Suzanne Laferté

Mature dendritic cells pulsed with exosomes stimulate efficient cytotoxic T‐lymphocyte responses and antitumour immunity

Exosomes (EXO) derived from dendritic cells (DC), which express major histocompatibility complex (MHC) and costimulatory molecules, have been used for antitumour vaccines. However, they are still less effective by showing only prophylatic immunity in animal models or very limited immune responses in clinical trials. In this study, we showed that ovalbumin (OVA) protein‐pulsed DC (DCOVA)‐derived EXO (EXOOVA) displayed MHC class I–OVA I peptide (pMHC I) complexes, CD11c, CD40, CD80, CCR7, DEC205, Toll‐like receptor 4 (TLR4), TLR9, MyD88 and DC‐SIGN molecules, but at a lower level than DCOVA. EXOOVA can be taken up by DC through LFA‐1/CD54 and C‐type lectin/mannose (glucosamine)‐rich C‐type lectin receptor (CLR) interactions. Mature DC pulsed with EXOOVA, which were referred to as mDCEXO, expressed a higher level of pMHC I, MHC II, and costimulatory CD40, CD54 and CD80 than DCOVA. The mDCEXO could more strongly stimulate OVA‐specific CD8+ T‐cell proliferation in vitro and in vivo, and more efficiently induce OVA‐specific cytotoxic T‐lymphocyte responses, antitumour immunity and CD8+ T‐cell memory in vivo than EXOOVA and DCOVA. In addition, mDCEXO could also more efficiently eradicate established tumours. Therefore, mature DC pulsed with EXO may represent a new, highly effective DC‐based vaccine for the induction of antitumour immunity.

Tumor-associated antigen 90K/Mac-2-binding protein : Possible role in colon cancer

The tumor‐associated antigen 90K (TAA90K)/Mac‐2‐binding protein implicated in cancer progression and metastasis is modified by β1‐6 branched N‐linked oligosaccharides in colon cancer cells, glycans shown to contribute to cancer metastasis. To elucidate the role of TAA90K in colon cancer, we examined its expression and function in human colon tumors and colon carcinoma cell lines. Immunohistochemical analyses of colon tumors revealed elevated expression of TAA90K in all samples analyzed compared to normal colon. To examine the function of TAA90K in colon cancer, we carried out protein and cell binding assays using TAA90K‐His purified from HT‐29 cells colon carcinoma cells infected with recombinant vaccinia virus expressing TAA90K containing a C‐terminal poly‐histidine tag. TAA90K‐His bound to fibronectin, collagen IV, laminins‐1, ‐5, and ‐10 and galectin‐3 (Mac‐2) but poorly to collagen I and galectin‐1. As expected, binding of TAA90K to galectin‐3 was dependent on carbohydrate since it was inhibitable by lactose and asiolofetuin, and a TAA90K‐His glycoform purified from HT‐29 cells treated with the glycosylation inhibitor 1‐deoxymannojirimycin bound poorly to galectin‐3. Unlike TAA90K isolated from other cell types, TAA90K‐His isolated from colon cancer cells failed to mediate adhesion of colon cancer and normal cell lines, possibly due to cell‐type specific glycosylation of TAA90K‐His and/or its putative cellular receptor. However, at low concentrations, TAA90K‐His enhanced galectin‐3‐mediated HT‐29 cell adhesion while at high concentrations, it inhibited cell adhesion. Thus, a possible mechanism by which TAA90K may contribute to colon cancer progression is by modulating tumor cell adhesion to extracellular proteins, including galectin‐3. J. Cell. Biochem. 98: 1351–1366, 2006.

