Neutrophils activated by membrane attack complexes increase the permeability of melanoma blood vessels.

The microenvironment of malignant melanomas defines the properties of tumor blood vessels and regulates infiltration and vascular dissemination of immune and cancer cells, respectively. Previous research in other cancer entities suggested the complement system as an essential part of the tumor microenvironment. Here, we confirm activation of the complement system in samples of melanoma patients and murine melanomas. We identified the tumor endothelium as the starting point of the complement cascade. Generation of complement-derived C5a promoted the recruitment of neutrophils. Upon contact with the vascular endothelium, neutrophils were further activated by complement membrane attack complexes (MACs). MAC-activated neutrophils release neutrophil extracellular traps (NETs). Close to the blood vessel wall, NETs opened the endothelial barrier as indicated by an enhanced vascular leakage. This facilitated the entrance of melanoma cells into the circulation and their systemic spread. Depletion of neutrophils or lack of MAC formation in complement component 6 (C6)-deficient animals protected the vascular endothelium and prevented vascular intravasation of melanoma cells. Our data suggest that inhibition of MAC-mediated neutrophil activation is a potent strategy to abolish hematogenous dissemination in melanoma.

FAP-retargeted Ad5 enables in vivo gene delivery to stromal cells in the tumor microenvironment.

Fibroblast activation protein (FAP) is a cell surface serine protease that is highly expressed on reactive stromal fibroblasts, such as cancer-associated fibroblasts (CAFs), and generally absent in healthy adult tissues. FAP expression in the tumor stroma has been detected in more than 90% of all carcinomas, rendering CAFs excellent target cells for a tumor site-specific adenoviral delivery of cancer therapeutics. Here, we present a tropism-modified human adenovirus 5 (Ad5) vector that targets FAP through trivalent, designed ankyrin repeat protein-based retargeting adapters. We describe the development and validation of these adapters via cell-based screening assays and demonstrate adapter-mediated Ad5 retargeting to FAP+ fibroblasts in vitro and in vivo. We further show efficient in vivo delivery and in situ production of a therapeutic payload by CAFs in the tumor microenvironment (TME), resulting in attenuated tumor growth. We thus propose using our FAP-Ad5 vector to convert CAFs into a "biofactory," secreting encoded cancer therapeutics into the TME to enable a safe and effective cancer treatment.

Breastfed at Tiffany's.

The importance of breast milk for the growing infant is undisputed; breastfeeding decreases infantile mortality by tenfold and decreases the incidence of infectious diseases. Despite its recognized benefits, the structural richness of breast milk has also impeded the characterization of the multiple effects of milk components on infant physiology. However, the important roles of some components of breast milk are beginning to be dissected. For instance, molecules such as immunoglobulin A (IgA) and milk oligosaccharides protect from gastrointestinal infections and influence the development of the gut microbiota. Deciphering the complex composition of breast milk brings to light multifaceted contributions that combine to make breast milk the ultimate personalized medicine.

Analysis of serum glycome by lectin microarrays for prostate cancer patients - a search for aberrant glycoforms.

This is the first work focused on glycoprofiling of whole N- and O- glycome using lectins in an array format applied for analysis of serum samples from healthy individuals, benign prostate hyperplasia (BPH) patients, and prostate cancer (PCa) patients. Lectin microarray was prepared using traditional lectins with the incorporation of 2 recombinant bacterial lectins and 3 human lectins (17 lectins in total). Clinical validation of glycans as biomarkers was done in two studies: discrimination of healthy individuals with BPH patients vs. PCa patients (C vs. PCa) and discrimination of healthy individuals vs. BPH and PCa patients (H vs. PCond). Single lectins (17 lectins) and a combination of two lectins (136 binary lectin combinations) were applied in the clinical validation of glycan biomarkers providing 153 AUC values from ROC curves for both studies (C vs. PCa and H vs. PCond). Potential N- and O-glycans as biomarkers were identified and possible carriers of these glycans are shortly discussed.

Heparanase in Cancer Metastasis - Heparin as a Potential Inhibitor of Cell Adhesion Molecules.

