Epithelial and mesenchymal cancer cells block adipose stem cells differentiation

The tumour microenvironment consists of different factors that can contribute to neoplastic transformation, tumour growth and invasion by soluble factors, signalling molecules, and extracellular matrix. Adipose tissue is a rich source of multipotent mesenchymal stem cells termed adipose stem cells (ASCs). Studies showed that MSCs can promote tumour progression and metastasis, while others reported that MSCs suppress tumour growth. Therefore, molecular mechanisms that link MSCs to tumour cells growth are poorly understood. The aim of this study is to establish the effect of tumour microenvironment on normal human mesenchymal stem cells using co-cultures of osteosarcoma and breast cancer cells with ASCs. SAOS2, osteosarcoma cell line and MCF7, breast cancer cell line, were co-cultured with ASCs using an insert of 0,4μm at same cell density for 7, 14 and 21 days. Growth curves, stemness gene expression including OCT4, Sox2, and Nanog, leptin expression by RT-PCR, mesenchymal markers expression including CD34, CD29, CD90 and vimentin by flow cytometry, CD324, Twist and Snail, EMT-related markers, by RT-PCR were analyzed. In addition, angiogenic and adipogenic differentiation assays were performed after 21 days of co-culture. In addition, SMAD and p-SMAD2/3 were assayed by western blotting. Angiogenic gene expression was also analyzed by RT-PCR. The results showed that both SAOS2 and MCF7 cells induced an increase of ASCs proliferation respect to ASCs cultured alone. CD90, CD29 and vimentin expressions were similar in all tested conditions, with a mean percentage of 98%. CD324, an epithelial marker, was not expressed as well as no change of Twist and Snail was detectable. Interestingly, SAOS2 and MCF7 cells induced a variation of CD34 expression both at molecular and proteic levels. In particular, at 7 days, MCF7 induced an increase of CD34 gene expression, at 14 days a decrease and at 21 days a strong de novo increase with respect to ASCs cultured alone: the latter was also confirmed by flow cytometry. On the contrary, at 7 days, SAOS2 induced a decrease of CD34 gene expression, but at 14 and 21 days, induced a strong increase by RT-PCR. At proteic level, SAOS2 induced an increase of CD34 in culture time. For stemness genes, both MCF7 and SAOS2 induced an increase of OCT4, Nanog and Sox2 with respect to ASCs cultured alone and a decrease of angiogenic factors, including CD31, PDGFα, PDGFRα, PDGFRβ with respect to ASCs cultured alone, with a significant increase of VEGF at 21 days (p<0.001). Leptin was increased in all conditions, whereas both SMAD and pSMAD2/3 increased only on ASCs were cultured alone. After 21 days of coculture, only ASCs cocultured were able to differentiate both in adipocytes and endothelial cells. Taken together, our results indicate that both mesenchymal and epithelial cancer cells are able to maintain the stemness of ASCs upregulating CD34 expression and inhibiting the angiogenic differentiation

Characterization and gene expression of cancer stem cells grown as sarcospheres from human primary sarcomas

This study aimed to identify, isolate and characterize cancer stem cells from human primary sarcomas. We performed cytometric analyses for stemness and differentiation antigens including CD29, CD34, CD44, CD90, CD117 and CD133 on 21 human primary sarcomas on the day of surgery. From sarcoma biopsies, we obtained two chondrosarcoma stabilized cell lines and two osteosarcoma stabilized cell lines on which spheres formation, side population profile, stemness gene expression and in vivo and in vitro assays were performed. On a chondrosarcoma cell line, the whole genoma microarray analyses were performed (sarcospheres versus adherent cells). All samples expressed the CD133, CD44 and CD29 markers. We selected a CD133+ subpopulation from stabilized cell lines that displayed the capacity to grow as sarcospheres able to initiate and sustain tumour growth in NOD/SCID mice, to express stemness genes including OCT3/4, Nanog, Sox2 and Nestin and to differentiate into mesenchymal lineages. Microarray analyses pointed out a huge gene expression difference between sarcospheres and adherent cells. About 2000 genes, including ones related to cell cycle, stemness and epigenetic regulation, resulted to be very differentially expressed in the two population considered. With signed ratio of 2,806 genes were over-expressed and 1,029 under-expressed on sarcospheres when compared with adherent cells. The most highly overexpressed gene were thioredoxin interacting protein (fold +25) that modulates the cellular redox state, growth differentiation factor 15 (fold +10), member of the TGF–β superfamily , histone cluster 1, H2ad (fold +9) and solute carrier family 2 member 14 that facilitate glucose transport (fold +8,9). Our findings evidence the existence of cancer stem cells in human primary bone sarcomas and highlight CD133 as pivotal marker for identification of these cells. This may be of primary importance in the development of new therapeutic strategies and new prognostic procedures against these highly aggressive and metastatic tumours