Integrin α3β1 expressed by human colon cancer cells is a major carrier of oncodevelopmental carbohydrate epitopes

Oncodevelopmental carbohydrate epitopes are a common feature of human colorectal carcinoma, yet their biological significance remains unclear. We have shown previously that monoclonal antibody (MAb) 3A7, which recognizes a determinant on type 2 chain blood group A and B oligosaccharides, detects oncodevelopmental changes in azoxymethane‐induced rat colon tumors and some human colon cancer cell lines. (Laferté S et al. [1995] J. Cell. Biochem. 57:101–119). In this study, we set out to purify gp140, the major glycoprotein carrier of the 3A7 epitope expressed by human colon cancer cells, as a first step towards elucidating the contribution of the 3A7 epitope and its major glycoprotein carrier to colon cancer progression. To this end, gp140 was purified from HT29 cells and used for the preparation of polypeptide‐specific monoclonal antibodies. Five monoclonal antibodies (7A8, 7B11, 8C7, 8H7, and 11D4) immunoprecipitated a 3A7‐immunoreactive glycoprotein complex of 140 kDa which was subsequently identified by partial protein sequencing as α3β1 integrin. Flow cytometric analysis of Chinese hamster ovary (CHO) cells expressing different human integrin chains revealed that MAbs 7A8 and 7B11 detect the α3 integrin subunit whereas MAbs 8C7 and 8H7 detect the β1 integrin subunit. MAb 11D4, which did not bind to any of the CHO transfectants, detected type 2 chain blood group A determinant. Flow cytometric analysis of a panel of human colon carcinoma cell lines obtained from blood group A, AB, or B individuals revealed a direct correlation between cell‐surface expression of the 3A7 epitope and α3 integrin subunit, suggesting that α3β1 integrin is a preferred target of the 3A7 epitope in colon cancer cells. Using lectins and glycosidases to examine further the carbohydrate structure of α3β1 integrin, we demonstrated that it is a sialoglycoprotein containing both N‐ and O‐linked oligosaccharides. In addition, both α3 and β1 integrin subunits express β1–6 branched Asn‐linked oligosaccharides and short poly‐N‐acetyllactosamine units (Galβ1–4GlcNAc‐R; n ≤ 3), glycans previously implicated in cancer metastasis.Thus, α3β1 integrin expressed by human colon carcinoma cells is a major carrier of oncodevelopmental carbohydrate epitopes whose presence may modulate tumor cell adhesion, migration, and/or invasion. J. Cell. Biochem. 72:189–209, 1999.

The tumor-associated antigen 90K/Mac-2-binding protein secreted by human colon carcinoma cells enhances extracellular levels of promatrilysin and is a novel substrate of matrix metalloproteinases-2, -7 (matrilysin) and -9: Implications of proteolytic cleavage.

BACKGROUND The tumor-associated antigen 90K (TAA90K)/Mac-2-binding protein is expressed at elevated level in cancerous tissues and associated with poor prognosis. Since TAA90K has been implicated in the restructuring of the extracellular matrix, we examined the functional relationship between colon cancer cell-derived TAA90K and the matrix metalloproteinase (MMP) promatrilysin (proMMP-7), and also tested whether TAA90K is a novel substrate for MMPs-2, -7 and -9. METHODS The effect of TAA90K on proMMP-7 levels in HT-29 conditioned media was quantified by enzyme-linked immunosorbent assays. Binding of TAA90K to MMPs, extracellular matrix proteins and galectin-3 was measured by solid-phase binding assays. Proteolytic cleavage of TAA90K by MMPs was documented by SDS-PAGE and protein sequencing analysis. RESULTS TAA90K enhanced extracellular levels of proMMP-7 in HT-29 cells. In addition, TAA90K was cleaved by MMPs-2, -7 and -9. MMP-7-mediated cleavage of TAA90K did not affect its binding to MMP-7, laminin-1, collagen IV and galectin-3 but reduced its interaction with fibronectin and laminin-10, and lowered the levels of proMMP-7 in the HT-29 medium. CONCLUSION TAA90K is a novel substrate for MMPs-2, -7 and -9 and modulates proMMP-7 levels in colon cancer cells. GENERAL SIGNIFICANCE Proteolytic cleavage of TAA90K may have functional implications in colon cancer.