Tumor progression associated with hematogenous metastatic spread is a multistep process based on a cross-talk between tumor and stromal cells in a tumor microenvironment. In the blood circulation, tumor cells interact with blood cells through receptors such as selectin and integrins that promote tumor cells survival. At the metastatic sites, heparanase secreted by tumor or stromal cells is an important modifier of the tumor microenvironment while promoting tumor invasiveness and angiogenesis. Heparin, particularly low molecular weight heparin, is used for treatment of cancer patients with evidence of hypercoagulability. However, in preclinical studies heparins was shown to contain other biological activities that affect cancer progression including inhibition of heparanase, selectins and integrins. While ongoing clinical trials are assessing inhibition of heparanase on cancer progression, the remaining biological activities of heparins inhibiting cells adhesion, through selectins and integrins remains largely unexplored. This chapter addresses the potential role of heparins in oncology with respect to their anti-heparanase and anti-adhesive activities and aims to discuss aspects relevant for broader therapeutic application of heparins.

Tunicate Heparan Sulfate Enriched in 2-Sulfated ß-Glucuronic Acid: Structure, Anticoagulant Activity, and Inhibitory Effect on the Binding of Human Colon Adenocarcinoma Cells to Immobilized P-Selectin.

Heparin or highly sulfated heparan sulfate (HS) has been described in different invertebrates. In ascidians (Chordata-Tunicata), these glycosaminoglycans occur in intracellular granules of oocyte accessory cells and circulating basophil-like cells, resembling mammalian mast cells and basophils, respectively. HS is also a component of the basement membrane of different ascidian organs. We have analyzed an HS isolated from the internal organs of the ascidian Phallusia nigra, using solution 1H/13C NMR spectroscopy, which allowed us to identify and quantify the monosaccharides found in this glycosaminoglycan. A variety of α-glucosamine units with distinct degrees of sulfation and N-acetylation were revealed. The hexuronic acid units occur both as α-iduronic acid and ß-glucuronic acid, with variable sulfation at the 2-position. A peculiar structural aspect of the tunicate HS is the high content of 2-sulfated ß-glucuronic acid, which accounts for one-third of the total hexuronic acid units. Another distinct aspect of this HS is the occurrence of high content of N-acetylated α-glucosamine units bearing a sulfate group at position 6. The unique ascidian HS is a potent inhibitor of the binding of human colon adenocarcinoma cells to immobilized P-selectin, being 11-fold more potent than mammalian heparin, but almost ineffective as an anticoagulant. Thus, the components of the HS structure required to inhibit coagulation and binding of tumor cells to P-selectin are distinct. Our results also suggest that the regulation of the pathway involved in the biosynthesis of glycosaminoglycans suffered variations during the evolution of chordates.

Gut microbiota modulate T cell trafficking into human colorectal cancer.

OBJECTIVE: Tumour-infiltrating lymphocytes (TILs) favour survival in human colorectal cancer (CRC). Chemotactic factors underlying their recruitment remain undefined. We investigated chemokines attracting T cells into human CRCs, their cellular sources and microenvironmental triggers. DESIGN: Expression of genes encoding immune cell markers, chemokines and bacterial 16S ribosomal RNA (16SrRNA) was assessed by quantitative reverse transcription-PCR in fresh CRC samples and corresponding tumour-free tissues. Chemokine receptor expression on TILs was evaluated by flow cytometry on cell suspensions from digested tissues. Chemokine production by CRC cells was evaluated in vitro and in vivo, on generation of intraperitoneal or intracecal tumour xenografts in immune-deficient mice. T cell trafficking was assessed on adoptive transfer of human TILs into tumour-bearing mice. Gut flora composition was analysed by 16SrRNA sequencing. RESULTS: CRC infiltration by distinct T cell subsets was associated with defined chemokine gene signatures, including CCL5, CXCL9 and CXCL10 for cytotoxic T lymphocytes and T-helper (Th)1 cells; CCL17, CCL22 and CXCL12 for Th1 and regulatory T cells; CXCL13 for follicular Th cells; and CCL20 and CCL17 for interleukin (IL)-17-producing Th cells. These chemokines were expressed by tumour cells on exposure to gut bacteria in vitro and in vivo. Their expression was significantly higher in intracecal than in intraperitoneal xenografts and was dramatically reduced by antibiotic treatment of tumour-bearing mice. In clinical samples, abundance of defined bacteria correlated with high chemokine expression, enhanced T cell infiltration and improved survival. CONCLUSIONS: Gut microbiota stimulate chemokine production by CRC cells, thus favouring recruitment of beneficial T cells into tumour tissues.

Selectins in cancer immunity.

