Self-Renewal and Pluripotency in Osteosarcoma Stem Cells' Chemoresistance: Notch, Hedgehog, and Wnt/ß-Catenin Interplay with Embryonic Markers.

Osteosarcoma is a highly malignant bone tumor derived from mesenchymal cells that contains self-renewing cancer stem cells (CSCs), which are responsible for tumor progression and chemotherapy resistance. Understanding the signaling pathways that regulate CSC self-renewal and survival is crucial for developing effective therapies. The Notch, Hedgehog, and Wnt/ß-Catenin developmental pathways, which are essential for self-renewal and differentiation of normal stem cells, have been identified as important regulators of osteosarcoma CSCs and also in the resistance to anticancer therapies. Targeting these pathways and their interactions with embryonic markers and the tumor microenvironment may be a promising therapeutic strategy to overcome chemoresistance and improve the prognosis for osteosarcoma patients. This review focuses on the role of Notch, Hedgehog, and Wnt/ß-Catenin signaling in regulating CSC self-renewal, pluripotency, and chemoresistance, and their potential as targets for anti-cancer therapies. We also discuss the relevance of embryonic markers, including SOX-2, Oct-4, NANOG, and KLF4, in osteosarcoma CSCs and their association with the aforementioned signaling pathways in overcoming drug resistance.

Chemoresistance-Related Stem Cell Signaling in Osteosarcoma and Its Plausible Contribution to Poor Therapeutic Response: A Discussion That Still Matters.

Osteosarcoma is amongst the most prevalent bone sarcomas and majorly afflicts children and adolescents. Therapeutic regimens based on the triad of doxorubicin, cisplatin and methotrexate have been used as the state-of-the-art approach to clinical treatment and management, with no significant improvements in the general outcomes since their inception in the early 1970s. This fact raises the following problematic questions: Why do some patients still relapse despite an initial good response to therapy? Why do nearly 30% of patients not respond to neoadjuvant therapies? Does residual persistent disease contribute to relapses and possible metastatic dissemination? Accumulating evidence suggests that chemoresistant cancer stem cells may be the major culprits contributing to those challenging clinical outcomes. Herein, we revisit the maneuvers that cancer stem cells devise for eluding cell killing by the classic cytotoxic therapies used in osteosarcoma, highlighting studies that demonstrate the complex crosstalk of signaling pathways that cancer stem cells can recruit to become chemoresistant.

The interplay between glioblastoma and microglia cells leads to endothelial cell monolayer dysfunction via the interleukin-6-induced JAK2/STAT3 pathway.

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor, with an average life expectancy of 12-15 months. GBM is highly infiltrated by microglial cells (MG) promoting tumor growth and invasiveness. Moreover, microglia activation and subsequent neuroinflammation seem to be involved in blood-brain barrier (BBB) dysfunction commonly observed in several central nervous system diseases, including brain tumors. Nevertheless, how the crosstalk between microglia and tumor cells interferes with BBB function is far from being clarified. Herein, we evaluated the effects of reciprocal interactions between MG and GBM cells in the barrier properties of brain endothelial cells (ECs), using an in vitro approach. The exposure of ECs to the inflammatory microenvironment mediated by MG-GBM crosstalk induced a decrease in the transendothelial electric resistance and an increase in permeability across the ECs (macromolecular flux of 4 kDa-fluorescein isothiocyanate and 70 kDa-Rhodamine B isothiocyanate-Dextran). These effects were accompanied by a downregulation of the intercellular junction proteins, ß-catenin and zonula occludens. Moreover, the dynamic interaction between microglia and tumor cells triggered the release of interleukin-6 (IL-6) by microglia and subsequent activation of the downstream Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) pathway. Interestingly, the depletion of IL-6 or the blockade of the JAK/STAT3 signaling with AG490 were able to prevent the EC hyperpermeability. Overall, we demonstrated that IL-6 released during MG-GBM crosstalk leads to barrier dysfunction through the activation of the JAK/STAT3 pathway in ECs and downregulation of intercellular junction proteins. These results provide new insights into the mechanisms underlying the disruption of BBB permeability in GBM.

Therapy-induced enrichment of cancer stem-like cells in solid human tumors: Where do we stand?

