Tumor-associated endothelial cells with cytogenetic abnormalities.

Tumor angiogenesis is necessary for solid tumor progression and metastasis. Tumor blood vessels have been shown to differ from normal counterparts, for example, by changes in morphology. An important concept in tumor angiogenesis is that tumor endothelial cells are assumed to be genetically normal, although these endothelial cells are structurally and functionally abnormal. However, we hypothesized that given the phenotypic differences between tumor and normal blood vessels, there may be genotypic alterations as well. Mouse endothelial cells were isolated from two different human tumor xenografts, melanoma and liposarcoma, and from two normal endothelial cell counterparts, skin and adipose. Tumor-associated endothelial cells expressed typical endothelial cell markers, such as CD31. They had relatively large, heterogeneous nuclei. Unexpectedly, tumor endothelial cells were cytogenetically abnormal. Fluorescence in situ hybridization (FISH) analysis showed that freshly isolated uncultured tumor endothelial cells were aneuploid and had abnormal multiple centrosomes. The degree of aneuploidy was exacerbated by passage in culture. Multicolor FISH indicated that the structural chromosomal aberrations in tumor endothelial cells were heterogeneous, indicating that the cytogenetic alterations were not clonal. There was no evidence of human tumor-derived chromosomal material in the mouse tumor endothelial cells. In marked contrast, freshly isolated normal skin and adipose endothelial cells were diploid, had normal centrosomes, and remained cytogenetically stable in culture even up to 20 passages. FISH analysis of tumor sections also showed endothelial cell aneuploidy. We conclude that tumor endothelial cells can acquire cytogenetic abnormalities while in the tumor microenvironment.

Myogenic potential of muscle side and main population cells after intravenous injection into sub-lethally irradiated mdx mice.

Muscle side population (SP) cells have demonstrated hematopoietic and myogenic activities in vivo upon intravenous (IV) injection into lethally irradiated mdx mice. In contrast, muscle main population (MP) cells were unable to rescue the bone marrow of lethally irradiated mice and, consequently, their in vivo myogenic potential could not be assessed using this method. In the current study, muscle SP or MP cells derived from syngeneic wild-type male mice were delivered to sub-lethally irradiated mdx female mice by single or serial IV injections. Recipient mice were euthanized 12 weeks after transplantation at which time the quadriceps and diaphragm muscles were analyzed for the presence of donor-derived cells. Mice injected with 10(4) muscle SP cells or with 10(6) MP cells appeared to have similar numbers of dystrophin-positive myofibers containing fused donor nuclei. Analysis of the remaining tissue via real-time quantitative PCR indicated that mice injected with muscle SP cells had a higher percentage of donor-derived Y-DNA in the quadriceps than mice injected with MP cells, suggesting that muscle SP cells may be enriched for progenitors able to engraft dystrophic skeletal muscles from the circulation. Although the overall engraftment did not reach therapeutically significant levels, these results indicate that further optimization of cell delivery techniques may lead to improved efficacy of cell-mediated therapy using muscle SP cells.

Endothelial progenitor cells in infantile hemangioma.

Infantile hemangioma is an endothelial tumor that grows rapidly after birth but slowly regresses during early childhood. Initial proliferation of hemangioma is characterized by clonal expansion of endothelial cells (ECs) and neovascularization. Here, we demonstrated mRNA encoding CD133-2, an important marker for endothelial progenitor cells (EPCs), predominantly in proliferating but not involuting or involuted hemangioma. Progenitor cells coexpressing CD133 and CD34 were detected by flow cytometry in 11 of 12 proliferating hemangioma specimens from children 3 to 24 months of age. Furthermore, in 4 proliferating hemangiomas, we showed that 0.14% to 1.6% of CD45(-) nucleated cells were EPCs that coexpressed CD133 and the EC marker KDR. This finding is consistent with the presence of KDR(+) immature ECs in proliferating hemangioma. Our results suggest that EPCs contribute to the early growth of hemangioma. To our knowledge, this is the first study to show direct evidence of EPCs in a human vascular tumor.

Role of bone marrow cell trafficking in replenishing skeletal muscle SP and MP cell populations.

The multipotent nature of skeletal muscle-derived side population cells is demonstrated by their myogenic and hematopoietic potential in vivo. However, whether muscle side population cells are derived from the bone marrow is unclear. To study the long-term contribution of the hematopoietic system to muscle side population, whole bone marrow cells from Ly5.1 males or from e-GFP transgenic male mice were transplanted into lethally irradiated Ly5.2 females. Long-term cell trafficking of donor bone marrow cells to muscle side population was monitored 17 times in a 34-week study. Fluorescence-activated cell sorter analyses were used to detect Ly5.1 and GFP(+) donor cells, which were confirmed by fluorescence in situ hybridization of the Y-chromosome. Analyses post-transplantation indicated that whereas cells of donor origin could be found in the muscle, donor bone marrow cells had contributed little to the muscle side population. Attempts to increase cell trafficking by induced muscle damage again confirmed that more than 90% of side population cells present in the muscle were derived from the host. These results demonstrate that muscle side population cells are not replenished by the bone marrow and suggest a non-hematopoietic origin for this cell population.