The Impact of A3AR Antagonism on the Differential Expression of Chemoresistance-Related Genes in Glioblastoma Stem-like Cells.

Glioblastoma (GB) is the most aggressive and common primary malignant tumor of the brain and central nervous system. Without treatment, the average patient survival time is about six months, which can be extended to fifteen months with multimodal therapies. The chemoresistance observed in GB is, in part, attributed to the presence of a subpopulation of glioblastoma-like stem cells (GSCs) that are characterized by heightened tumorigenic capacity and chemoresistance. GSCs are situated in hypoxic tumor niches, where they sustain and promote the stem-like phenotype and have also been correlated with high chemoresistance. GSCs have the particularity of generating high levels of extracellular adenosine (ADO), which causes the activation of the A3 adenosine receptor (A3AR) with a consequent increase in the expression and activity of genes related to chemoresistance. Therefore, targeting its components is a promising alternative for treating GB. This analysis determined genes that were up- and downregulated due to A3AR blockades under both normoxic and hypoxic conditions. In addition, possible candidates associated with chemoresistance that were positively regulated by hypoxia and negatively regulated by A3AR blockades in the same condition were analyzed. We detected three potential candidate genes that were regulated by the A3AR antagonist MRS1220 under hypoxic conditions: LIMD1, TRIB2, and TGFB1. Finally, the selected markers were correlated with hypoxia-inducible genes and with the expression of adenosine-producing ectonucleotidases. In conclusion, we detected that hypoxic conditions generate extensive differential gene expression in GSCs, increasing the expression of genes associated with chemoresistance. Furthermore, we observed that MRS1220 could regulate the expression of LIMD1, TRIB2, and TGFB1, which are involved in chemoresistance and correlate with a poor prognosis, hypoxia, and purinergic signaling.

MicroRNA-204 Regulates Angiogenesis and Vasculogenic Mimicry in CD44+/CD24- Breast Cancer Stem-like Cells.

Tumors have high requirements in terms of nutrients and oxygen. Angiogenesis is the classical mechanism for vessel formation. Tumoral vascularization has the function of nourishing the cancer cells to support tumor growth. Vasculogenic mimicry, a novel intratumoral microcirculation system, alludes to the ability of cancer cells to organize in three-dimensional (3D) channel-like architectures. It also supplies the tumors with nutrients and oxygen. Both mechanisms operate in a coordinated way; however, their functions in breast cancer stem-like cells and their regulation by microRNAs remain elusive. In the present study, we investigated the functional role of microRNA-204 (miR-204) on angiogenesis and vasculogenic mimicry in breast cancer stem-like cells. Using flow cytometry assays, we found that 86.1% of MDA-MB-231 and 92% of Hs-578t breast cancer cells showed the CD44+/CD24- immunophenotype representative of cancer stem-like cells (CSCs). The MDA-MB-231 subpopulation of CSCs exhibited the ability to form mammospheres, as expected. Interestingly, we found that the restoration of miR-204 expression in CSCs significantly inhibited the number and size of the mammospheres. Moreover, we found that MDA-MB-231 and Hs-578t CSCs efficiently undergo angiogenesis and hypoxia-induced vasculogenic mimicry in vitro. The transfection of precursor miR-204 in both CSCs was able to impair the angiogenesis in the HUVEC cell model, which was observed as a diminution in the number of polygons and sprouting cells. Remarkably, miR-204 mimics also resulted in the inhibition of vasculogenic mimicry formation in MDA-MB-231 and Hs-578t CSCs, with a significant reduction in the number of channel-like structures and branch points. Mechanistically, the effects of miR-204 were associated with a diminution of pro-angiogenic VEGFA and ß-catenin protein levels. In conclusion, our findings indicated that miR-204 abrogates the angiogenesis and vasculogenic mimicry development in breast cancer stem-like cells, suggesting that it could be a potential tool for breast cancer intervention based on microRNA replacement therapies.

Glioblastoma Microenvironment and Invasiveness: New Insights and Therapeutic Targets.

