Isolation, Culture and Characterization of Cancer Stem Cells from Primary Osteosarcoma

Osteosarcoma is a common childhood bone cancer with a poor survival rate. Osteosarcoma cancer stem cells (CSCs) contribute to the recurrence, drug resistance and metastasis of this disease. Previous evidence suggested that cancer cells are able to spontaneously turn into CSCs, thus it is crucial to simultaneously target osteosarcoma cells and CSCs. Our previous studies have demonstrated that salinomycin preferably eliminated osteosarcoma CSCs. In addition, amplification of the epidermal growth factor receptor (EGFR) is a common genetic aberration in osteosarcoma, and thus EGFR is a promising target in osteosarcoma. The present study aimed to develop EGFR aptamer-conjugated salinomycin-loaded polymer-lipid hybrid nanoparticles (EGFR-SNPs) to target both osteosarcoma cells and CSCs. The results revealed that EGFR was overexpressed in these cells, and that EGFR-SNPs possessed a small size of 95 nm, suitable drug encapsulation efficiency (63%) and sustained drug release over 120 h. EGFR-SNPs targeted EGFR-overexpressing osteosarcoma cells and CSCs, resulting in an enhanced cytotoxic effect compared with non-targeted SNPs and salinomycin. Notably, EGFR-SNPs was able to reduce the osteosarcoma tumorsphere formation rate and proportion of CD133+ osteosarcoma CSCs in the osteosarcoma cell lines more effectively compared with SNPs and salinomycin, suggesting that EGFR-SNPs effectively reduced the proportion of osteosarcoma CSCs. In conclusion, the interaction of EGFR aptamers and EGFR is a potential approach to promote the effective delivery of salinomycin to osteosarcoma. The study results suggested that EGFR-SNPs represents a promising approach to target osteosarcoma cells and CSCs.

Targeted salinomycin delivery with EGFR and CD133 aptamers based dual-ligand lipid-polymer nanoparticles to both osteosarcoma cells and cancer stem cells

Pulmonary metastasis is the leading cause of death in osteosarcoma (OS), the most common malignant bone tumor in children and young adults. The molecular mechanisms that regulate the formation of metastases in OS remain mostly unknown. A subset of cancer stem cells (CSCs) with metastatic potential has been identified in breast, colon and pancreatic cancers. Therefore, we hypothesize that CSCs drive the metastatic process in OS by their ability to migrate, adapt to a different microenvironment, and subsequently generate distal metastatic lesions in the lungs. Using a novel mutant p53 mouse model of metastatic OS, which has been developed in Dr. Jason Yustein's laboratory, we have identified putative OS CSCs in primary bone tumors and metastatic lesions. Cancer stem cells have been cultured from solid tumors growing as sphere-like structures under serum-free conditions. This method has been validated as a surrogate of their self-renewal capacity. We have observed that murine tumor cells obtained from both primary bone tumors and pulmonary nodules developed sarcospheres. Notably, sarcophere-forming efficiency was significantly increased (2-3 times) in tumor cells obtained from metastatic lesions. Aldehyde dehydrogenase 1 (ALDH1) activity has been used as a functional stem cell marker to isolate CSCs in numerous solid tumors. We have found that both primary bone tumors and metastatic pulmonary nodules contain ALDH Hi (stem-like cells). The high expression of ALDH1 in murine OS tumors ranged from 0.7 to 17%. Further analysis of derived cell lines generated from primary OS tumor, circulating tumor cells (CTCs) and metastatic tumors showed that distal metastases have 4-5 times more ALDH Hi cells than CTCs and up to 10 times more ALDH Hi cells than the primary bone tumors. The isolation of OS CSCs by fluorescent activated cell sorting (FACS) facilitates the analysis of the molecular pathways and biological process that regulates this tumor cell compartment. Our initial data has shown that the Insulin-like Growth Factor (IGF) family members were differentially expressed between OS primary and metastatic cells. Furthermore, we are actively utilizing these mouse derived cell lines to perform orthotopic transplants in immunocompetent (syngeneic) mice aiming to better understand the biology and genetics of CSCs in OS. Subsequent studies will be performed to determine the functional significance of the unique OS CSC genetic alterations. In summary, we have identified putative metastatic OS CSCc in a mutant p53 mouse model of metastatic OS. Our preliminary data has shown that CSC content seems to be increased in metastases. Our studies using an OS mouse model that parallels the biological behavior of human disease in addition to provide freshly isolated tumor specimens constitute a strong pre-clinical model to test the effect of novel anti-cancer therapies in the CSC compartment. Citation Format: Nino Rainusso, Lyazat Kurenbekova, Lawrence Donehower, Jeffrey Rosen, Jason Yustein. Characterization of metastatic cancer stem cells in osteosarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C80.

