In Vivo Evolution of Tumor-Derived Endothelial Cells

The growth of a malignant tumor beyond a certain, limited size requires that it first develop an independent blood supply. In addition to providing metabolic support, this neovasculature also allows tumor cells to access the systemic circulation, thus facilitating metastatic dissemination. The neovasculature may originate either from normal blood vessels in close physical proximity to the tumor and/or from the recruitment of bone marrow-derived endothelial cell (EC) precursors. Recent studies have shown that human tumor vasculature ECs may also arise directly from tumor cells themselves and that the two populations have highly similar or identical karyotypes. We now show that, during the course of serial in vivo passage, these tumor-derived ECs (TDECs) progressively acquire more pronounced EC-like properties. These include higher-level expression of EC-specific genes and proteins, a greater capacity for EC-like behavior in vitro, and a markedly enhanced propensity to incorporate into the tumor vasculature. In addition, both vessel density and size are significantly increased in neoplasms derived from mixtures of tumor cells and serially passaged TDECs. A comparison of early- and late-passage TDECs using whole-genome single nucleotide polymorphism profiling showed the latter cells to have apparently evolved by a process of clonal expansion of a population with a distinct pattern of interstitial chromosomal gains and losses affecting a relatively small number of genes. The majority of these have established roles in vascular development, tumor suppression or epithelial-mesenchymal transition. These studies provide direct evidence that TDECs have a strong evolutionary capacity as a result of their inherent genomic instability. Consequently such cells might be capable of escaping anti-angiogenic cancer therapies by generating resistant populations

Permanently blocked stem cells derived from breast cancer cell lines

Cancer stem cells (CSCs) are thought to be resistant to standard chemotherapeutic drugs and the inimical conditions of the tumor microenvironment. Obtaining CSCs in sufficient quantities and maintaining their undifferentiated state have been major hurdles to their further characterization and to the identification of new pharmaceuticals that preferentially target these cells. We describe here the tagging of CSC-like populations from four human breast cancer cell lines with green fluorescent protein (GFP) under the control of the Oct3/4 stem cell-specific promoter. As expected, GFP was expressed by the CSC-enriched populations. However, an unanticipated result was that these cells remained blocked in a CSC-like state and tended to be resistant to chemotherapeutic drugs as well as acidotic and hypoxic conditions. These CSC-like cells possessed several other in vitro attributes of CSCs and were able to reproducibly generate tumors in immunocompromised mice from as few as 100 cells. Moreover, the tumors derived from these cells were comprised almost exclusively of pure CSCs. The ability of the Oct3/4 promoter to block CSC differentiation underscores its potential general utility for obtaining highly purified CSC populations, although the mechanism by which it does so remains undefined and subject to further study. Nonetheless, such stable cell lines should be extremely valuable tools for studying basic questions pertaining to CSC biology and for the initial identification of novel CSC-specific chemotherapeutic agents, which can then be verified in primary CSCs

EC-specific marker expression increases in serially-passaged H460-derived TDECs.

<p>(A) Cell surface staining for hCD31. TDECs from the indicated serial passages were isolated and the cell surface expression of the EC-specific marker hCD31 was then assessed by flow cytometry. (B) AcLDL uptake, E-lectin binding and anti-human vWF (hvWF) and anti-human ESAM (hESAM) immuno-staining were assessed in early and late TDECs using confocal microscopy (16). HUVECs were included as positive controls. Nuclei were stained with DAPI (blue). H&E staining under light microscopy was used to compare the morphologies of H460 tumor cells, TDECs, and HUVECs. Images were obtained at 20−40× magnification. (C) Expression of CD31 and vWF assessed by immuno-blotting. Early- and late-passage TDECs were examined by immuno-blotting for hCD31 and hvWF, as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037138#pone.0037138-Sajithlal1" target="_blank">[16]</a>. All lanes in the hCD31 blot were from the same X-ray film. β-actin levels were measured on the same samples and served as a loading control. (D) qRT-PCR was used to assess mRNA expression for the EC markers ESAM, CD31, and VWF in late passage TDECs relative to early passage TDECs. Values shown represent the average of at least triplicate reactions and <i>p</i> values were from a standard Student’s <i>t</i> test.</p

Summary of genomic alterations in late passage H460 TDECs.

<p>The nucleotide coordinates of genomic alterations in SP5 and SP6 TDECs were determined by the first and last SNPs that differed from those of early passage TDECs and the precise locations were obtained using the Ensembl Genome Browser 57 database.</p

Increased tumor re-populating ability of serially-passaged TDECs.

<p>(A) Human and murine TAECs were isolated from initial H460 lung cancer xenografts by CD31 immuno-affinity purification <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037138#pone.0037138-Sajithlal1" target="_blank">[16]</a>. The latter were then eliminated by propagating the cells in G-418 and the resultant GFP+, G418-resistant human TDECs were then serially passaged <i>in vivo</i> with a 20-fold excess of non-GFP-tagged H460 tumor cells. At the indicated passage numbers, the percent of GFP+ TDECs was determined 1–2 days after isolation and before the addition of G-418. The results shown represent the average values obtained from 3–5 fields (±SEM) with a total of 738, 761, 128, and 296 cells counted for SP1, SP2, SP4, and SP6, respectively. The statistical analysis was performed using a two-tailed Student’s <i>t</i> test (**, <i>p</i><0.01; ***, <i>p</i><0.0001). (B) Photomicrographs of typical blood vessels from H460 tumor xenografts established as described in <i>A</i> for serial passages SP1 and SP5. Frozen sections were visualized for GFP and stained for human-specific CD31 (hCD31) (red) and DAPI (blue). Images were taken at 20× magnification. (C) Live animal imaging of tumor xenografts arising in a representative mouse co-inoculated with an equal number of dsRed-tagged SP0 TDECs and GFP-tagged SP6 TDECs plus a 20-fold excess of untagged H460 tumor cells. (D) Quantification of the percent red SP1 versus the percent green SP7 TDECs isolated from three tumors. The graph represents the mean values (±SEM) with the <i>p</i> value determined using a one-tailed Student’s <i>t</i> test.</p