Truncated O-glycosylation in metastatic triple-negative breast cancer reveals a gene expression signature associated with extracellular matrix and proteolysis.

Breast cancer (BC) is the leading cause of death by cancer in women worldwide. Triple-negative (TN) BC constitutes aggressive and highly metastatic tumors associated with shorter overall survival of patients compared to other BC subtypes. The Tn antigen, a glycoconjugated structure resulting from an incomplete O-glycosylation process, is highly expressed in different adenocarcinomas, including BC. It also favors cancer growth, immunoregulation, and metastasis in TNBC. This work describes the differentially expressed genes (DEGs) associated with BC aggressiveness and metastasis in an incomplete O-glycosylated TNBC cell model. We studied the transcriptome of a TNBC model constituted by the metastatic murine 4T1 cell line that overexpresses the Tn antigen due to a mutation in one of the steps of the O-glycosylation pathway. We analyzed and compared the results with the parental wild-type cell line and with a Tn-negative cell clone that was poorly metastatic and less aggressive than the 4T1 parental cell line. To gain insight into the generated expression data, we performed a gene set analysis. Biological processes associated with cancer development and metastasis, immune evasion, and leukocyte recruitment were highly enriched among functional terms of DEGs. Furthermore, different highly O-glycosylated protein-coding genes, such as mmp9, ecm1 and ankyrin-2, were upregulated in 4T1/Tn+ tumor cells. The altered biological processes and DEGs that promote tumor growth, invasion and immunomodulation might explain the aggressive properties of 4T1/Tn+ tumor cells. These results support the hypothesis that incomplete O-glycosylation that leads to the expression of the Tn antigen, which might regulate activity or interaction of different molecules, promotes cancer development and immunoregulation.

Trypanosoma cruzi-Derived Molecules Induce Anti-Tumour Protection by Favouring Both Innate and Adaptive Immune Responses.

Lung cancer remains the leading cause of cancer mortality worldwide. Thus, the development of strategies against this type of cancer is of high value. Parasite infections can correlate with lower cancer incidence in humans and their use as vaccines has been recently explored in preclinical models. In this study, we investigated whether immunisations with a Trypanosoma cruzi lysate from epimastigotes protect from lung tumour growth in mice. We also explore the role of parasite glycans in the induction of the protective immune response. A pre-clinical murine cancer model using the lung tumour cell line LL/2 was used to evaluate the anti-tumour potential, both in preventive and therapeutic settings, of a T. cruzi epimastigote-derived protein lysate. Immunisation with the parasite lysate prevents tumour growth and induces both humoral and cellular anti-tumour immune responses to LL-2 cancer cells. The induced immunity and tumour protection were associated with the activation of natural killer (NK) cells, the production of interferon-γ (IFN-γ) and tumour cell cytotoxicity. We also show that mannose residues in the T. cruzi lysate induce Toll-like receptor (TLR) signalling. The evaluated T. cruzi lysate possesses anti-tumour properties likely by activating innate and adaptive immunity in a process where carbohydrates seem to be essential.

Macrophage Gal/GalNAc lectin 2 (MGL2)+ peritoneal antigen presenting cells during Fasciola hepatica infection are essential for regulatory T cell induction.

Fasciola hepatica, one of the agents that causes fasciolosis, modulates the host immune system to allow parasite survival in the host. F. hepatica expresses carbohydrate-containing glycoconjugates that are decoded by C-type lectin receptors, such as Dectin-1, mannose receptor, DC-SIGN and MGL, that are mainly present on myeloid antigen presenting cells (APCs) and can mediate immunoregulatory properties on T cells. In particular, Macrophage Gal/GalNAc lectin 2 (MGL2) expands modified Th2 immune responses, while suppressing Th1 polarization, upon recognition of GalNAc-glycosylated parasite components. In this study, by using MGL2-DTR transgenic mice that encode human diphtheria toxin receptor in MGL2+ cells, we demonstrate the role of peritoneal APCs during F. hepatica infection in favoring parasite survival. This process might be mediated by the induction of splenic Tregs in vivo, since the depletion of MGL2+ cells conferred mice with partial resistance to the infection and abrogated the increase of CD4+/CD25+ FoxP3+ Tregs induced by the parasite. Therefore, MGL2+ cells are critical determinants of F. hepatica infection and could constitute immune checkpoints to control parasite infection.