In the published article, an unintentional error occurred in the caption of Figure 1: [Article in the Journal of Cellular Biochemistry. https://doi.org/10.1002/jcb.29863]. Article first published online: 2 November 2020. The caption of Figure 1: “Isolation and characterization of cancer stem cells from TNBC lines MDA-MB-231 and MDA-MB-468. (A,B) FACS sorting of cancer stem cells as CD44 + and CD24 − cells from MDA-MB-231 and MDA-MB-468. (C,D) Phase-contrast images of sorted cancer stem cells in culture. (E,F) Population doubling time (PDT) of sorted cancer stem cells. (G,H) Immunocytochemistry of WT and CSC population from MDA-MB-231. (I,J) Immunocytochemistry of WT and CSC population from MDA-MB-468. Scale bar = 100 μm. Error bars indicate mean ± SD, and represent at least three independent experiments. The differences between more than two groups were analyzed by oneway ANOVA followed by post hoc test (Bonferroni correction). The p > .05 (ns), <.05 (*), .01 (**), <.001 (***) were considered as significant. Error bar represents the ±standard error of the mean (SEM). ANOVA, analysis of variance; CSC, cancer stem cell; FACS, fluorescent activated cell sorting; MDA, malondialdehyde; TNBC, triple-negative breast cancer” should be read as: “Isolation and characterization of cancer stem cells from TNBC lines MDA-MB-231 and MDA-MB-468. (A,F) FACS sorting of cancer stem cells as CD44 + and CD24 − cells from MDA-MB-231 and MDA-MB-468. (B, G) Phase-contrast images of sorted cancer stem cells in culture. (C, H) Population doubling time (PDT) of sorted cancer stem cells. (D, E) Immunocytochemistry of WT and CSC population fromMDA-MB-231. (I, J) Immunocytochemistry of WT and CSC population from MDA-MB-468. Scale bar = 100μm. Error bars indicate mean ± SD, and represent at least three independent experiments. The differences between more than two groups were analyzed by one-way ANOVA followed by post hoc test (Bonferroni correction). The p > .05 (ns), <.05 (*), .01 (**), <.001 (***) were considered as significant. Error bar represents the ±standard error of the mean (SEM). ANOVA, analysis of variance; CSC, cancer stem cell; FACS, fluorescent activated cell sorting; TNBC, triple-negative breast cancer” The authors regret this error.

Cancer Stem Cell–Directed Therapies: Recent Data From the Laboratory and Clinic

Perspectives on cancer stem cells in osteosarcoma

Osteosarcoma is the most common primary malignant tumor of bone in children and adolescents and advanced osteosarcoma patients with evidence of metastasis share a poor prognosis. Osteosarcoma frequently gains resistance to standard therapies highlighting the need for improved treatment regimens and identification of novel therapeutic targets. The composition of cancer stem cells is one of the attractive theoretical studies in osteosarcoma biology. It has been proposed that osteosarcomas are composed of a group of functionally heterogeneous cells. Only a small number of osteosarcoma cells have stem cell characteristics, with self-renewal and differentiation capacities, which facilitates in the generation of different types of osteosarcoma cells. These osteosarcoma stem cells play a decisive role in maintaining the proliferation, invasion, recurrence, and metastasis of tumors. Previous research suggests that osteosarcoma formation may result from the development of cancer stem cells by the deregulation of normal self-renewal pathways of tissue stem cells. The discovery of cancer stem cells in osteosarcoma offers a new approach to understanding the biology of these conditions. Further study is needed to understand both normal and osteosarcoma stem cell development and various signaling pathways which appear to regulate self-renewal of stem cells and CSCs, with dysregulation of these signaling pathways leading to tumorigenesis. Ultimately, new prognostic and predictive markers, as well as targeted therapeutic strategies, may be developed to force osteosarcoma into better outcome or permanent remission. In this review we will discuss the research advance of cancer stem cells in osteosarcoma such as commonly used isolation techniques for osteosarcoma stem cells as well as the identified biochemical and molecular markers and we share our viewpoints on controversy in research of osteosarcoma stem cells.