Selectins are vascular adhesion molecules that mediate physiological responses such as inflammation, immunity and hemostasis. During cancer progression, selectins promote various steps enabling the interactions between tumor cells and the blood constituents, including platelets, endothelial cells and leukocytes. Selectins are carbohydrate-binding molecules that bind to sialylated, fucosylated glycan structures. The increased selectin ligand expression on tumor cells correlates with enhanced metastasis and poor prognosis for cancer patients. While, many studies focused on the role of selectin as a mediator of tumor cell adhesion and extravasation during metastasis, there is evidence for selectins to activate signaling cascade that regulates immune responses within a tumor microenvironment. L-Selectin binding induces activation of leukocytes, which can be further modulated by selectin-mediated interactions with platelets and endothelial cells. Selectin ligand on leukocytes, PSGL-1, triggers intracellular signaling in leukocytes that are induced through platelet's P-selectin or endothelial E-selectin binding. In this review, I summarize the evidence for selectin-induced immune modulation in cancer progression that represents a possible target for controlling tumor immunity.

Sulfated fucans and a sulfated galactan from sea urchins as potent inhibitors of selectin-dependent hematogenous metastasis.

Metastasis is responsible for the majority of cancer-associated deaths, though only a very small number of tumor cells are able to efficiently complete all the steps of that process. Tumor cell survival in the bloodstream is one of the limiting aspects of the metastatic cascade. The formation of tumor cell-platelet complexes that promote tumor cell survival is facilitated by the binding of P-selectin on activated platelets to sialyl Lewis-containing oligosaccharides on the surface of tumor cells. Inhibition of this interaction has been shown to attenuate metastasis. Heparin is a potent selectin inhibitor and is capable to block platelet-tumor cell complex formation, thereby attenuating metastasis. Similarly, other sulfated polysaccharides isolated from marine invertebrates attenuate metastasis by a P-selectin-mediated mechanism. In this work, we investigated the selectin-dependent antimetastatic activity of sea urchin sulfated polysaccharides with slight structural differences: a sulfated fucan from Strongylocentrotus franciscanus; a sulfated fucan from Strongylocentrotus droebachiensis; and a sulfated galactan from Echinometra lucunter. The results demonstrate that these fucans and the galactan have different antiselectin activities despite being very similar molecules. Therefore, they may be interesting tools for studies on the structure-function relationship or even for future treatments.

Stromal Expression of Activated Leukocyte Cell Adhesion Molecule Promotes Lung Tumor Growth and Metastasis.

Activated leukocyte cell adhesion molecule (ALCAM) is expressed on various cell types, including leukocytes, endothelial cells, and certain tumor cells. Although ALCAM expression on tumor cells has been linked to tumor invasion and metastatic spread, the contribution of ALCAM expressed in cells forming the tumor stroma to cancer progression has not been investigated. In this study, ALCAM-deficient (ALCAM-/-) mice were used to evaluate the role of ALCAM in lung tumor growth and metastasis. ALCAM-/- mice displayed an altered blood vascular network in the lung and the diaphragm, indicative of an angiogenetic defect. The absence of ALCAM expression in cells forming the stromal tumor microenvironment profoundly affected lung tumor growth in three different i.v. metastasis models. In the case of Lewis lung carcinoma (LLC), an additional defect in tumor cell homing to the lungs and a resulting reduction in the number of lung tumor nodules were observed. Similarly, when LLC cells were implanted subcutaneously for the study of spontaneous tumor cell metastasis, the rate of LLC metastasis to the lungs was profoundly reduced in ALCAM-/- mice. Taken together, our work demonstrates for the first time the in vivo contribution of ALCAM to angiogenesis and reveals a novel role of stromally expressed ALCAM in supporting tumor growth and metastatic spread.

Milk oligosaccharide sialyl(α2,3)lactose activates intestinal CD11c+ cells through TLR4.

Breast milk oligosaccharides shape the intestinal environment by affecting mucosal immunity and bacterial colonization. To clarify the role of milk oligosaccharide sialyl(α2,3)lactose (3SL) in intestinal physiology and disease, we investigated colitis development in Il10(-/-) mice exposed to normal or 3SL-deficient milk during lactation. Onset and progression of intestinal inflammation were delayed in Il10(-/-) mice deficient for the α2,3 sialyltransferase 4 (ST3GAL4) responsible for 3SL biosynthesis. The proinflammatory role of 3SL was confirmed by showing that oral supplementation of newborn Il10(-/-);St3gal4(-/-) mice with 3SL increased colitis severity. Conversely, fostering of newborn Il10(-/-) mice to lactating St3gal4(-/-) mothers reduced colitis severity. 3SL directly stimulated mesenteric lymph node CD11c(+) dendritic cells and induced production of cytokines required for expansion of TH1 and TH17 T cells. The stimulatory effect of 3SL was attenuated in Tlr4-deficient CD11c(+) cells, demonstrating that 3SL induces inflammation through Toll-like receptor 4 (TLR4) signaling. Thus, 3SL directly modulates mucosal immunity, which increases susceptibility to colitis.