Lung Tumor Cells with Different Tn Antigen Expression Present Distinctive Immunomodulatory Properties.

Lung cancer is the first leading cause of cancer-related deaths in the world. Aberrant glycosylation in lung tumors leads to the expression of tumor-associated carbohydrate structures, such as the Tn antigen, consisting of N-acetyl-galactosamine (GalNAc) linked to a serine or threonine residue in proteins (α-GalNAc-O-Ser/Thr). The Tn antigen can be recognized by the Macrophage Galactose/GalNAc lectin (MGL), which mediates various immune regulatory and tolerogenic functions, mainly by reprogramming the maturation of function of dendritic cells (DCs). In this work, we generated two different Tn-expressing variants from the Lewis-type lung murine cancer cell line LL/2, which showed different alterations in the O-glycosylation pathways that influenced the interaction with mouse MGL2 and the immunomodulatory properties of DCs. Thus, the identification of the biological programs triggered by Tn+ cancer cells might contribute to an improved understanding of the molecular mechanisms elicited by MGL-dependent immune regulatory circuits.

Liver function markers and haematological dynamics during acute and chronic phases of experimental Fasciola hepatica infection in cattle treated with triclabendazole.

Fasciola hepatica, a worldwide-distributed liver fluke, is one of the causative agents of fasciolosis, a zoonotic disease that affects livestock and humans. In livestock, fasciolosis causes huge economic losses worldwide, reducing animal fertility, milk production, weight gain and condemnation of livers. In spite of the availability of drugs, such as triclabendazole (TCZ), for the treatment of fasciolosis, they do not necessarily prevent liver damage or parasite reinfection and can eventually increase parasite resistance. The aim of this research was to relate the hepatic function, haematological parameters, leukocyte counts in circulation and parasite egg shedding during F. hepatica acute and chronic phases of infection in cattle as well as to determine how these parameters change with TCZ-treatment of chronically infected cattle. Our results show that increased levels of serum aspartate aminotransferase (AST) and gamma glutamyltransferase (GGT) were detected in early stages of the experimental infection. Moreover, high circulating eosinophil count and plateletcrit levels were correlated with fluke number in livers from infected cattle. On the other hand, although TCZ-treatment in the chronic phase of infection reduced parasite burden and damage in the liver, it was not able to completely avoid them. In conclusion, our work sheds light into the physiopathological mechanisms induced during fluke infection in cattle, revealing the complexity of the host response to the infection, together with the effects of TCZ-treatment in chronically infected animals.

The tumor-associated Tn antigen fosters lung metastasis and recruitment of regulatory T cells in triple negative breast cancer.

Cancer is a leading cause of death worldwide, accounting for nearly 10 million deaths. Among breast cancers (BC) subtypes, triple-negative (TN) BC is characterized by metastatic progression and poor patient prognosis. Although, TNBC is initially sensitive to chemotherapy, many TNBC patients rapidly develop resistance, at which point metastatic disease is highly lethal. Cancer cells present phenotypic changes or molecular signatures that distinguish them from healthy cells. The Tn antigen (GalNAc-O-Thr/Ser), which constitutes a powerful tool as tumor marker, was recently reported to contribute to tumor growth. However, its role in BC-derived metastasis has not yet been addressed. In this work, we generated a pre-clinical orthotopic Tn+ model of metastatic TNBC, which mimics the patient surgical treatment and is useful to study the role of Tn in metastasis and immunoregulation. We obtained two different cell clones, which differed in their Tn antigen expression: a high Tn-expressing and a non-expressing clone. Interestingly, the Tn-positive cell line generated significantly larger tumors and higher degree of lung metastases associated with a lower survival rate than the Tn-negative and parental cell line. Furthermore, we also found that both tumors and draining-lymph nodes from Tn+-tumor-bearing mice presented a higher frequency of CD4+ FoxP3+ T cells, while their splenocytes expressed higher levels of IL-10. In conclusion, this work suggests that the Tn antigen participates in breast tumor growth and spreading, favoring metastases to the lungs that are associated with an immunoregulatory state, suggesting that Tn-based immunotherapy could be a strategy of choice to treat these tumors.

