Gap junction communication between autologous endothelial and tumor cells induce cross-recognition and elimination by specific CTL.

Cellular interactions in the tumor stroma play a major role in cancer progression but can also induce tumor rejection. To explore the role of endothelial cells in these interactions, we used an in vitro three-dimensional collagen matrix model containing a cytotoxic T lymphocyte CTL clone (M4.48), autologous tumor cells (M4T), and an endothelial cell (M4E) line that are all derived from the same tumor. We demonstrate in this study that specific killing of the endothelial cells by the CTL clone required the autologous tumor cells and involved Ag cross-presentation. The formation of gap junctions between endothelial and tumor cells is required for antigenic peptide transfer to endothelial cells that are then recognized and eliminated by CTL. Our results indicate that gap junctions facilitate an effective CTL-mediated destruction of endothelial cells from the tumor microenvironment that may contribute to the control of tumor progression.

Selective human endothelial cell activation by chemokines as a guide to cell homing.

An original model of organo-specific, immortalized and stabilized endothelial cell lines was used to delineate the part played by some chemokines (CCL21, CX3CL1, CCL5 and CXCL12) and their receptors in endothelium organo-specificity. Chemokine receptor expression and chemokine presentation were investigated on organo-specific human endothelial cell lines. Although the chemokines showed distinct binding patterns for the various endothelial cell lines, these were not correlated with the expression of the corresponding receptors (CX3CR1, CXCR4, CCR5 and CCR7). Experiments with CCL21 on peripheral lymph node endothelial cells demonstrated that the chemokine did not co-localize with its receptor but was associated with extracellular matrix components. The specific activity of chemokines was clearly shown to be related to the endothelial cell origin. Indeed, CX3CL1 and CCL21 promoted lymphocyte recruitment by endothelial cells from the appendix and peripheral lymph nodes, respectively, while CX3CL1 pro-angiogenic activity was restricted to endothelial cells from the appendix and skin. The high specificity of the chemokine/endothelium interaction allowed the design of a direct in vitro endothelial cell targeting assay. This unique cellular model demonstrated a fundamental role for chemokines in conferring on the endothelium its organo-specificity and its potential for tissue targeting through the selective binding, presentation and activation properties of chemokines.

Flow cytometric assay for quantitative and qualitative evaluation of adhesive interactions of tumor cells with endothelial cells.

The purpose of the study was to develop a flow cytometric assay for quantitative determination of adhesive interactions of human endothelial cells (ECs) with tumor cells. EC lines established from human lymph node, appendix, lung, skin and intestine microvessels, labeled with PKH26-GL fluorescent dye, were grown to confluency in 24-well TC plates. Human colon adenocarcinoma cell suspension was overlaid onto labeled ECs, and allowed to adhere for 20 min at 4 degrees C under static conditions. Non-adhering cells were collected first, and adhering tumor cells together with ECs were detached from the culture plate. Collected cell fractions were evaluated by flow cytometry. Results were re-calculated as a ratio (R) of adhering colon carcinoma cells per one EC. We demonstrated that immortalized human microvascular ECs preserved their organ specificity. Colon carcinoma cells adhere preferentially to ECs of intestine origin. The immunofluorescent staining of adhering and non-adhering cancer cell subpopulations has revealed an augmented level of Lewis(x) antigen on adhering cancer cells. The organ specificity of endothelial cell interactions with colon carcinoma cells demonstrated in static conditions was verified and confirmed with flow adhesion assay. The method elaborated is suitable for quantifying of tumor cells adhering to ECs, with simultaneous evaluation of cell surface phenotypic markers of both partner cells participating in adhesive interactions. Validated by comparison to dynamic shear stress adhesion assay in blood flow reconstituted conditions this assay greatly facilitates evaluation of tumor cell-endothelial cell mutual interactions taking place during metastatic process.

Cell model of inflammation.

Chemical and physical stimuli trigger a cutaneous response by first inducing the main epidermal cells, keratinocytes, to produce specific mediators that are responsible for the initiation of skin inflammation. Activation modulates cell communication, namely leucocyte recruitment and blood-to-skin extravasation through the selective barrier of the vascular ECs (endothelial cells). In the present study, we describe an in vitro model which takes into account the various steps of human skin inflammation, from keratinocyte activation to the adhesion of leucocytes to dermal capillary ECs. Human adult keratinocytes were subjected to stress by exposure to UV irradiation or neuropeptides, then the conditioned culture medium was used to mimic the natural micro-environmental conditions for dermal ECs. A relevant in vitro model must include appropriate cells from the skin. This is shown in the present study by the selective reaction of dermal ECs compared with EC lines from distinct origins, in terms of leucocyte recruitment, sensitivity to stress and nature of the stress-induced secreted mediators. This simplified model is suitable for the screening of anti-inflammatory molecules whose activity requires the presence of various skin cells.