The development of local recurrence and metastatic disease, most probably attributable to the intrinsic or acquired resistance of tumor cells to standard therapy, still constitute the major clinical problem preventing the cure of cancer patients. Despite progress in the research of new therapeutic targets and compounds, resistant cells displaying stem-like properties seem to play a leading role in therapeutic failures and to be the culprit cells responsible for associated tumor recurrence. A whole new plethora of research studies suggest that drug-tolerant cancer stem cells may be induced by conventional cancer chemotherapeutics such as doxorubicin, cisplatinum and ionizing radiation. This phenotypic plasticity and transition from a differentiated to stem-like cell state associates with the activation of diverse stem cell self-renewal (e.g. Notch, Hedgehog, Wnt), drug efflux (e.g. ABC transporters) and survival-related pathways (e.g. TGF-ß, ERK, AKT), which may confer resistance and treatment failures in solid tumors. Therefore, combined therapeutic strategies aiming to simultaneously target drug-sensitive tumor cells and their capacity of phenotypic switching may lead to survival benefits and meaningful disease remissions. This knowledge can be applicable to the clinic and contribute to better therapeutic outcomes and prevent tumor recurrence.

Osteosarcoma Stem Cells Have Active Wnt/ß-catenin and Overexpress SOX2 and KLF4.

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/ß-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/ß-catenin signaling as evidenced by increased nuclear ß-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.

Therapeutic implications of an enriched cancer stem-like cell population in a human osteosarcoma cell line.

BACKGROUND: Osteosarcoma is a bone-forming tumor of mesenchymal origin that presents a clinical pattern that is consistent with the cancer stem cell model. Cells with stem-like properties (CSCs) have been identified in several tumors and hypothesized as the responsible for the relative resistance to therapy and tumor relapses. In this study, we aimed to identify and characterize CSCs populations in a human osteosarcoma cell line and to explore their role in the responsiveness to conventional therapies. METHODS: CSCs were isolated from the human MNNG/HOS cell line using the sphere formation assay and characterized in terms of self-renewal, mesenchymal stem cell properties, expression of pluripotency markers and ABC transporters, metabolic activity and tumorigenicity. Cell's sensitivity to conventional chemotherapeutic agents and to irradiation was analyzed and related with cell cycle-induced alterations and apoptosis. RESULTS: The isolated CSCs were found to possess self-renewal and multipotential differentiation capabilities, express markers of pluripotent embryonic stem cells Oct4 and Nanog and the ABC transporters P-glycoprotein and BCRP, exhibit low metabolic activity and induce tumors in athymic mice. Compared with parental MNNG/HOS cells, CSCs were relatively more resistant to both chemotherapy and irradiation. None of the treatments have induced significant cell-cycle alterations and apoptosis in CSCs. CONCLUSIONS: MNNG/HOS osteosarcoma cells contain a stem-like cell population relatively resistant to conventional chemotherapeutic agents and irradiation. This resistant phenotype appears to be related with some stem features, namely the high expression of the drug efflux transporters P-glycoprotein and BCRP and their quiescent nature, which may provide a biological basis for resistance to therapy and recurrence commonly observed in osteosarcoma.

Therapeutic potential of the metabolic modulator Metformin on osteosarcoma cancer stem-like cells.

PURPOSE: Osteosarcoma is the most common primary bone tumour appearing in children and adolescents. Recent studies demonstrate that osteosarcoma possesses a stem-like cell subset, so-called cancer stem-like cells, refractory to conventional chemotherapeutics and pointed out as responsible for relapses frequently observed in osteosarcoma patients. Here, we explored the therapeutic potential of Metformin on osteosarcoma stem-like cells, alone and as a chemosensitizer of doxorubicin. METHODS: Stem-like cells were isolated from human osteosarcoma cell lines, MNNG/HOS and MG-63, using the sphere-forming assay. Metformin cytotoxicity alone and combined with doxorubicin were evaluated using MTT/BrdU assays. Protein levels of AMPK and AKT were evaluated by Western Blot. Cellular metabolic status was assessed based on [18F]-FDG uptake and lactate production measurements. Sphere-forming efficiency and expression of pluripotency transcription factors analysed by qRT-PCR were tested as readout of Metformin effects on stemness features. RESULTS: Metformin induced a concentration-dependent decrease in the metabolic activity and proliferation of sphere-forming cells and improved doxorubicin-induced cytotoxicity. This drug also down-regulated the expression of master regulators of pluripotency (OCT4, SOX2, NANOG), and decreased spheres' self-renewal ability. Metformin effects on mitochondria led to the activation and phosphorylation of the energetic sensor AMPK along with an upregulation of the pro-survival AKT pathway in both cell populations. Furthermore, Metformin-induced mitochondrial stress increased [18F]-FDG uptake and lactate production in parental cells but not in the quiescent stem-like cells, suggesting the inability of the latter to cope with the energy crisis induced by metformin. CONCLUSIONS: This preclinical study suggests that Metformin may be a potentially useful therapeutic agent and chemosensitizer of osteosarcoma stem-like cells to doxorubicin.