Heme-Oxygenase-1 Attenuates Oxidative Functions of Antigen Presenting Cells and Promotes Regulatory T Cell Differentiation during Fasciola hepatica Infection.

Fasciola hepatica is a fluke that infects livestock and humans causing fasciolosis, a zoonotic disease of increasing importance due to its worldwide distribution and high economic losses. The parasite regulates the host immune system by inducing a strong Th2 and regulatory T (Treg) cell immune response through mechanisms that might involve the expression or activity of heme-oxygenase-1 (HO-1), the rate-limiting enzyme in the catabolism of free heme that also has immunoregulatory and antioxidant properties. In this paper, we show that F. hepatica-infected mice upregulate HO-1 on peritoneal antigen-presenting cells (APC), which produce decreased levels of both reactive oxygen and nitrogen species (ROS/RNS). The presence of these cells was associated with increased levels of regulatory T cells (Tregs). Blocking the IL-10 receptor (IL-10R) during parasite infection demonstrated that the presence of splenic Tregs and peritoneal APC expressing HO-1 were both dependent on IL-10 activity. Furthermore, IL-10R neutralization as well as pharmacological treatment with the HO-1 inhibitor SnPP protected mice from parasite infection and allowed peritoneal APC to produce significantly higher ROS/RNS levels than those detected in cells from infected control mice. Finally, parasite infection carried out in gp91phox knockout mice with inactive NADPH oxidase was associated with decreased levels of peritoneal HO-1+ cells and splenic Tregs, and partially protected mice from the hepatic damage induced by the parasite, revealing the complexity of the molecular mechanisms involving ROS production that participate in the complex pathology induced by this helminth. Altogether, these results contribute to the elucidation of the immunoregulatory and antioxidant role of HO-1 induced by F. hepatica in the host, providing alternative checkpoints that might control fasciolosis.

Polypeptide-GalNAc-Transferase-13 Shows Prognostic Impact in Breast Cancer.

Breast cancer is a public health concern and is currently the fifth cause of mortality worldwide. Identification of different biological subtypes is essential for clinical management; therefore, the role of pathologists is essential and useful tools for immunohistochemistry diagnosis are needed. Polypeptide-GalNAc-transferases are emerging novel biomarkers related to cancer behavior and GalNAc-T13, correlated with aggressiveness in some tumors, is an interesting candidate. Few monoclonal antibodies reacting with native proteins, and not affected by fixation and paraffin embedding, have been reported. The aim of this work was to develop a useful monoclonal antibody anti-GalNAc-T13 and to assess its potential significance in breast cancer diagnosis. We evaluated 6 human breast cancer cell lines, 338 primary breast tumors and 48 metastatic lymph nodes and looked for clinical significance correlating GalNAc-T13 expression with patients' clinical features and survival. We found high GalNAc-T13 expression in 43.8% of the cases and observed a significant higher expression in metastatic lymph nodes, correlating with worse overall survival. We hypothesized several possible molecular mechanisms and their implications. We conclude that GalNAc-T13 may be a novel biomarker in breast cancer, useful for routine pathological diagnosis. Elucidation of molecular mechanisms related to aggressiveness should contribute to understand the role of GalNAc-T13 in breast cancer biology.

The Tn antigen promotes lung tumor growth by fostering immunosuppression and angiogenesis via interaction with Macrophage Galactose-type lectin 2 (MGL2).

