Cecile Artaud

Chondroitin sulfate glycosaminoglycans as major P‐selectin ligands on metastatic breast cancer cell lines

The metastatic breast cancer cell line, 4T1, abundantly expresses the oligosaccharide sialylated Lewis x (sLex). SLex oligosaccharide on tumor cells can be recognized by E‐ and P‐selectin, contributing to tumor metastatic process. We observed that both selectins reacted with this cell line. However, contrary to the E‐selectin reactivity, which was sLex dependent, P‐selectin reactivity with this cell line was sLex‐independent. The sLex‐Neg variant of the 4T1 cell line with markedly diminished expression of sLex and lack of sLea, provided a unique opportunity to characterize P‐selectin ligands and their contribution to metastasis in the absence of overlapping selectin ligands and E‐selectin binding. We observed that P‐selectin binding was Ca2+‐independent and sulfation‐dependent. We found that P‐selectin reacted primarily with cell surface chondroitin sulfate (CS) proteoglycans, which were abundantly and stably expressed on the surface of the 4T1 cell line. P‐selectin binding to the 4T1 cells was inhibited by heparin and CS glycosaminoglycans (GAGs). Moreover, Heparin administration significantly inhibited experimental lung metastasis. In addition, the data suggest that surface CS GAG chains were involved in P‐selectin mediated adhesion of the 4T1 cells to murine platelets and human umbilical vein endothelial cells. The data suggest that CS GAGs are also the major P‐selectin‐reactive ligands on the surface of human MDA‐MET cells. The results warrant conducting clinical studies on the involvement of cell surface CS chains in breast cancer metastasis and evaluation of various CS types and their biosynthetic pathways as target for development of treatment strategies for antimetastatic therapy of this disease.

A mimic of tumor rejection antigen-associated carbohydrates mediates an antitumor cellular response.

Tumor-associated carbohydrate antigens are typically perceived as inadequate targets for generating tumor-specific cellular responses. Lectin profile reactivity and crystallographic studies demonstrate that MHC class I molecules can present to the immune system posttranslationally modified cytosolic peptides carrying O-β-linked N-acetylglucosamine (GlcNAc). Here we report that a peptide surrogate of GlcNAc can facilitate an in vivo tumor-specific cellular response to established Meth A tumors that display native O-GlcNAc glycoproteins on the tumor cell surface. Peptide immunization of tumor-bearing mice had a moderate effect on tumor regression. Inclusion of interleukin 12 in the immunization regimen stimulated complete elimination of tumor cells in all of the mice tested, whereas interleukin 12 administration alone afforded no tumor growth inhibition. Adoptive transfer of immune T cells into tumor-bearing nude mice indicates a role for CD8+ T cells in tumor regression. This work postulates that peptide mimetics of glycosylated tumor rejection antigens might be further developed for immune therapy of cancer.

Reduction of Spontaneous Metastases through Induction of Carbohydrate Cross-Reactive Apoptotic Antibodies

The selective targeting of tumor-associated carbohydrate Ags by the induction of serum Abs that trigger apoptosis of tumor cells as a means to reduce circulating tumor cells and micrometastases would be an advantage in cancer vaccine development. Some plant lectins like Griffonia simplicifolia lectin I and wheat germ agglutinin mediate the apoptosis of tumor cells. We investigated the possibility of using these lectins as templates to select peptide mimotopes of tumor-associated carbohydrate Ags as immunogens to generate cross-reactive Abs capable of mediating apoptosis of tumor cells. In this study, we show that immunization with a mimotope selected based on its reactivity with Griffonia simplicifolia lectin I and wheat germ agglutinin induced serum IgM Abs in mice that mediated the apoptosis of murine 4T1 and human MCF7 cell lines in vitro, paralleling the apoptotic activity of the lectins. Vaccine-induced anti-carbohydrate Abs reduced the outgrowth of micrometastases in the 4T1 spontaneous tumor model, significantly increasing survival time of tumor-bearing animals. This finding parallels suggestions that carbohydrate-reactive IgM with apoptotic activity may have merit in the adjuvant setting if the right carbohydrate-associated targets are identified.

Deficiency in surface expression of E-selectin ligand promotes lung colonization in a mouse model of breast cancer

Expression of sialyl Lewisx (sLex) and sLea on tumor cells is thought to facilitate metastasis by promoting cell adhesion to selectins on vascular endothelial cells. Experiments supporting this concept usually bypass the early steps of the metastatic process by employing tumor cells that are injected directly into the blood. We investigated the relative role of sLex oligosaccharide in the dissemination of breast carcinoma, employing a spontaneous murine metastasis model. An sLex deficient subpopulation of the 4T1 mammary carcinoma cell line was produced by negative selection using the sLex‐reactive KM93 MAb. This subpopulation was negative for E‐selectin binding but retained P‐selectin binding. Both sLex‐negative and ‐positive cells grew at the same rate; however, sLex‐negative cells spread more efficiently on plates and had greater motility in wound‐scratch assays. Mice inoculated in the mammary fat pad with sLex‐negative and ‐positive variants produced lung metastases. However, the number of lung metastases was significantly increased in the group inoculated with the sLex‐negative variant (p = 0.0031), indicating that negative selection for the sLex epitope resulted in enrichment for a subpopulation of cells with a high metastatic phenotype. Cell variants demonstrated significant differences in cellular morphology and pattern of tumor growth in primary and secondary tumor sites. These results strongly suggest that loss of sLex may facilitate the metastatic process by contributing to escape from the primary tumor mass.

Carbohydrate Mimetic Peptides Augment Carbohydrate-Reactive Immune Responses in the Absence of Immune Pathology

Among the most challenging of clinical targets for cancer immunotherapy are Tumor Associated Carbohydrate Antigens (TACAs). To augment immune responses to TACA we are developing carbohydrate mimetic peptides (CMPs) that are sufficiently potent to activate broad-spectrum anti-tumor reactivity. However, the activation of immune responses against terminal mono- and disaccharide constituents of TACA raises concerns regarding the balance between “tumor destruction” and “tissue damage”, as mono- and disaccharides are also expressed on normal tissue. To support the development of CMPs for clinical trial testing, we demonstrate in preclinical safety assessment studies in mice that vaccination with CMPs can enhance responses to TACAs without mediating tissue damage to normal cells expressing TACA. BALB/c mice were immunized with CMPs that mimic TACAs reactive with Griffonia simplicifolia lectin 1 (GS-I), and tissue reactivity of serum antibodies were compared with the tissue staining profile of GS-I. Tissues from CMP immunized mice were analyzed using hematoxylin and eosin stain, and Luxol-fast blue staining for myelination. Western blots of membranes from murine mammary 4T1 cells, syngeneic with BALB/c mice, were also compared using GS-I, immunized serum antibodies, and naive serum antibodies. CMP immunization enhanced glycan reactivities with no evidence of pathological autoimmunity in any immunized mice demonstrating that tissue damage is not an inevitable consequence of TACA reactive responses.