Emilio Ciusani

We investigated in vitro the properties of selected populations of cancer stem-like cells defined as tumorospheres that were obtained from human glioblastoma. We also assessed their potential and capability of differentiating into mature cells of the central nervous system. In vivo, their tumorigenicity was confirmed after transplantation into the brain of non-obese diabetic/severe combined immunodeficient (NOD-SCID) mice. The angiogenic potential of tumorospheres and glioblastoma-derived cells grown as adherent cells was revealed by evaluating the release of angiogenic factors such as vascular endothelial growth factor and CXCL12 by ELISA, as well as by rat aortic ring assay. The proliferative response of tumorospheres in the presence of CXCL12 was observed for the first time. Multidrug resistance-associated proteins 1 and 3 as well as other molecules conferring multidrug resistance were higher when compared with primary adherent cells derived from the same tumor. Finally, we obtained cells from the tumor developing after grafting that clearly expressed the putative neural stem cell marker CD133 as shown by FACS analysis and also nestin and CXCR4. The cells' positivity for glial fibrillary acidic protein was very low. Moreover these cells preserved their angiogenic potential. We conclude that human glioblastoma could contain tumor cell subsets with angiogenic and chemoresistance properties and that this chemoresistance potential is highly preserved by immature cells whereas the angiogenic potential is, to a higher extent, a property of mature cells. A better understanding of the features of these cell subsets may favor the development of more specifically targeted therapies.

The association of primary sclerosing cholangitis (PSC) to HLA class II genes was studied by comparing patients from five different European populations. Deduced HLA-DRB1, DQA1, DQB1 haplotypes of 256 PSC patients from England, Italy, Norway, Spain and Sweden were compared to those observed in 764 ethnically-matched controls. Increased frequencies of the DRB1*03, DQA1*0501, DQB1*02 (RR=3.0, P<0.00001) and the DRB1*13, DQA1*0103, DQB1*0603 haplotypes (RR=2.4, P<0.0001) were observed in all five patient groups. A total of 16% of the PSC patients were homozygous for the DRB1*03, DQA1*0501, DQB1*02 haplotype compared to 1% of the controls (RR=20, P<0.0001). The DRB1*04, DQA1*03, DQB1*0302 haplotype was significantly reduced in frequency(RR=0.4, P<0.00001). Among Norwegian, Swedish and British patients that did not carry neither the DRB1*03, DQA1*0501, DQB1*02 nor the DRB1*13, DQA1*0103, DQB1*0603 haplotype, an increased frequency of the DRB1*15, DQA1*0102, DQB1*0602 haplotype was observed (RR=2.0, P<0.0001). Thus, PSC was found to be positively associated to three different HLA class II haplotypes (i.e. the DRB1*03, DQA1*0501, DQB1*02, the DRB1*15, DQA1*0102, DQB1*0602 and the DRB1*13, DQA1*0103, DQB1*0603 haplotypes) and negatively associated to one HLA class II haplotype (i.e. the DRB1*04, DQB1*0302 haplotype).

BACKGROUND: Mesenchymal stromal cells may represent an ideal candidate to deliver anti-cancer drugs. In a previous study, we demonstrated that exposure of mouse bone marrow derived stromal cells to Doxorubicin led them to acquire anti-proliferative potential towards co-cultured haematopoietic stem cells (HSCs). We thus hypothesized whether freshly isolated human bone marrow Mesenchymal stem cells (hMSCs) and mature murine stromal cells (SR4987 line) primed in vitro with anti-cancer drugs and then localized near cancer cells, could inhibit proliferation. METHODS AND PRINCIPAL FINDINGS: Paclitaxel (PTX) was used to prime culture of hMSCs and SR4987. Incorporation of PTX into hMSCs was studied by using FICT-labelled-PTX and analyzed by FACS and confocal microscopy. Release of PTX in culture medium by PTX primed hMSCs (hMSCsPTX) was investigated by HPLC. Culture of Endothelial cells (ECs) and aorta ring assay were used to test the anti-angiogenic activity of hMSCsPTX and PTX primed SR4987(SR4987PTX), while anti-tumor activity was tested in vitro on the proliferation of different tumor cell lines and in vivo by co-transplanting hMSCsPTX and SR4987PTX with cancer cells in mice. Nevertheless, despite a loss of cells due to chemo-induced apoptosis, both hMSCs and SR4987 were able to rapidly incorporate PTX and could slowly release PTX in the culture medium in a time dependent manner. PTX primed cells acquired a potent anti-tumor and anti-angiogenic activity in vitro that was dose dependent, and demonstrable by using their conditioned medium or by co-culture assay. Finally, hMSCsPTX and SR4987PTX co-injected with human cancer cells (DU145 and U87MG) and mouse melanoma cells (B16) in immunodeficient and in syngenic mice significantly delayed tumor takes and reduced tumor growth. CONCLUSIONS: These data demonstrate, for the first time, that without any genetic manipulation, mesenchymal stromal cells can uptake and subsequently slowly release PTX. This may lead to potential new tools to increase efficacy of cancer therapy.