Glioblastoma (GBM) is the most common and malignant primary brain cancer in adults. Without treatment the mean patient survival is approximately 6 months, which can be extended to 15 months with the use of multimodal therapies. The low effectiveness of GBM therapies is mainly due to the tumor infiltration into the healthy brain tissue, which depends on GBM cells' interaction with the tumor microenvironment (TME). The interaction of GBM cells with the TME involves cellular components such as stem-like cells, glia, endothelial cells, and non-cellular components such as the extracellular matrix, enhanced hypoxia, and soluble factors such as adenosine, which promote GBM's invasiveness. However, here we highlight the role of 3D patient-derived glioblastoma organoids cultures as a new platform for study of the modeling of TME and invasiveness. In this review, the mechanisms involved in GBM-microenvironment interaction are described and discussed, proposing potential prognosis biomarkers and new therapeutic targets.

The low affinity A2B adenosine receptor enhances migratory and invasive capacity in vitro and angiogenesis in vivo of glioblastoma stem-like cells.

Glioblastoma (GBM) is the most common and deadly malignant brain tumor, with a median survival of 15 to 17 months for a patient. GBM contains a cellular subpopulation known as GBM stem-like cells (GSCs) that persist in hypoxic niches and are capable of infiltrating into healthy brain tissue. For this reason, GSCs are considered one of the main culprits for GBM recurrence. A hypoxic microenvironment increases extracellular adenosine levels, activating the low affinity A2B adenosine receptor (A2BAR). Adenosine, through A2BAR, is capable of modulating invasiveness. However, its role in the invasion/migration of hypoxic-GSCs is still unknown. This study aims to understand the importance of A2BAR in modulating the migratory/invasive capacity of GSCs under hypoxia. Data analysis from The Cancer Genome Atlas (TCGA) program correlates A2BAR expression with high-grade glioma and hypoxic necrotic areas. U87MG and primary culture-derived GSCs under hypoxic conditions (0.5% O2) increased A2BAR mRNA and protein levels. As expected, the migratory and invasive capacity of GSCs increased under hypoxia, which was counteracted by blocking A2BAR, through the downregulation of MMP9 activity and epithelial-mesenchymal transition marker expression. Finally, in a xenograft mouse model, we demonstrate that treatment with MRS1754 did not affect the tumor volume but could decrease blood vessel formation and VEGF expression. Our results suggest that extracellular adenosine, through the activation of A2BAR, enhances the migratory and invasive capacity of GSCs in vitro under hypoxic conditions. Targeting A2BAR can be an effective therapy for GBM recurrence.

Adapt to Persist: Glioblastoma Microenvironment and Epigenetic Regulation on Cell Plasticity.

Glioblastoma (GBM) is the most frequent and aggressive brain tumor, characterized by great resistance to treatments, as well as inter- and intra-tumoral heterogeneity. GBM exhibits infiltration, vascularization and hypoxia-associated necrosis, characteristics that shape a unique microenvironment in which diverse cell types are integrated. A subpopulation of cells denominated GBM stem-like cells (GSCs) exhibits multipotency and self-renewal capacity. GSCs are considered the conductors of tumor progression due to their high tumorigenic capacity, enhanced proliferation, invasion and therapeutic resistance compared to non-GSCs cells. GSCs have been classified into two molecular subtypes: proneural and mesenchymal, the latter showing a more aggressive phenotype. Tumor microenvironment and therapy can induce a proneural-to-mesenchymal transition, as a mechanism of adaptation and resistance to treatments. In addition, GSCs can transition between quiescent and proliferative substates, allowing them to persist in different niches and adapt to different stages of tumor progression. Three niches have been described for GSCs: hypoxic/necrotic, invasive and perivascular, enhancing metabolic changes and cellular interactions shaping GSCs phenotype through metabolic changes and cellular interactions that favor their stemness. The phenotypic flexibility of GSCs to adapt to each niche is modulated by dynamic epigenetic modifications. Methylases, demethylases and histone deacetylase are deregulated in GSCs, allowing them to unlock transcriptional programs that are necessary for cell survival and plasticity. In this review, we described the effects of GSCs plasticity on GBM progression, discussing the role of GSCs niches on modulating their phenotype. Finally, we described epigenetic alterations in GSCs that are important for stemness, cell fate and therapeutic resistance.

