Osteosarcoma is a bone tumor, displaying significant cellular and histological heterogeneity and a complex genetic phenotype. Although multiple studies strongly suggest the presence of cancer stem cells in osteosarcoma, a consensus on their characterization is still missing. We used a combination of functional assays (sphere-forming, Aldefluor, and side-population) for identification of cancer stem cell populations in osteosarcoma cell lines. Expression of stemness-related transcription factors, quiescent nature, in vivo tumorigenicity, and Wnt/β-catenin activation were evaluated. We show that different cancer stem cell populations may co-exist in osteosarcoma cell lines exhibiting distinct functional properties. Osteosarcoma spheres are slowly-proliferating populations, overexpress SOX2, and KLF4 stemness-related genes and have enhanced tumorigenic potential. Additionally, spheres show specific activation of Wnt/β-catenin signaling as evidenced by increased nuclear β-catenin, TCF/LEF activity, and AXIN2 expression, in a subset of the cell lines. Aldefluor-positive populations were detected in all osteosarcoma cell lines and overexpress SOX2, but not KLF4. The side-population phenotype is correlated with ABCG2 drug-efflux transporter expression. Distinct functional methods seem to identify cancer stem cells with dissimilar characteristics. Intrinsic heterogeneity may exist within osteosarcoma cancer stem cells and can have implications on the design of targeted therapies aiming to eradicate these cells within tumors. J. Cell. Physiol. 231: 876-886, 2016. © 2015 Wiley Periodicals, Inc.

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Myxoid liposarcoma (MLS) is a soft tissue sarcoma characterized by a recurrent t(12;16) translocation. Although tumors are initially radio- and chemosensitive, the management of inoperable or metastatic MLS can be challenging. Therefore, our aim was to identify novel targets for systemic therapy. We performed an in vitro high-throughput drug screen using three MLS cell lines (402091, 1765092, DL-221), which were treated with 273 different drugs at four different concentrations. Cell lines and tissue microarrays were used for validation. As expected, all cell lines revealed a strong growth inhibition to conventional chemotherapeutic agents, such as anthracyclines and taxanes. A good response was observed to compounds interfering with Src and the mTOR pathway, which are known to be affected in these tumors. Moreover, BIRC5 was important for MLS survival because a strong inhibitory effect was seen at low concentration using the survivin inhibitor YM155, and siRNA for BIRC5 decreased cell viability. Immunohistochemistry revealed abundant expression of survivin restricted to the nucleus in all 32 tested primary tumor specimens. Inhibition of survivin in 402-91 and 1765-92 by YM155 increased the percentage S-phase but did not induce apoptosis, which warrants further investigation before application in the treatment of metastatic MLS. Thus, using a 273-compound drug screen, we confirmed previously identified targets (mTOR, Src) in MLS and demonstrate survivin as essential for MLS survival.

We have developed a “one‐tube” triple staining procedure that allows the identification of intratumor phenotypic subpopulations by FCM. Solid tumors were dissociated by a combined mechanical/enzymatic method. Ovarian ascites tumor cell aggregates were enzymatically dissociated using trypsin. An antikeratin 8/18 MAb was used to label the epithelial fraction of these tumor samples. A second MAb directed against the leukocyte common antigen (LCA) was applied to identify nonneoplastic DNA‐diploid cells. Other MAbs used as a second marker were directed against a tumor‐associated surface, a cytoplasmic, or a nuclear antigen. Cells were stained using subclass‐specific fluorescein‐isothiocyanate (FITC) or R‐phycoerythrin (PE)‐conjugated antibodies. DNA was stained with propidium iodide (PI). Triply stained samples were measured on a standard bench‐top flow cytometer (FACScan). Keratin 8/18‐positive cells, LCA‐positive cells, and DNA could be simultaneously detected in dissociated breast carcinomas, mixed Müllerian tumors, and ovarian ascites specimen for refining DNA index (DI) calculations and S phase fraction (SPF) determination. Coefficients of variation (CV) of the G0G1 peak of the DNA histograms obtained ranged from 2.55% to 4.64% and from 2.71% to 4.71% for the DNA‐diploid and ‐aneuploid fractions, respectively. In DNA‐diploid tumors, antigen expression (HER‐2/Neu, proliferating cell nuclear antigen) could be analyzed without interference of fluorescence signals from nonneoplastic cells. Neoplastic tumor subpopulations were clearly identified based on both DNA‐ploidy status and heterogeneity of antigen expression. The present method offers new possibilities for multiparameter DNA FCM on clinical samples and enables the identification of intratumor neoplastic subpopulations based on antigen expression and DNA‐ploidy status. © 1996 Wiley‐Liss, Inc.

Invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC) are the most frequently occurring histological subtypes of breast cancer, accounting for 80–90% and 10–15% of the total cases, respectively. At the time of diagnosis and surgical resection of the primary tumour, most patients do not have clinical signs of metastases, but bone micrometastases may already be present. Our aim was to develop a novel preclinical ILC model of spontaneous bone micrometastasis. We used murine invasive lobular breast carcinoma cells (KEP) that were generated by targeted deletion of E-cadherin and p53 in a conditional K14cre;Cdh1(F/F);Trp53(F/F) mouse model of de novo mammary tumour formation. After surgical resection of the growing orthotopically implanted KEP cells, distant metastases were formed. In contrast to other orthotopic breast cancer models, KEP cells readily formed skeletal metastases with minimal lung involvement. Continuous treatment with SD-208 (60 mg/kg per day), an orally available TGFβ receptor I kinase inhibitor, increased the tumour growth at the primary site and increased the number of distant metastases. Furthermore, when SD-208 treatment was started after surgical resection of the orthotopic tumour, increased bone colonisation was also observed (versus vehicle). Both our in vitro and in vivo data show that SD-208 treatment reduced TGFβ signalling, inhibited apoptosis, and increased proliferation. In conclusion, we have demonstrated that orthotopic implantation of murine ILC cells represent a new breast cancer model of minimal residual disease in vivo, which comprises key steps of the metastatic cascade. The cancer cells are sensitive to the anti-tumour effects of TGFβ. Our in vivo model is ideally suited for functional studies and evaluation of new pharmacological intervention strategies that may target one or more steps along the metastatic cascade of events. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.