BackgroundAs an important stress-response mechanism, autophagy plays crucial role in the tumor formation and drug resistance of cancer cells including osteosarcoma (OS). OS cancer stem cells (CSCs) also are considered a key factor of tumorigenesis, drug resistance and tumor recurrence. However, the relationship between autophagy and OS CSCs still remains unclear.MethodsCD271+ OS CSCs and CD271- OS cells were isolated by magnetic activated cell sorting. The autophagy level was evaluated by the mRNA expression of autophagy genes, the protein level of LC3II and p62, and the mean number of GFP-LC3 dot per cell. Lentivirus-delivered specific shRNA was utilized to inhibit the corresponding gene expression. The cell viability was examined with CCK8 assay. The cell proliferation level was detected with BrdU staining assay. Cell death was determined by Annexin V/PI double staining of fluorescence activated cell sorting, lactate dehydrogenase release and caspase-3 activity. Tumorigenicity ability was evaluated by colony and sphere formation assay, the protein expression of stemness markers and tumor formation in nude mice.ResultsOur data indicated that CD271+ OS CSCs had a similar basic autophagy level with CD271- OS cells. Autophagy deficiency had no observable effects on the levels of cell proliferation and death both in CD271+ and CD271- OS cells under normal condition. However, CD271+ OS cells showed a higher autophagy activity than CD271- OS cells under hypoxia and low nutrient (LH) condition. Moreover, autophagy-deficient CD271+ OS cells lost the advantage of tolerance to LH condition compared to CD271- OS cells. Meanwhile, autophagy deficiency enhanced the sensitivity to chemotherapeutics in the CD271+ cells to the comparable level in the CD271- cells. More importantly, deficient-autophagy decreased the protein expression of stemness markers and caused the disappearance of the superiority in tumorigenicity in vitro and vivo in CD271+ OS cells.ConclusionThe results above demonstrated that autophagy contributes to the stem-like features of CD271+ OS CSCs. Inhibition of autophagy is a promising strategy in the CSCs-targeting OS therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12929-016-0297-5) contains supplementary material, which is available to authorized users.

Chemoresistance, tumor progression, and metastasis are features that are frequently seen in cancer that have been associated with cancer stem cells (CSCs). These cells are a promising target in the future of cancer therapy but remain largely unknown. Deregulation of pathways that govern stemness in non-tumorigenic stem cells (SCs), such as Notch, Wnt, and Hedgehog pathways, has been described in CSC pathogenesis, but it is necessary to conduct further studies to discover potential new therapeutic targets. In addition, some markers for the identification and characterization of CSCs have been suggested, but the search for specific CSC markers in many cancer types is still under development. In addition, methods for CSC cultivation are also under development, with great heterogeneity existing in the protocols used. This review focuses on the most recent aspects of the identification, characterization, cultivation, and targeting of human CSCs, highlighting the advances achieved in the clinical implementation of therapies targeting CSCs and remarking those potential areas where more research is still required.

Background. To review the current progress in osteosarcoma stem cells, including isolation and identification, special cell surface markers, relationship between drug-resistance and metastasis, and the involving signal pathways. Methods. A review of the literature encompassing osteosarcoma stem cells was performed. Results. Although the cancer stem cells hypothesis was first proposed about 50 years ago, it is only in the last 10 years that advances in stem cell biology have provided increasing experimental evidence supporting this hypothesis. It has been postulated that within a tumor, a minor subpopulation of cells, termed cancer stem cells (CSC), drive the self-renewal and differentiation that account for the initiation, proliferation, metastasis, therapeutic resistance and recurrence of cancer. The CSC hypothesis opens up a novel conceptual approach for curing tumors that selectively kills CSCs, making it possible to eradicate cancer. Currently, osteosarcoma stem cells have been isolated and identified using various methods. Given the specific stem cell features, the study of CSCs has important implications in osteosarcoma prevention, detection and treatment, especially in curing early metastasis and preventing drug resistance. Focusing on their stem-like character, CSCs can be appropriately targeted by identifying links between the cells and their microenvironment. Conclusion. All of this research is in its infancy – many problems still exist. Further studies are needed to search for specific targeted therapies for osteosarcoma, in-depth study of mechanism of drug resistance, identifying the role that CSCs play in tumor metastasis, and demonstrate the imbalance of specific pathways in osteosarcoma stem cells.

