Spindle cell overgrowth in tumor cultures has long been appreciated in carcinoma, where these cells are thought to be derived from stromal fibroblasts (32
). To our knowledge, the origin of these cells and their MSC properties have never been investigated. In sarcoma, morphological distinction between benign and malignant cells is more difficult. Mesenchymal cell-like morphology in sarcoma primary cultures has been noted in early studies from this institution (34
). Attempts to culture sarcoma samples often yield non-tumor-forming, cytogenetically normal cultures; this has been proposed as an explanation for the paucity of sarcoma cell lines(35
We now demonstrate that sarcoma-derived cultures have properties of MSCs, namely surface marker expression, morphology, and in vitro
differentiation potential. The finding of benign MSCs in sarcoma was unexpected. Despite the accumulating evidence for MSC origin of sarcoma, there are no published reports describing the expression of MSC markers in sarcoma or the isolation of MSCs from sarcoma cell lines or primary sarcoma samples. Adherent cells from single-cell digested liposarcoma surgical specimens have been shown to differentiate along the adipocyte lineage with the addition of a PPARγ agonist(36
), but bone and cartilage differentiation potential or MSC marker expression was not investigated in these cultures. In retrospect, some of these cultures may have been benign SDBMSCs we describe in the current study.
In fact, even in carcinoma, where tumor-associated fibroblasts are well-studied(38
), their relationship to MSCs remains unclear. Heterologous bone-marrow-derived MSCs have been shown to stimulate tumor metastasis(40
) and home to tumors in mice when injected intravenously(41
). However, fibroblasts isolated from human carcinoma specimens have never been shown to have MSC properties such as differentiation into mesenchymal lineages and surface marker expression.
Brune et al recently reported in abstract form the isolation of MSCs from osteosarcoma using a similar approach to ours. They also find that osteosarcoma-derived MSCs are cytogenetically normal and therefore likely benign(42
). The authors conclude that the benign MSCs likely represent “tumor stroma”. Another abstract reports isolation of MSCs from glioma(43
). To our knowledge, these are the only studies reporting isolation of MSCs from human tumors of any histology.
Suva et al used a cancer stem cell marker, CD133, to isolate a tumor-initiating fraction in Ewing sarcoma. CD133+ cells demonstrated MSC-like differentiation and overexpressed FLI1, indicative of the presence of the EWS-FLI1 translocation (44
). We have not analyzed any Ewing sarcoma samples in the current study. However, in a flow cytometric analysis of over 30 single-cell digested surgical sarcoma samples of various histologies, we saw no CD133-expressing cells when CD45+ and CD31+ cells are excluded (data not shown).
Selective survival of benign MSCs in vitro
suggests that our culture conditions do not adequately mimic the tissue microenvironment where sarcoma cells clearly outgrow benign cells. In fact, human stromal cells including endothelial cells and pericytes are quickly replaced by mouse vasculature in tumor xenografts, suggesting that in the mouse environment human malignant cells survive, while human benign cells do not(45
The presence of benign MSCs within sarcomas underscores the need to analyze primary tumor cultures for the presence of benign cells. In studies attempting to identify the cancer stem cell fractions in sarcoma(46
), it is important to ensure that tumor-initiating ability of individual tumor fractions is normalized to the numbers of malignant cells using a technique such as interphase FISH. Similarly, expression profiling on whole tumor preparations may detect the MSC signature of benign cells.
The tumor microenvironment is increasingly recognized as a novel therapeutic target(47
). Tumor stroma is broadly defined to include tumor microvasculature (endothelial cells and pericytes), tumor-associated fibroblasts and tumor-infiltrating hematopoetic cells(38
). In sarcoma, benign cells include hematopoetic cells, endothelial cells, stromal fibroblasts recognized by their lack of nuclear atypia, and pericytes. We find that sarcoma-derived benign MSCs have properties of pericytes, such as the expression of pericyte markers and cooperation with endothelial cells in tube formation assays. Given the recent finding that normal tissue pericytes have MSC properties, we hypothesize that in sarcoma, benign MSCs also reside in a perivascular location. Indeed, we show that one MSC marker, CD105, is expressed by endothelial cells and pericytes in a variety of sarcomas, but not in malignant cells.
Some surgical sarcoma samples we analyzed did not yield any MSC cultures, such as 334, 904, 355, 394 (). One of them (355) has been irradiated. Due to a small number of samples we are unable to make correlations between the yield of MSC cultures or their benign vs malignant nature and clinical characteristics of the tumor.
Two roles of pericytes in sarcoma can be envisioned. First, as the cells in direct contact with malignant sarcoma cells, pericytes may constitute a previously unrecognized stromal supporting element. In accordance with this model, we demonstrate that under low-serum conditions, SDBMSCs as well as pericytes from healthy donors markedly stimulate the growth of sarcoma cell lines. In other malignancies such as leukemia, stromal cocultures are well-established and have been used to study leukemia-stroma interactions. To our knowledge, this is the first demonstration of such a stromal co-culture model in sarcoma. Such a coculture system may represent a more physiologically relevant model for evaluating therapeutic agents in vitro than sarcoma cell lines grown alone in high serum. Some agents may exhibit activity in sarcoma not by targeting the malignant cells but by abrogating the supporting influence of the pericytes. Such agents may be identified through a high-thoughput screen using our co-culture model; this effort is currently underway in our laboratory. Potential therapeutic strategies to target pericytes in sarcoma may involve antibodies against CD105(31
), which we show to be largely restricted to sarcoma vasculature, and endosialin which is expressed on tumor pericytes and malignant cells(24
Second, malignant sarcoma cells share features of pericytes, such as differentiation along mesenchymal lineages and expression of pericyte markers such as NG2(50
) and endosialin(24
), pointing to the pericyte as a candidate cell of origin of sarcoma. Experiments to address this model are under way in our laboratory.