulation of ANXA2 in HEKmGAS clones resulted in loss of matrixmetalloproteinases (MMPs) and re-formation of rounded spheroids, suggesting that high levels of CS-ANXA2/MMPs may impact spheroid morphology. Downregulation of DCLK1 significantly attenuated activation of ß-catenin, with loss of proliferation of HEKmGAS and HEKC cells, suggesting that DCLK1 is required for maintaining proliferation of cells. Our results suggest the novel possibility that transformed stem cells, unlike nontransformed stem cells, coexpress stem cell markers DCLK1 and CD44 with CS-ANXA2.

Clathrin mediates endocytosis of progastrin and activates MAPKs: role of cell surface annexin A2.

Cell-surface-associated annexin A2 (CS-ANXA2) is a nonconventional "receptor" for progastrin; expression levels of both are elevated in colon cancers, and downregulation of either reduces tumorigenic potential of cells. We recently reported internalization of progastrin in target cells. Here, mechanisms mediating internalization of progastrin were examined. Initially, we confirmed that cell-surface ANXA2 mediates binding and internalization of progastrin in intestinal cells. Progastrin, covalently linked to sepharose beads, failed to activate p38MAPK/ERKs, suggesting internalization of progastrin was required for eliciting biological effects; importantly annexin A2 expression and availability of CS-ANXA2 were required for internalization of progastrin. Clathrin expression and formation of clathrin-coated pits were critically required for endocytotic internalization of progastrin; in the absence of clathrin, progastrin failed to activate p38MAPK/ERKs. Downregulation of caveolin had no effect on binding or internalization of progastrin. We therefore demonstrate for the first time that progastrin binds CS-ANXA2 and is rapidly internalized via clathrin-mediated endocytotic pathway, resulting in activation of MAPKinases. Targeting clathrin-mediated endocytosis of progastrin may thus inhibit previously reported co-carcinogenic/tumorigenic effects of progastrin on intestinal cells.

Annexin A2 mediates up-regulation of NF-κB, ß-catenin, and stem cell in response to progastrin in mice and HEK-293 cells.

BACKGROUND & AIMS: Prograstrin induces proliferation in colon crypts by activating p65nuclear factor-κB (NF-κB) (p65) and ß-catenin. We investigated whether Annexin A2 (AnxA2), a progastrin receptor, activates NF-κB and ß-catenin in vivo. METHODS: ANXA2-null (ANXA2(-/-)) and wild-type (ANXA2(+/+)) mice were studied, along with clones of progastrin-responsive HEK-293 cells that stably expressed full-length progastrin (HEK-mGAS) or an empty vector (HEK-C). Small interfering RNA was used to down-regulate AnxA2, p65NF-κB, and ß-catenin in cells. RESULTS: Proliferation and activation of p65 and ß-catenin increased significantly in HEK-mGAS compared with HEK-C clones. HEK-mGAS cells had a 2- to 4-fold increase in relative levels of c-Myc, cyclooxygenase (COX)-2, CyclinD1, double cortin CAM kinase-like 1 (DCAMKL+1), and CD44, compared with HEK-C clones. Down-regulation of AnxA2 in HEK-mGAS clones reduced activation of NF-κB and ß-catenin, as well as levels of DCAMKL+1. Surprisingly, down-regulation of ß-catenin had no effect on activation of p65NF-κB, whereas down-regulation of p65 significantly reduced activation of ß-catenin in HEK-mGAS clones. Loss of either p65 or ß-catenin significantly reduced proliferation of HEK-mGAS clones, indicating that both factors are required for the proliferative effects of progastrin. Lengths of colon crypts and levels of p65, ß-catenin, DCAMKL+1, and CD44 were significantly higher in ANXA2(+/+) mice compared with either ANXA2(-/-) mice given progastrin or ANXA2(+/+) and ANXA2(-/-) mice given saline. CONCLUSIONS: AnxA2 expression is required for the biologic effects of progastrin in vivo and in vitro and mediates the stimulatory effect of progastrin on p65NF-κ, ß-catenin, and the putative stem cell markers DCAMKL+1 and CD44. AnxA2 might therefore mediate the hyperproliferative and cocarcinogenic effects of progastrin.