Ex vivo evaluation of tumor cell specific drug responses in malignant pleural effusions.

The effect of chemotherapy may be improved by combining the most effective drugs based on testing the sensitivity of the individual tumor ex vivo. Such estimations of tumor cells from effusions have so far not been implemented in the clinical routine as a basis for individualized choice of therapy. One obstacle for such analyses is the admixture of benign cells that might obscure the results. In this paper we test and compare two ways of performing the analysis specifically on tumor cells. First we enrich the tumor cells, using antibody labeled magnetic separation, and measure the effects of subsequent drug exposure with the metabolic activity assays WST-1 and alamar blue. The second way of estimating drug effects specifically on tumor cells employs multi parameter flow cytometry, measuring apoptosis with the propidium iodide / AnnexinV technique and, particularly for pemetrexed, possible effects on cell cycle progression in immunologically identified tumor cells. The two techniques produce similar results, indicating a possible use in personalized medicine. The possible predictive role of the analysis remains to be shown.

HSCT recipients have specific tolerance to MSC but not to the MSC donor.

Multipotent mesenchymal stromal cells (MSC) are increasingly used to treat refractory graft-versus-host-disease and other complications after hematopoietic stem cell transplantation (HSCT). We evaluated immunogenicity of HLA-mismatched MSC infused posttransplant to HSCT recipients. Recipient lymphocyte response to MSC and peripheral blood lymphocytes (PBL) from the MSC or third party donors was measured before and after infusion. In vitro primary and rechallenge lymphocyte responses of healthy individuals to MSC and to PBL from the MSC donor were similarly studied. HSCT recipients given MSC responded to third party allostimuli, but showed no response to infused MSC before and upto 6 months after infusion, whereas maintaining an alloresponse to the MSC donor. This indicates immune unresponsiveness restricted to MSC, as the HSCT recipient was not tolerized to the MSC donor. In vitro, we confirmed that MSC failed to prime responder lymphocytes to rechallenge with PBL from the MSC donor, and lymphocytes primed with MSC donor and rechallenged with MSC only showed weak responses at high stimulator-responder ratios. Although MSC up-regulated lymphocyte gene expression of CD25, IFN-gamma, FoxP3, CTLA-4, and IL-10, they failed in both unprimed and primed responders to induce CD25+ (activated) or CD57+ (effector) CD4+ or CD8+ T-lymphocyte subsets and only inconsistently induced FoxP3+ regulatory T-lymphocytes. These results show for the first time that infused MSC are only weakly immunogenic in humans and validate the clinical use of MSC from HLA-mismatched donors.