This study evaluates the antitumor immune response induced by human hydatic cyst fluid (HCF) in an animal model of colon carcinoma. We found that anti-HCF antibodies were able to identify cell surface and intracellular antigens in CT26 colon cancer cells. In prophylactic tumor challenge experiments, HCF vaccination was found to be protective against tumor formation for 40% of the mice (P = 0.01). In the therapeutic setting, HCF vaccination induced tumor regression in 40% of vaccinated mice (P = 0.05). This vaccination generated memory immune responses that protected surviving mice from tumor rechallenge, implicating the development of an adaptive immune response in this process. We performed a proteomic analysis of CT26 antigens recognized by anti-HCF antibodies to analyze the immune cross-reactivity between E. granulosus (HCF) and CT26 colon cancer cells. We identified two proteins: mortalin and creatine kinase M-type. Interestingly, CT26 mortalin displays 60% homology with E. granulosus hsp70. In conclusion, our data demonstrate the capacity of HCF vaccination to induce antitumor immunity which protects from tumor growth in an animal model. This new antitumor strategy could open new horizons in the development of highly immunogenic anticancer vaccines.

Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease, has anticancer effects mediated, at least in part, by parasitederived products which inhibit growth of tumor cells. We investigated whether immunity to T. cruzi antigens could induce antitumor activity, using two rat models which reproduce human carcinogenesis: colon cancer induced by 1,2-dimethylhydrazine (DMH), and mammary cancer induced by N-nitroso-N-methylurea (NMU). We found that vaccination with T. cruzi epimastigote lysates strongly inhibits tumor development in both animal models. Rats immunized with T. cruzi antigens induce activation of both CD4 1 and CD8 1T cells and splenocytes from these animals showed higher cytotoxic responses against tumors as compared to rats receiving adjuvant alone. Tumor-associated immune responses included increasing number of CD11b/c 1 His48 2 MHC II 1 cells corresponding to macrophages and/or dendritic cells, which exhibited augmented NADPH-oxidase activity. We also found that T. cruzi lysate vaccination developed antibodies specific for colon and mammary rat cancer cells, which were capable of mediating antibody-dependent cellular cytotoxicity (ADCC) in vitro. Anti-T. cruzi antibodies cross-reacted with human colon and breast cancer cell lines and recognized 41/60 (68%) colon cancer and 38/63 (60%) breast cancer samples in a series of 123 human tumors. Our results suggest that T. cruzi antigens can evoke an integrated antitumor response involving both the cellular and humoral components of the immune response and provide novel insights into the understanding of the intricate relationship between parasite infection and tumor growth.

Galectin-3 (Gal-3) is a multifunctional protein that plays different roles in cancer biology. To better understand the role of Gal-3 and its ligands during colon carcinogenesis, we studied its expression in tumors induced in rats treated with 1,2-dimethylhydrazine (DMH) and in human tissues. Normal colon from untreated rats showed no staining using two specific monoclonal antibodies. In contrast, morphologically normal colon from DMH-treated rats and dysplastic aberrant crypt foci were strongly stained, indicating that increased Gal-3 expression is an early event during the neoplastic transformation in colon cells. Gal-3 was weakly expressed in adenocarcinomas. Overall, the Gal-3 expression pattern observed in the DMH rat model closely resembles that displayed by human colon stained with the same antibodies. We also found that Gal-3 phosphorylation diminishes in serines while increasing in tyrosines during rat colon carcinogenesis. Finally, we showed that Gal-3-ligands expression is strikingly similar in rat and human malignant colon and in non-malignant tissues. In conclusion, the DMH-induced rat colon cancer model displays expression patterns of Gal-3 and its ligands very similar to those observed in human samples. This animal model should contribute to clarifying the role of Gal-3 in colon carcinogenesis and also to finding effective preventive cancer agents based on Gal-3 targeting.