Pharmacological targeting of the ephrin receptor kinase signalling by GLPG1790 in vitro and in vivo reverts oncophenotype, induces myogenic differentiation and radiosensitizes embryonal rhabdomyosarcoma cells

EPH (erythropoietin-producing hepatocellular) receptors are clinically relevant targets in several malignancies. This report describes the effects of GLPG1790, a new potent pan-EPH inhibitor, in human embryonal rhabdomyosarcoma (ERMS) cell lines

Cancer stem cells in head and neck tumors: evidence for metastatic spread and treatment resistance

The major challenge in the management of patients with oral squamous cell carcinomas (OSCC) is the development of resistance to therapy leading to disseminated disease. Since cancer stem cells (CSC) have emerged as important players in OSCC metastasis, our objectives were to explore the implications of CSC in OSCC tumor progression, invasion and response to conventional therapies. Methods: A panel of well-characterized cell lines originated from the most common sites in the head and neck area was used. Cells were cultured as floating spheres or under normal adherent conditions and analyzed for CD44, ALDH, CD24, CD29, CD56 by flow cytometry, PCR arrays for genes related to stemness, metastasis and EMT . We also investigated sLeX expression, known to play a key-role in many cancers metastasis by promoting tumor cells binding to endothelial E-selectin. We analyzed the tumorigenic potential of OSCC cells by invasion assays and in vivo OSCC experimental models comparatively to CSC cells. Moreover resistance to cisplatin and radiation was assessed by annexin V/PI assay and by colony forming assay. Results: The highest levels of sLeX expression were found in cell lines originated from oral cavity (9%-47%) compared to other head and neck locations (0.1%-7%). Cells grown as spheres were 95-100% positive for sLeX compared to 10-40% of adherent counterpart. Although sLeX+ and sLeX- cells were both able to form spheres, sLeX+ spheres were predominant and larger. Flow cytometry and PCR arrays indicated that the spheres were highly enriched in CSC and metastatic markers. Consistently, the spheres showed increased invasive and tumorigenic potential, and resistance to conventional chemotherapy and radiations. Conclusion: these studies are the first to unveil a novel link between sLeX expression, stem cell formation and metastatic spread in OSCC, and provide supportive evidence for CSC resistance to treatment. Understanding the mechanisms of tumor invasion and metastasis will improve patient outcome and survival

Methylation and epigenetic modification by 5’ azacytidine and valproic acid treatment increase stemness attributes in bone sarcoma cell lines

