Generation of highly pure fusions of colorectal carcinoma and antigen-presenting cells.

purposeThe induction of potent T cell responses against tumors is the goal of tumor immunotherapy. One approach is the fusion of antigen-presenting cells (APCs) with tumor cells. Hybrid cells combine the antigenicity of tumors with the immunostimulatory capacity of APCs. However, contaminating unfused cells present in the fusion reaction may prevent the induction of antitumoral immune responses. Here, we present a simple and effective protocol to substantially elevate the purity of hybrid cells.

methodsColorectal tumor cell lines and CD40-activated B cells as APCs were fused using polyethylene glycol. Important parameters including cell numbers, concentrations, and handling and detection procedures were optimized. Combination of these optimized fusion conditions with both magnetic cell sorting and selective adherence delivered very pure preparations of APC/tumor cell hybrids. The T cell stimulatory capacity of these hybrids was tested using ELISpot.

resultsThe optimization of the fusion resulted in maximal fusion efficiencies of 31.6% (n = 10, range 13.5-46.6%). Prelabeling of APCs with magnetic beads allowed for easy elimination of up to 94.3% of unfused tumor cells from the cell mixture by magnetic separation. Hybrid cell capacity to firmly adhere to plastic was then used to remove unfused B cells from the remaining cell mixture by simple washing. The obtained 85.0% pure hybrids cells readily induced antitumoral T cell responses.

conclusionsOur protocol delivers pure hybrid cell preparations with strong immunostimulatory potential. In subsequent experiments, the ability of hybrid cells to stimulate specific antitumoral T cell responses must be tested in vivo.