Tn is a tumor-associated carbohydrate antigen that constitutes both a diagnostic tool and an immunotherapeutic target. It originates from interruption of the mucin O-glycosylation pathway through defects involving, at least in part, alterations in core-1 synthase activity, which is highly dependent on Cosmc, a folding chaperone. Tn antigen is recognized by the Macrophage Galactose-type Lectin (MGL), a C-type lectin receptor present on dendritic cells and macrophages. Specific interactions between Tn and MGL shape anti-tumoral immune responses by regulating several innate and adaptive immune cell programs. In this work, we generated and characterized a variant of the lung cancer murine cell line LL/2 that expresses Tn by mutation of the Cosmc chaperone gene (Tn+ LL/2). We confirmed Tn expression by lectin glycophenotyping and specific anti-Tn antibodies, verified abrogation of T-synthase activity in these cells, and confirmed its recognition by the murine MGL2 receptor. Interestingly, Tn+ LL/2 cells were more aggressive in vivo, resulting in larger and highly vascularized tumors than those generated from wild type Tn- LL/2 cells. In addition, Tn+ tumors exhibited an increase in CD11c+ F4/80+ cells with high expression of MGL2, together with an augmented expression of IL-10 in infiltrating CD4+ and CD8+ T cells. Importantly, this immunosuppressive microenvironment was dependent on the presence of MGL2+ cells, since depletion of these cells abrogated tumor growth, vascularization and recruitment of IL-10+ T cells. Altogether, our results suggest that expression of Tn in tumor cells and its interaction with MGL2-expressing CD11c+F4/80+ cells promote immunosuppression and angiogenesis, thus favoring tumor progression.

Eosinophils Control Liver Damage by Modulating Immune Responses Against Fasciola hepatica.

Eosinophils are granulocytes that participate in the defense against helminth parasites and in hypersensitivity reactions. More recently, eosinophils were shown to have other immunomodulatory functions, such as tissue reparation, metabolism regulation, and suppression of Th1 and Th17 immune responses. In the context of parasitic helminth infections, eosinophils have a controversial role, as they can be beneficial or detrimental for the host. In this work, we investigate the role of eosinophils in an experimental infection in mice with the trematode parasite Fasciola hepatica, which causes substantial economical losses around the world due to the infection of livestock. We demonstrate that eosinophils are recruited to the peritoneal cavity and liver from F. hepatica-infected mice and this recruitment is associated with increased levels of CCL11, TSLP, and IL-5. Moreover, the characterization of peritoneal and hepatic eosinophils from F. hepatica-infected mice showed that they express distinctive molecules of activation and cell migration. Depletion of eosinophils with an anti-Siglec-F antibody provoked more severe clinical signs and increased liver damage than control animals which were accompanied by an increase in the production of IL-10 by hepatic and splenic CD4+ T cells. In addition, we also report that eosinophils participate in the modulation of humoral immune responses during F. hepatica infection, contributing to their degranulation. In conclusion, we demonstrate that eosinophils are beneficial for the host during F. hepatica infection, by limiting the production of IL-10 by specific CD4+ T cells and favoring eosinophil degranulation induced by specific antibodies. This work contributes to a better understanding of the role of eosinophils in parasitic helminth infections.

Revisiting the human polypeptide GalNAc-T1 and T13 paralogs.