IWR-1, a tankyrase inhibitor, attenuates Wnt/ß-catenin signaling in cancer stem-like cells and inhibits in vivo the growth of a subcutaneous human osteosarcoma xenograft.

Wnt/ß-catenin or canonical Wnt signaling pathway regulates the self-renewal of cancer stem-like cells (CSCs) and is involved in tumor progression and chemotherapy resistance. Previously, we reported that this pathway is activated in a subset of osteosarcoma CSCs and that doxorubicin induced stemness properties in differentiated cells through Wnt/ß-catenin activation. Here, we investigated whether pharmacological Wnt/ß-catenin inhibition, using a tankyrase inhibitor (IWR-1), might constitute a strategy to target CSCs and improve chemotherapy efficacy in osteosarcoma. IWR-1 was specifically cytotoxic for osteosarcoma CSCs. IWR-1 impaired spheres' self-renewal capacity by compromising landmark steps of the canonical Wnt signaling, namely translocation of ß-catenin to the nucleus and subsequent TCF/LEF activation and expression of Wnt/ß-catenin downstream targets. IWR-1 also hampered the activity and expression of key stemness-related markers. In vitro, IWR-1 induced apoptosis of osteosarcoma spheres and combined with doxorubicin elicited synergistic cytotoxicity, reversing spheres' resistance to this drug. In vivo, IWR-1 co-administration with doxorubicin substantially decreased tumor progression, associated with specific down-regulation of TCF/LEF transcriptional activity, nuclear ß-catenin and expression of the putative CSC marker Sox2. We suggest that targeting the Wnt/ß-catenin pathway can eliminate CSCs populations in osteosarcoma. Combining conventional chemotherapy with Wnt/ß-catenin inhibition may ameliorate therapeutic outcomes, by eradicating the aggressive osteosarcoma CSCs and reducing drug resistance.

Stroma-derived IL-6, G-CSF and Activin-A mediated dedifferentiation of lung carcinoma cells into cancer stem cells.

Cancer stem cells (CSCs) are a small population of resistant cells inhabiting the tumors. Although comprising only nearly 3% of the tumor mass, these cells were demonstrated to orchestrate tumorigenesis and differentiation, underlie tumors' heterogeneity and mediate therapy resistance and tumor relapse. Here we show that CSCs may be formed by dedifferentiation of terminally differentiated tumor cells under stress conditions. Using a elegant co-culture cellular system, we were able to prove that nutrients and oxygen deprivation activated non-malignant stromal fibroblasts, which in turn established with tumor cells a paracrine loop mediated by Interleukine-6 (IL-6), Activin-A and Granulocyte colony-stimulating factor (G-CSF), that drove subsequent tumor formation and cellular dedifferentiation. However, by scavenging these cytokines from the media and/or blocking exosomes' mediated communication it was possible to abrogate dedifferentiation thus turning these mechanisms into potential therapeutic targets against cancer progression.

Multidrug resistance in cancer: its mechanism and its modulation.

One of the major problems related with the curative treatment of cancer patients is resistance against anticancer drugs. This resistance, which may occur from the beginning or is evident only later as an acquired phenomenon, is due to the action of drug transporters. These transmembrane proteins belong to the ATP-binding cassette (ABC) transporters which reduce bioavailability of drugs, but also determine the elimination of xenobiotics into bile, urine and feces. The present review summarizes recent knowledge in this area, highlighting the mechanism of action of these transporters, its clinical significance and its possible modulation. Novel approaches to overcome multidrug resistance include agents which inhibit or circumvent this efflux mechanism. For the latter category developments in nanomedicine may be of consequence. However, in spite of considerable progress in research regarding multidrug resistance, the phase of efficacious clinical use of this knowledge has not been reached yet.

Phthalocyanine Labels for Near-Infrared Fluorescence Imaging of Solid Tumors.

Diamagnetic metal complexes of phthalocyanines with n-butoxyl groups in all the α-benzo positions of the macrocycle skeleton, MPc(OBu)8, have strong near-infrared absorptions and intense fluorescences that are Stokes shifted by more than 15 nm. Interestingly, the silicon complex 6 is also remarkably photostable and nontoxic. The use of 6 in the fluorescence imaging of BALB/c mice bearing a 4T1-luc2 tumor in the mammary fat pad unambiguously revealed the presence of the tumor when it was only 1 mm in diameter and was not visible with the naked eye. Compound 6 has an intrinsic ability to accumulate in the tumor, adequate spectroscopic properties, and excellent stability to function as a NIR fluorescent label in the early detection of tumors.