Osteosarcoma is the most common primary malignant bone cancer in children and adolescents. Emerging evidence has suggested that the capability of a tumor to grow is driven by a small subset of cells within a tumor, termed cancer stem cells (CSCs). Although several methods have been explored to identify or enrich CSCs in osteosarcoma, these methods sometimes seem impractical, and chemotherapy enrichment for CSCs in osteosarcoma is rarely investigated. In the present study, we found that short exposure to chemotherapy could change the morphology of osteosarcoma cells and increase sarcosphere formation in vitro, as well as increase tumor formation in vivo. Furthermore, methotrexate (MTX)-resistant U2OS/MTX300 osteosarcoma cells were larger in size and grew much more tightly than parental U2OS cells. More importantly, U2OS/MTX300 cells possessed a higher potential to generate sarcospheres in serum-free conditions compared to parental U2OS cells. Also, U2OS/MTX300 cells exhibited the side population (SP) phenotype and expressed CSC surface markers CD117 and Stro-1. Notably, U2OS/MTX300 cells showed a substantially higher tumorigenicity in nude mice relative to U2OS cells. Therefore, we conclude that chemotherapy enrichment is a feasible and practical way to enrich osteosarcoma stem cells.

Osteosarcoma is the most common primary malignant bone cancer in children and adolescents. Emerging evidence has suggested that the capability of a tumor to grow is driven by a small subset of cells within a tumor, termed cancer stem cells (CSCs). Although several methods have been explored to identify or enrich CSCs in osteosarcoma, these methods sometimes seem impractical, and chemotherapy enrichment for CSCs in osteosarcoma is rarely investigated. In the present study, we found that short exposure to chemotherapy could change the morphology of osteosarcoma cells and increase sarcosphere formation , as well as increase tumor formation . Furthermore, methotrexate (MTX) resistant U2OS/MTX300 osteosarcoma cells were larger in size and grew much more tightly than parental U2OS cells. More importantly, U2OS/MTX300 cells possessed a higher potential to generate sarcospheres in serum free conditions compared to parental U2OS cells. Also, U2OS/MTX300 cells exhibited the side population (SP) phenotype and expressed CSC surface markers CD117 and Stro 1. Notably, U2OS/ MTX300 cells showed a substantially higher tumorigenicity in nude mice relative to U2OS cells. Therefore, we conclude that chemotherapy enrichment is a feasible and practical way to enrich osteosarcoma stem cells.

Background: Cancer as the most malignant disease still demands the more effective therapeutics methods although continuous efforts have been input to cancer therapies. Recent studies indicate that only a small subset of cancer cells termed cancer stem cells (CSCs) contributes to tumor progression, metastasis and drug-resistance. Drugs targeting to CSCs might become more effective approaches to cancer therapy. To date, isolation and characterization of CSCs have largely depended on surface markers which are not specific or well-recognized for individual tumor types. Our goal is to develop methods for isolating and characterizing a set of lung CSCs based on the specific binding peptides for CSCs without any recognized definitive specific surface markers. Materials and Methods: Stem-like cells were enriched from non-small cell lung cancer (NSCLC) cell line H460 by sphere-forming assay. Characterized these stem-like cells by colony-forming assay, drug-resistance assay and soft agar assay. Screening and identification of specific binding peptides for CSCs were performed with bacteria surface display methods. The affinity between the specific binding peptides and CSCs was determined with fluorescence microscope and flow cytometric analysis. The specificity of peptides binding with CSCs was examined through comparing the binding capacity of peptides with either H460 cells or other cell lines including stem cells. The isolation of CSCs was performed using specific fluorescent peptides with FACS machine. The further characterization of selected CSCs including exploring the source and tumorigenesis mechanism of CSCs was performed through miRNA analysis. Results: Stem-like cells divided and formed tumor spheres in a couple of weeks, while most of cancer cells died in the serum-free medium. These stem-like cells were able to grow indefinitely as tumor spheres in long-term culture (more than 20 passages). Meanwhile, sphere cells could form colonies in the soft agar plates and exhibit the drug resistance capacity, which indicated that these sphere cells behaved as CSCs. Three peptides binding specifically to those CSCs were identified with bacteria surface display method. They could seldom bind with other cell types including H460 cells, H1299 sphere cells, HLF cells, MSCs and ESCs. One selected peptides named HCBP-1 exhibited highly specific binding capacity to H460 CSCs. The apparent dissociation constant (KD) was 6.4 × 10-7 M with a flow cytometry-based assay. In the peptides binding subgroup of H460 cells, at least 10 miRNAs presented the different expression rate from the H460 cells. Conclusions: In our study, a panel of specific binding peptides was identified with bacteria surface display method which could be used for isolation and characterization of CSCs without definitive CSCs markers. Furthermore, this new method could be potentially expanded to characterize and isolate CSCs in any types of cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A54. Citation Format: Yimin Zhu, Anxin Wang. Isolation and characterization of cancer stem cells with peptides. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A54.