Antitumor properties of a new non-anticoagulant heparin analog from the mollusk Nodipecten nodosus: Effect on P-selectin, heparanase, metastasis and cellular recruitment.

Inflammation and cancer are related pathologies acting synergistically to promote tumor progression. In both, hematogenous metastasis and inflammation, P-selectin participates in interactions involving tumor cells, platelets, leukocytes and endothelium. Heparin has been shown to inhibit P-selectin and as a consequence it blunts metastasis and inflammation. Some heparin analogs obtained from marine invertebrates are P-selectin inhibitors and do not induce bleeding effects. The present work focuses on the P-selectin blocking activity of a unique heparan sulfate (HS) from the bivalve mollusk Nodipecten nodosus. Initially, we showed that the mollusk HS inhibited LS180 colon carcinoma cell adhesion to immobilized P-selectin in a dose-dependent manner. In addition, we demonstrated that this glycan attenuates leukocyte rolling on activated endothelium and inflammatory cell recruitment in thioglycollate-induced peritonitis in mice. Biochemical analysis indicated that the invertebrate glycan also inhibits heparanase, a key player in cell invasion and metastasis. Experimental metastasis of Lewis lung carcinoma cells was drastically attenuated by the mollusk HS through a mechanism involving inhibition of platelet-tumor-cell complex formation in blood vessels. These data suggest that the mollusk HS is a potential alternative to heparin for inhibiting P-selectin-mediated events such as metastasis and inflammatory cell recruitment.

Ontogenetic regulation of leukocyte recruitment in mouse yolk sac vessels.

In adult mammals, leukocyte recruitment follows a well-defined cascade of adhesion events enabling leukocytes to leave the circulatory system and transmigrate into tissue. Currently, it is unclear whether leukocyte recruitment proceeds in a similar fashion during fetal development. Considering the fact that the incidence of neonatal sepsis increases dramatically with decreasing gestational age in humans, we hypothesized that leukocyte recruitment may be acquired only late during fetal ontogeny. To test this, we developed a fetal intravital microscopy model in pregnant mice and, using LysEGFP (neutrophil reporter) mice, investigated leukocyte recruitment during fetal development. We show that fetal blood neutrophils acquire the ability to roll and adhere on inflamed yolk sac vessels during late fetal development, whereas at earlier embryonic stages (before day E15), rolling and adhesion were essentially absent. Accordingly, flow chamber experiments showed that fetal EGFP(+) blood cells underwent efficient adhesion only when they were harvested on or after E15. Fluorescence-activated cell sorter analysis on EGFP(+) fetal blood cells revealed that surface expression of CXCR2 and less pronounced P-selectin glycoprotein ligand-1 (PSGL-1) begin to increase only late in fetal life. Taken together, our findings demonstrate that inflammation-induced leukocyte recruitment is ontogenetically regulated and enables efficient neutrophil trafficking only during late fetal life.

Tumor Cell-Intrinsic c-Myb Upregulation Stimulates Antitumor Immunity in a Murine Colorectal Cancer Model.

The transcription factor c-Myb is overexpressed in many different types of solid tumors, including colorectal cancer. However, its exact role in tumorigenesis is unclear. In this study, we show that tumor-intrinsic c-Myb expression in mouse models of colon cancer and melanoma suppresses tumor growth. Although no differences in proliferation, apoptosis, and angiogenesis of tumors were evident in tumors with distinct levels of c-Myb expression, we observed changes in intratumoral immune cell infiltrates. MC38 tumors with upregulated c-Myb expression showed increased numbers of CD103+ dendritic cells and eosinophils, but decreased tumor-associated macrophages (TAM). Concomitantly, an increase in the number of activated cytotoxic CD8+ T cells upon c-Myb upregulation was observed, which correlated with a pro-inflammatory tumor microenvironment and increased numbers of M1 polarized TAMs. Mechanistically, c-Myb upregulation in immunogenic MC38 colon cancer cells resulted in enhanced expression of immunomodulatory genes, including those encoding ß2-microglobulin and IFNß, and decreased expression of the gene encoding the chemokine receptor CCR2. The increased numbers of activated cytotoxic CD8+ T cells contributed to tumor growth attenuation. In poorly immunogenic CT26, LLC, and B16-BL6 tumor cells, c-Myb upregulation did not affect the immunomodulatory gene expression. Despite this, c-Myb upregulation led to reduced B16-BL6 tumor growth but it did not affect tumor growth of CT26 and LLC tumors. Altogether, we postulate that c-Myb functions as a tumor suppressor in a tumor cell-type specific manner and modulates antitumor immunity.