Cervical Cancer Stem-Like Cell Transcriptome Profiles Predict Response to Chemoradiotherapy.

Cervical cancer (CC) represents a major global health issue, particularly impacting women from resource constrained regions worldwide. Treatment refractoriness to standard chemoradiotheraphy has identified cancer stem cells as critical coordinators behind the biological mechanisms of resistance, contributing to CC recurrence. In this work, we evaluated differential gene expression in cervical cancer stem-like cells (CCSC) as biomarkers related to intrinsic chemoradioresistance in CC. A total of 31 patients with locally advanced CC and referred to Mário Penna Institute (Belo Horizonte, Brazil) from August 2017 to May 2018 were recruited for the study. Fluorescence-activated cell sorting was used to enrich CD34+/CD45- CCSC from tumor biopsies. Transcriptome was performed using ultra-low input RNA sequencing and differentially expressed genes (DEGs) using Log2 fold differences and adjusted p-value < 0.05 were determined. The analysis returned 1050 DEGs when comparing the Non-Responder (NR) (n=10) and Responder (R) (n=21) groups to chemoradiotherapy. These included a wide-ranging pattern of underexpressed coding genes in the NR vs. R patients and a panel of lncRNAs and miRNAs with implications for CC tumorigenesis. A panel of biomarkers was selected using the rank-based AUC (Area Under the ROC Curve) and pAUC (partial AUC) measurements for diagnostic sensitivity and specificity. Genes overlapping between the 21 highest AUC and pAUC loci revealed seven genes with a strong capacity for identifying NR vs. R patients (ILF2, RBM22P2, ACO16722.1, AL360175.1 and AC092354.1), of which four also returned significant survival Hazard Ratios. This study identifies DEG signatures that provide potential biomarkers in CC prognosis and treatment outcome, as well as identifies potential alternative targets for cancer therapy.

Decreased Equilibrative Nucleoside Transporter 1 (ENT1) Activity Contributes to the High Extracellular Adenosine Levels in Mesenchymal Glioblastoma Stem-Like Cells.

Glioblastoma multiforme is one of the most malignant types of cancer. This is mainly due to a cell subpopulation with an extremely aggressive potential, called glioblastoma stem-like cells (GSCs). These cells produce high levels of extracellular adenosine which has been associated with increased chemoresistance, migration, and invasion in glioblastoma. In this study, we attempted to elucidate the mechanisms that control extracellular adenosine levels in GSC subtypes. By using primary and U87MG-derived GSCs, we associated increased extracellular adenosine with the mesenchymal phenotype. [3H]-adenosine uptake occurred mainly through the equilibrative nucleoside transporters (ENTs) in GSCs, but mesenchymal GSCs have lower expression and ENT1-mediated uptake activity than proneural GSCs. By analyzing expression and enzymatic activity, we determined that ecto-5'-nucleotidase (CD73) is predominantly expressed in proneural GSCs, driving AMPase activity. While in mesenchymal GSCs, both CD73 and Prostatic Acid Phosphatase (PAP) contribute to the AMP (adenosine monophosphate) hydrolysis. We did not observe significant differences between the expression of proteins involved in the metabolization of adenosine among the GCSs subtypes. In conclusion, the lower expression and activity of the ENT1 transporter in mesenchymal GSCs contributes to the high level of extracellular adenosine that these GSCs present.

In Vitro Employment of Recombinant Taenia solium Calreticulin as a Novel Strategy Against Breast and Ovarian Cancer Stem-like Cells.