Cancer stem cells in osteosarcoma

Cancer stem cells (CSC) are thought to be a sub-population of cancer cells, which are the main tumorigenic cells in the tumors. Although chemoradiotherapy may kill the majority of rapidly dividing cells, CSC-like cell populations may be left behind due to their low turnover and infrequent cell cycling. In 2007, Prince et al. reported the presence of head and neck cancer stem cells for the first time. In the current study, UTTSCC74A and UTTSCC74B head and neck cancer cell lines as material derived from the primary tumor and their lymph node metastasis, respectively, were used. FACS was used for isolation of cancer stem cells using antibodies against CD44 and ALDH1. However we could not get specific response from CD44 antigen ie can not isolate cancer stem cell. However ALDH1 antibody selection gave us isolation of cancer stem cells from both of UTTSCC74A and B cell lines though much less cancer stem cells existed in UTTSCC74B cell line. The cells were first cultured in normal DMEM with 10% FBS under the condition of 5% CO2. Then the cells were changed into stem cell medium and cultured. Cancer stem cells were isolated using antibody against ALDH1 and these cells were cultured in stem cell medium and its characteristics were confirmed through sphere formation as well as immunofluorescence staining with antibody against ALDH1 and control antibody against DAPI. Expressions of sox-2, oct-4 and klf-4 were confirmed. After characterization of cancer stem cell from head and neck cancer cell line was confirmed, microarray analysis were performed using Gene Chip PrimeView Human expression Array (U133-HG133/ 47,000 transcript). Each group of ALDH1+ (cancer stem) and - cells isolated from each of UTTSCC74A and UTTSCC74B cell lines were exposed to microarray analysis. RNA extraction and cDNA construction from each sub group were prepared for microarray analysis. Comparison of UTTSCC74A ALDH1+ and - provided increased expression of 2037 and decreased expression in 2263 genes in various ratios. Similarly comparison of UTTSCC74B ALDH1+ and - provided increased expression of 6349 and decreased expression in 5322 genes in various ratios. 26 genes with most up and down regulation were selected and confirmation was done through real-time RT-PCR designing custom primers. Similar results were taken as in microarray analysis. From these 26 genes with confirmation of their expressions, 5 up regulated and 5 downregulated genes were selected for further analysis. We currently prepare expression plasmids of these genes and continue their in vitro functional analysis. Thus, the identification of the cancer-stem cell related signalling pathways in head and neck cancer stem cell would provide valuable information for a better understanding of cancer stem cell properties, molecular mechanisms of carcinogenesis, invasion and metastasis in this cancer type. By this way, effective therapeutic options targeting CSCs may be developed. Citation Format: Mehmet Gunduz, Omer Faruk Hatipoglu, Esra Gunduz, Elif Nihan Cetin, Eyyup Uctepe, Sadık Cigdem, Reidar Grenman, Muradiye Acar. Characterization of cancer stem cell properties and identification of invasion as well as metastatic process in head and neck cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1399. doi:10.1158/1538-7445.AM2015-1399