Polypeptide GalNAc-transferases (GalNAc-Ts) constitute a family of 20 human glycosyltransferases (comprising 9 subfamilies), which initiate mucin-type O-glycosylation. The O-glycoproteome is thought to be differentially regulated via the different substrate specificities and expression patterns of each GalNAc-T isoforms. Here, we present a comprehensive in vitro analysis of the peptide substrate specificity of GalNAc-T13, showing that it essentially overlaps with the ubiquitous expressed GalNAc-T1 isoform found in the same subfamily as T13. We have also identified and partially characterized nine splice variants of GalNAc-T13, which add further complexity to the GalNAc-T family. Two variants with changes in their lectin domains were characterized by in vitro glycosylation assays, and one (Δ39Ex9) was inactive while the second one (Ex10b) had essentially unaltered activity. We used reverse transcription-polymerase chain reaction analysis of human neuroblastoma cell lines, normal brain and a small panel of neuroblastoma tumors to demonstrate that several splice variants (Ex10b, ΔEx9, ΔEx2-7 and ΔEx6/8-39bpEx9) were highly expressed in tumor cell lines compared with normal brain, although the functional implications remain to be unveiled. In summary, the GalNAc-T13 isoform is predicted to function similarly to GalNAc-T1 against peptide substrates in vivo, in contrast to a prior report, but is unique by being selectively expressed in the brain.

Trypanosoma cruzi extracts elicit protective immune response against chemically induced colon and mammary cancers.

Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease, has anticancer effects mediated, at least in part, by parasite-derived 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(+) and CD8(+) T 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(+) His48(-) MHC II(+) 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.

Antitumor activity of human hydatid cyst fluid in a murine model of colon cancer.

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.

Mucin-like peptides from Echinococcus granulosus induce antitumor activity.

There is substantial evidence suggesting that certain parasites can have antitumor properties. We evaluated mucin peptides derived from the helminth Echinococcus granulosus (denominated Egmuc) as potential inducers of antitumor activity. We present data showing that Egmuc peptides were capable of inducing an increase of activated NK cells in the spleen of immunized mice, a fact that was correlated with the capacity of splenocytes to mediate killing of tumor cells. We demonstrated that Egmuc peptides enhance LPS-induced maturation of dendritic cells in vitro by increasing the production of IL-12p40p70 and IL-6 and that Egmuc-treated DCs may activate NK cells, as judged by an increased expression of CD69. This evidence may contribute to the design of tumor vaccines and open new horizons in the use of parasite-derived molecules in the fight against cancer.

Mucin-type O-glycosylation in Mesocestoides vogae (syn. corti).

Protein glycosylation is an important post-translational modification underlying host-parasite interactions, which may determine the outcome of infection. Although Mesocestoides vogae represents an important model for investigating the various aspects of cestode biology, virtually no information is available about the structure and synthesis of glycans in this parasite. In this work, focused on the initiation pathway of mucin-type O-glycosylation in M. vogae, we characterized O-glycoproteins bearing the simple mucin-type cancer-associated Tn and sialyl-Tn antigens, and the expression and activity of ppGalNAc-T, the key enzyme responsible for the first step of mucin-type O-glycosylation. Using immunohistochemistry, Tn and sialyl-Tn antigens were detected mainly in the tegument (microtriches) and in parenchymal cells. Tn expression was also observed in lateral nerve cords. Both Tn and sialyl-Tn antigens were detected in in vitro cultured parasites. Based on their electrophoretic mobility, Tn- and sialyl-Tn-bearing glycoproteins from M. vogae were separated into several components of 22 to 60 kDa. The observation that Tn and sialyl-Tn glycoproteins remained in the 0.6N perchloric acid-soluble fraction suggested that they could be good candidates for characterizing mucin-type glycosylation in this parasite. O-glycoproteins were purified and initially characterized using a proteomic approach. Immunohistochemical analysis of the tissue distribution of ppGalNAc-T revealed that this enzyme is expressed in the sub-tegumental region and in the parenchyma of the parasite. In M. vogae cultured in vitro, ppGalNAc-T was mainly detected in the suckers. Using a panel of 8 acceptor substrate synthetic peptides, we found that M. vogae ppGalNAc-T preferentially glycosylate threonine residues, the best substrates being peptides derived from human mucin MUC1 and from Trypanosoma cruzi mucin. These results suggest that M. vogae might represent a useful model to study O-glycosylation, and provide new research avenues for future studies on the glycopathobiology of helminth parasites.