BACKGROUND AND AIMS: Calreticulin is a chaperone and master regulator of intracellular calcium homeostasis. Several additional functions have been discovered. Human and parasite calreticulin have been shown to suppress mammary tumor growth in vivo. Here, we explored the capacity of recombinant Taenia solium calreticulin (rTsCRT) to modulate cancer cell growth in vitro. METHODS: We used different concentrations of rTsCRT to treat cancer cell lines and analyzed viability and colony formation capacity. We also tested the combination of the IC20 or IC50 doses of rTsCRT and of the chemotherapeutic drug 5-fluorouracil on MCF7 and SKOV3 cell lines. As a control, the non-tumorigenic cell line MCF10-A was employed. The effect of the drug combinations was also assessed in cancer stem-like cells. Additionally, scavenger receptor ligands were employed to identify the role of this receptor in the rTsCRT anti-tumoral effect. RESULTS: rTsCRT has a dose-dependent in vitro anti-tumoral effect, being SKOV3 the most sensitive cell line followed by MCF7. When rTsCRT/5-fluorouracil were used, MCF7 and SKOV3 showed a 60% reduction in cell viability; colony formation capacity was also diminished. Treatment of cancer stem-like cells from MCF7 showed a higher reduction in cell viability, while those from SKOV3 were more sensitive to colony disaggregation. Finally, pharmacological inhibition of the scavenger receptor, abrogated the reduction in viability induced by rTsCRT in both the parental and stem-like cells. CONCLUSION: Our data suggest that rTsCRT alone or in combination with 5-fluorouracil inhibits the growth of breast and ovarian cancer cell lines through its interaction with scavenger receptors.

FK506 Attenuates the MRP1-Mediated Chemoresistant Phenotype in Glioblastoma Stem-Like Cells.

Poor response to current treatments for glioblastoma has been attributed to the presence of glioblastoma stem-like cells (GSCs). GSCs are able to expel antitumor drugs to the extracellular medium using the multidrug resistance-associated protein 1 (MRP1) transporter. Tacrolimus (FK506) has been identified as an MRP1 regulator in differentiated glioblastoma (GBM) cells (non-GSCs); however, the effect of FK506 on GSCs is currently unknown. The objective of the following research is to evaluate the effect of FK506 on the MRP1-related chemo-resistant phenotype of GSCs. For this, U87MG and C6 glioma cell lines were used to generate non-GSCs and GSCs. mRNA and MRP1-positive cells were evaluated by RT-qPCR and flow cytometry, respectively. A Carboxyfluorescein Diacetate (CFDA)-retention assay was performed to evaluate the MRP1 activity. Apoptosis and MTT assays were employed to evaluate the cytotoxic effects of FK506 plus Vincristine (MRP1 substrate). GSC-derived subcutaneous tumors were generated to evaluate the in vivo effect of FK506/Vincristine treatment. No differences in transcript levels and positive cells for MRP1 were observed in FK506-treated cells. Lesser cell viability, increased apoptosis, and CFDA-retention in the FK506/Vincristine-treated cells were observed. In vivo, the FK506/Vincristine treatment decreased the tumor size as well as ki67, Glial Fibrillary Acidic Protein (GFAP), and nestin expression. We conclude that FK506 confers a chemo-sensitive phenotype to MRP1-drug substrate in GSCs.

The Adenosine A3 Receptor Regulates Differentiation of Glioblastoma Stem-Like Cells to Endothelial Cells under Hypoxia.

Glioblastoma (GBM) is a neoplasm characterized by an extensive blood vessel network. Hypoxic niches of GBM can induce tumorigenic properties of a small cell subpopulation called Glioblastoma stem-like cells (GSCs) and can also increase extracellular adenosine generation which activates the A3 adenosine receptor (A3AR). Moreover, GSCs potentiates the persistent neovascularization in GBM. The aim of this study was to determine if A3AR blockade can reduce the vasculogenesis mediated by the differentiation of GSCs to Endothelial Cells (ECs) under hypoxia. We evaluated the expression of endothelial cell markers (CD31, CD34, CD144, and vWF) by fluorescence-activated cell sorting (FACS), and vascular endothelial growth factor (VEGF) secretion by ELISA using MRS1220 (A3AR antagonist) under hypoxia. We validate our results using U87MG-GSCs A3AR knockout (GSCsA3-KO). The effect of MRS1220 on blood vessel formation was evaluated in vivo using a subcutaneous GSCs-tumor model. GSCs increased extracellular adenosine production and A3AR expression under hypoxia. Hypoxia also increased the percentage of GSCs positive for endothelial cell markers and VEGF secretion, which was in turn prevented when using MRS1220 and in GSCsA3-KO. Finally, in vivo treatment with MRS1220 reduced tumor size and blood vessel formation. Blockade of A3AR decreases the differentiation of GSCs to ECs under hypoxia and in vivo blood vessel formation.

