Shared cell surface marker expression in mesenchymal stem cells and adult sarcomas

Abstract

Advanced adult soft-tissue sarcomas (STSs) are rare tumors with a dismal prognosis and limited systemic treatment options. STSs may originate from mesenchymal stem cells (MSCs); the latter have mainly been isolated from adult bone marrow as plastic-adherent cells with differentiation capacity into mesenchymal tissues. Recently, a panel of antibodies has been established that allows for the prospective isolation of primary MSCs with high selectivity. Similar to cancer stem cells in other malignancies, sarcoma stem cells may bear immunophenotypic similarity with the corresponding precursor, that is, MSCs. We therefore set out to establish the expression pattern of MSC markers in sarcoma cell lines and primary tumor samples by flow cytometry. In addition, fibroblasts from different sources were examined. The results document a significant amount of MSC markers shared by sarcoma cells. The expression pattern includes uniformly expressed markers, as well as MSC markers that only stained subpopulations of sarcoma cells. Expression of W5C5, W8B2 (tissue nonspecific alkaline phosphatase [TNAP]), CD344 (frizzled-4), and CD271 marked subpopulations displaying increased proliferation potential. Moreover, CD271+ cells displayed in vitro doxorubicin resistance and an increased capacity to form spheres under serum-free conditions. Interestingly, another set of antigens, including the bona fide progenitor cell markers CD117 and CD133, were not expressed. Comparative expression patterns of novel MSC markers in sarcoma cells, as well as fibroblasts and MSCs, are presented. Our data suggest a hierarchical cytoarchitecture of the most common adult type sarcomas and introduce W5C5, TNAP, CD344, and CD271 as potential sarcoma progenitor cell markers.

Figure 1.

Sarcoma cells with fibroblast morphology compared with MSCs, embryonic fibroblasts, and foreskin fibroblasts. To allow for a comparison of sarcomas with fibroblast morphology and MSCs, as well as embryonic and adult fibroblasts, the human fibrosarcoma cell line HT1080 and one case of MFS are shown side by side with MSCs, embryonic fibroblast cell line MRC5, and FF. The four functionally important markers W5C5, CD271, TNAP, and CD344 are depicted. Isotype controls are shown in gray, and specific antibody staining is shown in black. The same panels are shown as contour plots to allow for the comparative analysis of small subpopulations in supplemental online Figures 1–3. Abbreviations: FF, foreskin fibroblast; MFS, myxofibrosarcoma; MSC, mesenchymal stem cell; TNAP, tissue nonspecific alkaline phosphatase.

Figure 2.

Expression of W5C5, CD271, TNAP, and CD344 in leiomyosarcoma and rhabdomyosarcoma. Sarcomas with smooth and striated muscle morphology are shown side by side as follows: the human LMS cell line SK-LMS1, representative results of n = 3 cases of primary LMS, and representative data of n = 2 cases of primary rhabdomyosarcoma. Contour plots of the same data, as well as specific fluorescence index values, are shown in detail in supplemental online Figures 1 and 2. Abbreviations: LMS, leiomyosarcoma; RMS, rhabdomyosarcoma; TNAP, tissue nonspecific alkaline phosphatase.

Figure 3.

Expression of W5C5, CD271, TNAP, and CD344 on synovial sarcoma. One case of primary biphasic synovial sarcoma was analyzed and compared with the human synovial sarcoma cell line SW982. Exact data on subpopulations, as well as specific fluorescence index values, are depicted in supplemental online Figures 1 and 2. Abbreviations: Syn., synovial; TNAP, tissue nonspecific alkaline phosphatase.

Figure 4.

Comparative analysis of liposarcoma cell line and primary liposarcoma. The human liposarcoma cell line SW872 was compared with a case of primary liposarcoma. SW872 strongly expressed CD271, W5C5, and CD344 but very little TNAP (<0.5% of cells, see also supplemental online Fig. 1C). In contrast, primary liposarcoma cells strongly expressed TNAP but little CD344. Expression of CD271 and expression of W5C5 were comparable between cell line and primary sarcoma cells. Abbreviations: LS, liposarcoma; TNAP, tissue nonspecific alkaline phosphatase.

Figure 5.

Identification of subpopulations with high proliferative activity. (A): Compared with their negative counterparts and mock-sorted tumor cells (fibrosarcoma cell line HT1080), W5C5-positive, W8B2-positive, and CD344-positive subpopulations revealed increased proliferative potential as assessed in a metabolism-dependent assay using methyl tetrazolium salt as a substrate for mitochondrial enzymes. (B): CD271 is expressed by a highly proliferative cell population both in cell lines (HT1080, SK-LMS, SW872, SW982) and in primary tumor samples (leiomyosarcoma, chondrosarcoma, pleomorphic sarcoma NOS; two independent primary tumor samples each). (C): Summary of all proliferation assays as a ratio of CD271-positive versus CD271-negative cells (at a seeding density of 125 cells per well). Note the log-scaled axis of ordinates. Abbreviations: AU, arbitrary unit; FSC, forward scatter; neg, negative; NOS, not otherwise specified; pos, positive.

Figure 6.

Sarcosphere initiating capacity and doxorubicin resistance. (A): Representative sarcosphere grown in serum-free medium as described in Materials and Methods. (B): An increasing number of cells (8, 16, 31, 63, 125, 250, 500, and 1,000) were flow-sorted in 96-well flat-bottomed ultra-low attachment plates (Corning) and cultured under serum-free conditions. Note that sarcosphere initiating capacity is preserved down to a number of 63 CD271+ cells seeded as opposed to 250 CD271− cells. Mean values of quadruplicate experiments are shown. (C): CD271+ and CD271− cells were separated using magnetic activated cell sorting technology, and 2 × 10³ cells, respectively, were seeded in each well of white flat-bottomed 96-well plates. After 3 days of doxorubicin treatment, the plates were analyzed with the CellTiter-Glo luminescent cell viability assay (Promega GmbH) according to the manufacturer's instructions. Abbreviations: neg, negative; pos, positive.

Figure 7.

STS cell lines harbor differentiation capacity similar to MSCs. For in vitro differentiation, the indicated cell types were cultured and stained for adipogenesis (LipidTOX Green), chondrogenesis (Alcian blue), and osteogenesis (alizarin red S). The degree of differentiation was determined by light microscopy, and the rating was color-coded from no evidence of differentiation (empty box) to full differentiation capacity (++, dark green) as compared with MSCs. Abbreviations: MSC, mesenchymal stem cell; no, no evidence of differentiation.