Bone sarcoma is an aggressive malignancy with high mortality rate. Despite recent advances, the prognosis is still extremely poor. Bone sarcomas contain a small cell population with stem cell like properties, referred to as cancer stem cells (CSCs) expressing CD133 (Tirino et al, 2009; 2011). The biological relevance and regulatory mechanism of CD133 expression are not yet understood. The aim of this study is to elucidate mechanisms regulating aberrant expression of CD133 and stemness phenotype. Saos-2, MG63 and BS15 cell lines were treated with 0,5 mM valproic acid (VPA) and 3μM 5’azacytidine (5-AZA) for 48 hours alone and in combination. CD133 and stemness markers expression including OCT4, Sox2 and Nanog were analyzed by flow cytometry and real-time PCR. Vimentin and osteocalcin levels were also tested. Sarcospheres formation rate was assessed as spheres number/seed single cell number. After treatment with 5-AZA or VPA, the expression level of CD133 mRNA as well as of protein was significantly increased in all three cell lines. Also OCT4, Sox2 and Nanog, stemness markers, and vimentin, mesenchymal marker resulted to be upregulated after treatment by real time-PCR. On the contrary, the expression level of osteocalcin remained similar before and after treatment. Interestingly, combined treatment with 5-AZA and VPA induced an increase of CD133 expression in a synergistic manner in all three cell lines. In addition, sarcospheres formation rate was increased after drug treatment compared to untreated cells. Also in this case, the drug combination lead to synergistic increase of formation rate of spheres. In conclusion, our results indicate that DNA methylation is an important determinant of CD133 and stemness profile in human bone sarcomas and this mechanism may be associated with histone deacetilase inhibition

The role of autophagy in resistance to targeted therapies

Autophagy is a self-degradative cellular process, involved in stress response such as starvation, hypoxia, and oxidative stress. This mechanism balances macro-molecule recycling to regulate cell homeostasis. In cancer, autophagy play a role in the development and progression, while several studies describe it as one of the key processes in drug resistance. In the last years, in addition to standard anti-cancer treatments such as chemotherapies and irradiation, targeted therapy became one of the most adopted strategies in clinical practices, mainly due to high specificity and reduced side effects. However, similar to standard treatments, drug resistance is the main challenge in most patients. Here, we summarize recent studies that investigated the role of autophagy in drug resistance after targeted therapy in different types of cancers. We highlight positive results and limitations of pre-clinical and clinical studies in which autophagy inhibitors are used in combination with targeted therapies. Refereed/Peer-reviewe

Hyaluronan-Based Gel Promotes Human Dental Pulp Stem Cells Bone Differentiation by Activating YAP/TAZ Pathway.

Background: Hyaluronans exist in different forms, accordingly with molecular weight and degree of crosslinking. Here, we tested the capability to induce osteogenic differentiation in hDPSCs (human dental pulp stem cells) of three hyaluronans forms: linear pharmaceutical-grade hyaluronans at high and (HHA) low molecular weight (LHA) and hybrid cooperative complexes (HCC), containing both sizes. Methods: hDPSCs were treated with HHA, LHA, HCC for 7, 14 and 21 days. The effects of hyaluronans on osteogenic differentiation were evaluated by qRT-PCR and WB of osteogenic markers and by Alizarin Red S staining. To identify the involved pathway, CD44 was analyzed by immunofluorescence, and YAP/TAZ expression was measured by qRT-PCR. Moreover, YAP/TAZ inhibitor-1 was used, and the loss of function of YAP/TAZ was evaluated by qRT-PCR, WB and immunofluorescence. Results: We showed that all hyaluronans improves osteogenesis. Among these, HCC is the main inducer of osteogenesis, along with overexpression of bone related markers and upregulating CD44. We also found that this biological process is subordinate to the activation of YAP/TAZ pathway. Conclusions: We found that HA's molecular weight can have a relevant impact on HA performance for bone regeneration, and we unveil a new molecular mechanism by which HA acts on stem cells. Keywords: YAP/TAZ pathway; dental pulp stem cells; hyaluronic acid; osteogenic differentiation

Zoledronic acid-encapsulating self-assembling nanoparticles and doxorubicin: A combinatorial approach to overcome simultaneously chemoresistance and immunoresistance in breast tumors