Multidrug resistance in glioblastoma stem-like cells: Role of the hypoxic microenvironment and adenosine signaling.

Glioblastoma multiforme (GBM) is considered the most common and aggressive tumour of the central nervous system and is characterized for being highly chemoresistant. This property is mainly due to the activation of Multiple Drug Resistance (MDR) mechanisms that protect cancer cells from structurally and morphologically different drugs. Overexpression and increased ABC transporters activity is one of the most important MDR mechanisms at the clinical level, and both its expression and activity are elevated in GBM cells. Within the tumour, there is a subpopulation called glioblastoma stem-like cells (GSCs), which due to its high tumourigenic capacity and chemoresistance, have been postulated as the main responsible for tumour recurrence. The GSCs inhabit hypoxic tumour zones, niches that apart from maintaining and promoting stem phenotype have also been correlated with high chemoresistance. Of the signalling pathways activated during hypoxia, purinergic signalling has been highly associated to the induction of MDR mechanisms. Through its receptors, the nucleoside adenosine has been shown to promotes the chemoresistance mediated by ABC transporters. Therefore, targeting its components is a promising alternative for GBM treatment. In this review, we will discuss chemoresistance in GSCs and the effect of the hypoxic microenvironment and adenosine on MDR mechanisms.

Adenosine A3 receptor elicits chemoresistance mediated by multiple resistance-associated protein-1 in human glioblastoma stem-like cells.

MRP1 transporter correlates positively with glioma malignancy and the Multiple Drug Resistance (MDR) phenotype in Glioblastoma Multiforme (GBM). Evidence shows that the MRP1 transporter is controlled by the adenosine signalling axis. The aim of this study was to identify the role of adenosine on the MDR phenotype in Glioblastoma Stem-like Cells (GSCs), the cell population responsible for the tumorigenic and chemoresistance capabilities of this tumour. We found that GSCs have increased intrinsic capacity to generate extracellular adenosine, thus controlling MRP1 transporter expression and activity via activation of the adenosine A3 receptor (A3AR). We showed PI3K/Akt and MEK/ERK1/2 signaling pathways downstream A3AR to control MRP1 in GSCs. In vitro pharmacological blockade of A3AR had a chemosensitizing effect, enhancing the actions of antitumour drugs and decreasing cell viability and proliferation of GSCs. In addition, we produced an in vivo xenograft model by subcutaneous inoculation of human GSCs in NOD/SCID-IL2Rg null mice. Pharmacological blockade of A3AR generated a chemosensitizing effect, enhancing the effectiveness of the MRP1 transporter substrate, vincristine, reducing tumour size and the levels of CD44 and Nestin stem cell markers as well as the Ki-67 proliferation indicator. In conclusion, we demonstrated the chemosensitizing effect of A3AR blockade on GSCs.

Renal Subcapsular Space of Balb/c Nude Mice as a Route for Evaluating Subpopulations of Human Bladder Carcinoma Cells.

BACKGROUND/AIM: Subpopulations of bladder cancer (BC) cells have been found in tumors, with different abilities for malignancy and chemotherapy resistance. The BC cell line T24 has frequently been used to evaluate this phenomenon. Since technical limits exist in orthotopic procedures, we evaluated the renal subcapsular space as an alternative route for analyzing subpopulations of T24 BC cells in vivo. MATERIALS AND METHODS: Balb/c nude mice underwent renal subcapsular inoculation with T24 cells, suspended in two different volumes of PBS. Four weeks post-inoculation, histology and immunohistochemistry were carried out. RESULTS: In all the animals inoculated with a 10 µl volume of suspended cells, a pseudo-bladder structure in the renal subcapsular space was observed, with differential expression of mesenchymal and epithelial markers. T24 cells infiltrating the renal parenchyma towards the medulla and vessels were also observed. The volume used for inoculation was an important factor for the success of this technique. CONCLUSION: Renal subcapsular inoculation is an effective route for analyzing subpopulations and differentiation of T24 cells.