Chemotherapy induces stemness in osteosarcoma cells through activation of Wnt/ß-catenin signaling.

Development of resistance represents a major drawback in osteosarcoma treatment, despite improvements in overall survival. Treatment failure and tumor progression have been attributed to pre-existing drug-resistant clones commonly assigned to a cancer stem-like phenotype. Evidence suggests that non stem-like cells, when submitted to certain microenvironmental stimuli, can acquire a stemness phenotype thereby strengthening their capacity to handle with stressful conditions. Here, using osteosarcoma cell lines and a mouse xenograft model, we show that exposure to conventional chemotherapeutics induces a phenotypic cell transition toward a stem-like phenotype. This associates with activation of Wnt/ß-catenin signaling, up-regulation of pluripotency factors and detoxification systems (ABC transporters and Aldefluor activity) that ultimately leads to chemotherapy failure. Wnt/ß-catenin inhibition combined with doxorubicin, in the MNNG-HOS cells, prevented the up-regulation of factors linked to transition into a stem-like state and can be envisaged as a way to overcome adaptive resistance. Finally, the analysis of the public R2 database, containing microarray data information from diverse osteosarcoma tissues, revealed a correlation between expression of stemness markers and a worse response to chemotherapy, which provides evidence for drug-induced phenotypic stem cell state transitions in osteosarcoma.

Sensitizing osteosarcoma stem cells to doxorubicin-induced apoptosis through retention of doxorubicin and modulation of apoptotic-related proteins.

AIMS: Osteosarcoma is the most common pediatric bone malignancy with high propensity to metastasize and relapse. Emerging evidence suggest that osteosarcoma is sustained by a subset of self-renewing cancer stem like cells (CSCs) relying on mechanisms to evade apoptosis and survive in response to drugs-induced DNA damage. We proposed to decipher the mechanisms underlying the resistance of CSCs to doxorubicin-induced apoptosis. MAIN METHODS: CSCs were isolated using a sphere-forming assay and tested for sensitivity to doxorubicin-induced apoptosis, using MTT cell viability and BrdU proliferation assays, TUNEL staining and caspases 3/7 activity. Bcl-2 family proteins were analyzed by Western blot. Doxorubicin uptake was determined by confocal microscopy and bioluminescence imaging. KEY FINDINGS: We showed that osteosarcoma sphere stem-like cells expressed the multidrug-related efflux transporters P-glycoprotein and BCRP and are highly resistant to doxorubicin-induced apoptosis. Conversely after exposure to doxorubicin, these cells displayed an up-regulation of the anti-apoptotic proteins Bcl-2 and Bcl-xL with concomitant down-regulation of Bak and decreased caspase 3/7 activity. Inhibition of drug efflux transporters enhanced the cellular uptake of doxorubicin, being encompassed by an up-regulation the pro-apoptotic protein Bak and suppression of Bcl-2, favoring the commitment of CSCs towards apoptosis. SIGNIFICANCE: These results seemingly suggest that the high apoptotic threshold of CSCs to doxorubicin-induced cell dead stimuli is mainly dependent on the drug concentration reaching tumor cells that are governed by efflux transporter activity. Therefore, modulation of these transporters may be effective in potentiating the proapoptotic effects of doxorubicin, and emerges as an attractive strategy to sensitize osteosarcoma CSCs to chemotherapy.

Cationic lipophilic radiotracers for functional imaging of multidrug resistance.

The multidrug resistance (MDR) phenotype is frequently associated with the overexpression of transmembrane drug proteins such as the P-glycoprotein (Pgp) and/or multidrug resistance related protein-1 (MRP1). These proteins belong to the superfamily of the so-called ATP-binding cassette superfamily and act as drug efflux pumps of a broad range of chemotherapeutic agents commonly used in the treatment of malignancies. These proteins have been found to be overexpressed in both haematological and solid tumours and are considered as adverse prognostic factors. The ability to obtain in vivo and non-invasively information regarding the functional activity of MDR-related transporters, using probes that mimic the antineoplastic agents, provide a very useful tool in the clinical setting by determining the individual tumour susceptibility to chemotherapy. This previous knowledge could serve as a critical tool for optimizing chemotherapeutic protocols on a patient-specific basis. The emergence of non-invasive molecular imaging techniques using radiolabelled probes provides an interesting approach for functional assessment of the classical mechanism of MDR in cancer patients. Toward this objective, the clinically approved 99mTc-labelled cationic lipophilic complexes (sestamibi and tetrofosmin) have been characterized as transport substrates of Pgp and MRP1 and proposed as surrogate markers of chemotherapeutic agents for functional evaluation of MDR by single-photon emission tomography (SPECT). Here we review the potential applications of these agents in identifying drug resistance mechanisms based on functional assays and their potential as a tool for evaluating the efficacy of MDR inhibitors, using cellular and animal models of chemoresistance.