The resistance to chemotherapy and the tumor escape from host immunosurveillance are the main causes of the failure of anthracycline-based regimens in breast cancer, where an effective chemo-immunosensitizing strategy is lacking. The clinically used aminobisphosphonate zoledronic acid (ZA) reverses chemoresistance and immunoresistance in vitro. Previously we developed a nanoparticle-based zoledronic acid-containing formulation (NZ) that allowed a higher intratumor delivery of the drug compared with free ZA in vivo. We tested its efficacy in combination with doxorubicin in breast tumors refractory to chemotherapy and immune system recognition as a new combinatorial approach to produce chemo- and immunosensitization. NZ reduced the IC50 of doxorubicin in human and murine chemoresistant breast cancer cells and restored the doxorubicin efficacy against chemo-immunoresistant tumors implanted in immunocompetent mice. By reducing the metabolic flux through the mevalonate pathway, NZ lowered the activity of Ras/ERK1/2/HIF-1α axis and the expression of P-glycoprotein, decreased the glycolysis and the mitochondrial respiratory chain, induced a cytochrome c/caspase 9/caspase 3-dependent apoptosis, thus restoring the direct cytotoxic effects of doxorubicin on tumor cell. Moreover, NZ restored the doxorubicin-induced immunogenic cell death and reversed the tumorinduced immunosuppression due to the production of kynurenine, by inhibiting the STAT3/indoleamine 2,3 dioxygenase axis. These events increased the number of dendritic cells and decreased the number of immunosuppressive T-regulatory cells infiltrating the tumors. Our work proposes the use of nanoparticle encapsulating zoledronic acid as an effective tool overcoming at the same time chemoresistance and immunoresistance in breast tumors, thanks to the effects exerted on tumor cell and tumor-infiltrating immune cells

Human DPSCs fabricate vascularized woven bone tissue : a new tool in bone tissue engineering

Human dental pulp stem cells (hDPSCs) are mesenchymal stem cells that have been successfully used in human bone tissue engineering. To establish whether these cells can lead to a bone tissue ready to be grafted, we checked DPSCs for their osteogenic and angiogenic differentiation capabilities with the specific aim of obtaining a new tool for bone transplantation. Therefore, hDPSCs were specifically selected from the stromal-vascular dental pulp fraction, using appropriate markers, and cultured. Growth curves, expression of bone-related markers, calcification and angiogenesis as well as an in vivo transplantation assay were performed. We found that hDPSCs proliferate, differentiate into osteoblasts and express high levels of angiogenic genes, such as vascular endothelial growth factor and platelet-derived growth factor A. Human DPSCs, after 40 days of culture, give rise to a 3D structure resembling a woven fibrous bone. These woven bone (WB) samples were analysed using classic histology and synchrotron-based, X-ray phase-contrast microtomography and holotomography. WB showed histological and attractive physical qualities of bone with few areas of mineralization and neovessels. Such WB, when transplanted into rats, was remodelled into vascularized bone tissue. Taken together, our data lead to the assumption that WB samples, fabricated by DPSCs, constitute a noteworthy tool and do not need the use of scaffolds, and therefore they are ready for customized regeneration

Human DPSCs fabricate vascularized woven bone tissue: a new tool in bone tissue engineering

Human dental pulp stem cells (hDPSCs) are mesenchymal stem cells that have been successfully used in human bone tissue engineering. To establish whether these cells can lead to a bone tissue ready to be grafted, we checked DPSCs for their osteogenic and angiogenic differentiation capabilities with the specific aim of obtaining a new tool for bone transplantation. Therefore, hDPSCs were specifically selected from the stromal-vascular dental pulp fraction, using appropriate markers, and cultured. Growth curves, expression of bone-related markers, calcification and angiogenesis as well as an in vivo transplantation assay were performed. We found that hDPSCs proliferate, differentiate into osteoblasts and express high levels of angiogenic genes, such as vascular endothelial growth factor and platelet-derived growth factor A. Human DPSCs, after 40 days of culture, give rise to a 3D structure resembling a woven fibrous bone. These woven bone (WB) samples were analysed using classic histology and synchrotron-based, X-ray phase-contrast microtomography and holotomography. WB showed histological and attractive physical qualities of bone with few areas of mineralization and neovessels. Such WB, when transplanted into rats, was remodelled into vascularized bone tissue. Taken together, our data lead to the assumption that WB samples, fabricated by DPSCs, constitute a noteworthy tool and do not need the use of scaffolds, and therefore they are ready for customized regeneration