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Colorectal carcinoma (CRC) is the second leading cause of cancer mortality worldwide. O-glycosylated mucins at the cell surface of colonic mucosa exhibit alterations in cancer and are involved in fundamental biological processes, including invasion and metastasis. Certain members of the GalNAc-transferase family may be responsible for these changes and are being investigated as novel biomarkers of cancer. In the present study the prognostic significance of GalNAc-T6 was investigated in patients with CRC patients. GalNAc-T6 expression was observed in all three colon cancer cell lines analyzed by reverse transcription-polymerase chain reaction, immunofluorescence and flow cytometry. A cohort of 81 colon cancer specimens was analyzed by immunohistochemical staining using MAb T6.3. It was demonstrated that GalNAc-T6 was expressed in 35/81 (43%) cases of colon cancer but not in the normal colonic mucosa. No association was observed with the clinical-pathologic parameters. However, patients expressing GalNAc-T6 had a significantly increased overall survival (median, 58 months; P<0.001) compared with GalNAc-T6 negative patients, especially those with advanced disease. These results suggest that GalNAc-T6 expression predicts an improved outcome in patients with CRC. The molecular mechanism underlying the less aggressive behavior of colon cancer cells expressing GalNAc-T6 remains to be elucidated.

e14682 Background: Alterations in the O-glycosylation is one of the most common changes during colon carcinogenesis, which leads to the expression of short O-glycan antigens (Tn, sialyl-Tn, Tk, and core 6). These structures are associated with malignant behavior being actively investigated as targets for immunotherapy. The enzymes of GalNAc-T family regulate the initial step in mucins O-glycosylation and could be responsible for the altered glycosylation observed in cancer. The aim of this work was to evaluate the expression of GalNAc-T6 in colon cancer, and to determine its role as prognostic marker. Methods: We evaluated GalNAc-T6 expression in colon cell lines by immunocytochemistry, and in colon cancer tissue samples by immunohistochemistry using the monoclonal antibody T6.3 developed by us (Berois et al. J Histochem Cytochem. 2006). We analyzed 103 colon cancer samples and 10 normal colon tissues. Results: We found that GalNAc-T6 (usually expressed in normal placenta, trachea, pancreas and brain) is detected in colon cancer cell lines. GalNAc-T6 was also detected by immunohistochemistry in 50.5% of samples with cancer and no expression was found in normal colon tissue. The staining pattern was predominantly cytoplasmic. Multivariate analysis showed that GalNAc-T6 expression is an independent prognostic marker predicting improved survival in patients with positive tumors (p 0.014). Conclusions: GalNAc-T6 could be a new independent prognostic marker to predict better outcome in colon cancer patients.

e15060 Background: Abnormal O-glycosylation is one of the most common changes during colon carcinogenesis, leading to the expression of short truncated O-glycan antigens (such as Tn, sialyl-Tn, Tk, and core 6). These structures which are related with the malignant behavior are actively investigated as immunotherapeutic targets. The ppGalNAc-T family enzymes regulate the initial step of mucin O-glycosylation and could be responsible for the altered glycosylation observed in cancer. The objective of this work was to describe the abnormal expression of ppGalNAc-Ts in colon cancer comparing its relationship with incomplete O- glycosylated antigens. Methods: We studied the gene expression of ppGalNAc-Ts in colon cell lines by RT-PCR assays. Using immunohistochemistry we determined ppGalNAc-T6, Tn, sialyl-Tn, Tk, and core 6 expression in 64 colon cancer samples and in 10 normal colon tissues. Results: We found that ppGalNAc-T6 (an enzyme usually restricted to normal placenta, trachea, brain, and pancreas) is expressed by colon cancer cell lines. Using immunohistochemistry we detected ppGalNAc-T6 in 70.3% of cancer samples with no staining in normal colon tissues. Staining pattern was predominantly cytoplasmatic. Staining of Tn, STn, core 6 and Tk antigens was observed in 87,5%, 79,6%, 76,5% and 68.7% of tumors, respectively. We observed a statistically significant relationship between the enzyme expression and Tn antigen (p=0.009) and core 6 (p=0.001). No relationship was observed between the enzyme expression and sialyl-Tn (p = 0.406) and Tk (p= 0.18) antigens. Conclusions: ppGalNac-T6 is a new tumor marker for colon cancer and its expression is related with the accumulation of two O-glycosylated antigens such as Tn and core 6. This is the first evidence in human tissues suggesting that the abnormal expression of a ppGalNac transferase could be in the molecular basis of aberrant O-glycosylated antigens accumulation in cancer. No significant financial relationships to disclose.

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