P-glycoprotein versus MRP1 on transport kinetics of cationic lipophilic substrates: a comparative study using [99mTc]sestamibi and [99mTc]tetrofosmin.

The multidrug resistance (MDR) phenotype in cancer is closely related with the overexpression of P-glycoprotein (Pgp) and multidrug resistance protein-1 (MRP1). Although conferring resistance to a similar spectrum of drugs, these proteins present distinct transport mechanisms and have their own substrates. In this work, we compared the functional properties of Pgp and MRP1 in the transport kinetics of two cationic lipophilic tracers, [(99m)Tc]sestamibi and [(99m)Tc]tetrofosmin, in cellular models of resistance. Cellular transport kinetics of both tracers was evaluated in Small-cell lung cancer cell line H69 and in its drug-resistant sublines, H69LX4 and H69AR, overexpressing Pgp and MRP1, respectively. Studies were performed in the absence and in the presence of MDR modulators. Kinetic parameters extracted from time-activity curves were analyzed through receiver-operating characteristics curve analysis. The uptake and the efflux rate of both radiotracers were significantly higher (p < 0.05) in sensitive cells. However, MRP1 was more effective than Pgp in removing tracers from the intracellular medium. The addition of verapamil and PSC833 significantly reduced the efflux rate and restored the accumulation of both tracers in H69LX4 cells. Only verapamil was effective in the inhibition of MRP1; however, the effects were more pronounced with [(99m)Tc]sestamibi, when compared to [(99m)Tc]tetrofosmin. Outward transport of radiotracers by MRP1 was dependent on the intracellular glutathione levels. We concluded that both tracers can detect Pgp- and MRP1-mediated drug resistance, based on transport kinetics; however, MRP1 is more effective than Pgp on outward transport of radiotracers. We postulate that this finding can be useful to distinguish between the two resistance mechanisms.

Functional imaging of multidrug resistance in an orthotopic model of osteosarcoma using 99mTc-sestamibi.

PURPOSE: The purpose of this work was the development of an orthotopic model of osteosarcoma based on luciferase-expressing tumour cells for the in vivo imaging of multidrug resistance (MDR) with (99m)Tc-sestamibi. METHODS: Doxorubicin-sensitive (143B-luc(+)) and resistant (MNNG/HOS-luc(+)) osteosarcoma cell lines expressing different levels of P-glycoprotein and carrying a luciferase reporter gene were inoculated into the tibia of nude mice. Local tumour growth was monitored weekly by bioluminescence imaging and X-ray. After tumour growth, a (99m)Tc-sestamibi dynamic study was performed. A subset of animals was pre-treated with an MDR inhibitor (PSC833). Images were analysed for calculation of (99m)Tc-sestamibi washout half-life (t (1/2)), percentage washout rate (%WR) and tumour/non-tumour (T/NT) ratio. RESULTS: A progressively increasing bioluminescent signal was detected in the proximal tibia after 2 weeks. The t (1/2) of (99m)Tc-sestamibi was significantly shorter (p < 0.05) in drug-resistant MNNG/HOS-luc(+) tumours (t (1/2) = 87.3 +/- 15.7 min) than in drug-sensitive 143B-luc(+) tumours (t (1/2) = 161.0 +/- 47.4 min) and decreased significantly with PSC833 (t (1/2) = 173.0 +/- 24.5 min, p < 0.05). No significant effects of PSC833 were observed in 143B-luc(+) tumours. The T/NT ratio was significantly lower (p < 0.05) in MNNG/HOS-luc(+) tumours than in 143B-luc(+) tumours at early (1.55 +/- 0.22 vs 2.14 +/- 0.36) and delayed times (1.12 +/- 0.11 vs 1.62 +/- 0.33). PSC833 had no significant effects on the T/NT ratios of either tumour. CONCLUSION: The orthotopic injection of tumour cells provides an animal model suitable for functional imaging of MDR. In vivo bioluminescence imaging allows the non-invasive monitoring of tumour growth. The kinetic analysis of (99m)Tc-sestamibi washout provides information on the functional activity of MDR related to P-glycoprotein expression and its pharmacological inhibition in osteosarcoma.