Volatile anesthetics reduce invasion of colorectal cancer cells through down-regulation of matrix metalloproteinase-9.

BACKGROUND: Invasion of extracellular matrix is a hallmark of malignant tumors. Clamping maneuvers during cancer surgery reduce blood loss, but trigger reperfusion injury (RI). RI increases cancer recurrence in the reperfused organ through up-regulation of matrix metalloproteinase-9 (MMP-9). Interleukin-8 is an important cytokine in RI promoting accumulation of neutrophils, a major source of MMP-9. Volatile anesthetics were demonstrated to reduce RI. We hypothesized that these anesthetics might attenuate MMP-9 up-regulation and consequently tumor cell invasion in RI. METHODS: Isolated human neutrophils (n = 6) were preconditioned with sevoflurane or desflurane, followed by stimulation with interleukin-8, phorbol myristate acetate, or chemokine CXC-ligand 1 (CXCL1) to differentiate intracellular pathways. MMP-9 release and activity were quantified by enzyme-linked immunosorbent assay and zymography, respectively. CXC-receptor-2 (CXCR2) expression and phosphorylation of extracellular signal-regulated kinases 1/2 were assessed by flow cytometry. The impact of MMP-9 on the invasion of neutrophils and MC-38 colon cancer cells was assessed using Matrigel-coated filters (n = 6). RESULTS: Preconditioning reduced interleukin-8-induced MMP-9-release by 41% (±13, 5%, sevoflurane) and 40% (±13%, desflurane). This was also evident following stimulation of CXCR2 with CXCL1. No impact on phosphorylation of extracellular signal-regulated kinases 1/2 and MMP-9 release was observed with receptor-independent stimulation of protein kinase C with phorbol myristate acetate. Preconditioning reduced transmigration of neutrophils and MC-38 tumor cells to baseline levels. DISCUSSION: Volatile anesthetics impair neutrophil MMP-9 release and interfere with pathways downstream of CXCR2, but upstream of protein kinase C. Through down-regulation of MMP-9, volatile anesthetics decrease Matrigel breakdown and reduce subsequent migration of cancer cells in vitro.

Complete absence of the αGal xenoantigen and isoglobotrihexosylceramide in α1,3galactosyltransferase knock-out pigs.

BACKGROUND: Anti-Galα1,3Galß-R natural antibodies are responsible for hyperacute rejection in pig-to-primate xenotransplantation. Although the generation of pigs lacking the α1,3galactosyltransferase (GalT) has overcome hyperacute rejection, antibody-mediated rejection is still a problem. It is possible that other enzymes synthesize antigens similar to Galα1,3Gal epitopes that are recognized by xenoreactive antibodies. The glycosphingolipid isoglobotrihexosylceramide (iGb3) represents such a candidate expressing an alternative Galα1,3Gal epitope. The present work determined whether the terminal Galα1,3Gal disaccharide is completely absent in Immerge pigs lacking the GalT using several different highly sensitive methods. METHODS: The expression of Galα1,3Gal was evaluated using a panel of antibodies and lectins by flow cytometry and fluorescent microscopy; GalT activity was detected by an enzymatic assay; and ion trap mass spectroscopy of neutral cellular membranes extracted from aortic endothelial was used for the detection of sugar structures. Finally, the presence of iGb3 synthase mRNA was tested by RT-PCR in pig thymus, spleen, lymph node, kidney, lung, and liver tissue samples. RESULTS: Aortic endothelial cells derived from GalT knockout pigs expressed neither Galα1,3Gal nor iGb3 on their surface, and GalT enzymatic activity was also absent. Lectin staining showed an increase in the blood group H-type sugar structures present in GalT knockout cells as compared to wild-type pig aortic endothelial cells (PAEC). Mass spectroscopic analysis did not reveal Galα1,3Gal in membranes of GalT knockout PAEC; iGb3 was also totally absent, whereas a fucosylated form of iGb3 was detected at low levels in both pig aortic endothelial cell extracts. Isoglobotrihexosylceramide 3 synthase mRNA was expressed in all pig tissues tested whether derived from wild-type or GalT knockout animals. CONCLUSIONS: These results confirm unequivocally the absence of terminal Galα1,3Gal disaccharides in GalT knockout endothelial cells. Future work will have to focus on other mechanisms responsible for xenograft rejection, in particular non-Galα1,3Gal antibodies and cellular responses.

Selectins promote tumor metastasis.

Cancer metastasis is facilitated by cell-cell interactions between cancer cells and endothelial cells in distant tissues. In addition, cancer cell interactions with platelets and leukocytes contribute to cancer cell adhesion, extravasation, and the establishment of metastatic lesions. Selectins are carbohydrate-binding molecules that bind to sialylated, fucosylated glycan structures, and are found on endothelial cells, platelets and leukocytes. There are three members of the selectin family: P-selectin expressed on activated platelets and endothelial cells, L-selectin present on leukocytes and E-selectin expressed on activated endothelial cells. Besides the accepted roles of selectins in physiological processes, such as inflammation, immune response and hemostasis, there is accumulating evidence for the potential of selectins to contribute to a number of pathophysiological processes, including cancer metastasis. Cancer cell interactions with selectins are possible due to a frequent presence of carbohydrate determinants--selectin ligands on the cell surface of tumor cells from various type of cancer. The degree of selectin ligand expression by cancer cells is well correlated with metastasis and poor prognosis for cancer patients. Initial adhesion events of cancer cells facilitated by selectins result in activation of integrins, release of chemokines and are possibly associated with the formation of permissive metastatic microenvironment. While E-selectin has been evaluated as one of the initiating adhesion events during metastasis, it is becoming apparent that P-selectin and L-selectin-mediated interactions significantly contribute to this process as well. In this review we discuss the current evidence for selectins as potential facilitators of metastasis.

The Solute Carrier MFSD1 Decreases the Activation Status of ß1 Integrin and Thus Tumor Metastasis.

Solute carriers are increasingly recognized as participating in a plethora of pathologies, including cancer. We describe here the involvement of the orphan solute carrier Major Facilitator Superfamily Domain-containing protein 1 (MFSD1) in the regulation of tumor cell migration. Loss of MFSD1 enabled higher levels of metastasis in experimental and spontaneous metastasis mouse models. We identified an increased migratory potential in MFSD1-/- tumor cells which was mediated by increased focal adhesion turnover, reduced stability of mature inactive ß1 integrin, and the resulting increased integrin activation index. We show that MFSD1 promoted recycling to the cell surface of endocytosed inactive ß1 integrin and thereby protected ß1 integrin from proteolytic degradation; this led to dampening of the integrin activation index. Furthermore, downregulation of MFSD1 expression was observed during the early steps of tumorigenesis, and higher MFSD1 expression levels correlate with a better cancer patient prognosis. In sum, we describe a requirement for endolysosomal MFSD1 in efficient ß1 integrin recycling to suppress tumor cell dissemination.

Identification of TPM2 and CNN1 as Novel Prognostic Markers in Functionally Characterized Human Colon Cancer-Associated Stromal Cells.

Stromal infiltration is associated with poor prognosis in human colon cancers. However, the high heterogeneity of human tumor-associated stromal cells (TASCs) hampers a clear identification of specific markers of prognostic relevance. To address these issues, we established short-term cultures of TASCs and matched healthy mucosa-associated stromal cells (MASCs) from human primary colon cancers and, upon characterization of their phenotypic and functional profiles in vitro and in vivo, we identified differentially expressed markers by proteomic analysis and evaluated their prognostic significance. TASCs were characterized by higher proliferation and differentiation potential, and enhanced expression of mesenchymal stem cell markers, as compared to MASCs. TASC triggered epithelial-mesenchymal transition (EMT) in tumor cells in vitro and promoted their metastatic spread in vivo, as assessed in an orthotopic mouse model. Proteomic analysis of matched TASCs and MASCs identified a panel of markers preferentially expressed in TASCs. The expression of genes encoding two of them, calponin 1 (CNN1) and tropomyosin beta chain isoform 2 (TPM2), was significantly associated with poor outcome in independent databases and outperformed the prognostic significance of currently proposed TASC markers. The newly identified markers may improve prognostication of primary colon cancers and identification of patients at risk.