ICAM1 (CD54) Contributes to the Metastatic Capacity of Gastric Cancer Stem Cells.

Gastric cancer is the fourth leading cause of cancer deaths worldwide. The presence of chemoresistant cells has been used to explain this high mortality rate. These higher tumorigenic and chemoresistant cells involve cancer stem cells (CSCs), which have the potential for self-renewal, a cell differentiation capacity, and a greater tumorigenic capacity. Our research group identified gastric cancer stem cells (GCSCs) with the CD24+CD44+CD326+ICAM1+ immunophenotype isolated from gastric cancer patients. Interestingly, this GCSC immunophenotype was absent in cells isolated from healthy people, who presented a cell population with a CD24+CD44+CD326+ immunophenotype, lacking ICAM1. We aimed to explore the role of ICAM1 in these GCSCs; for this purpose, we isolated GCSCs from the AGS cell line and generated a GCSC line knockout for ICAM1 using CRISPR/iCas9, which we named GCSC-ICAM1KO. To assess the role of ICAM1 in the GCSCs, we analyzed the migration, invasion, and chemoresistance capabilities of the GCSCs using in vitro assays and evaluated the migratory, invasive, and tumorigenic properties in a zebrafish model. The in vitro analysis showed that ICAM1 regulated STAT3 activation (pSTAT3-ser727) in the GCSCs, which could contribute to the ability of GCSCs to migrate, invade, and metastasize. Interestingly, we demonstrated that the GCSC-ICAM1KO cells lost their capacity to migrate, invade, and metastasize, but they exhibited an increased resistance to a cisplatin treatment compared to their parental GCSCs; the GCSC-ICAM1KO cells also exhibited an increased tumorigenic capability in vivo.

Genetically modified ZIKA virus as a microRNA-sensitive oncolytic virus against central nervous system tumors.

Here we introduce a first-in-class microRNA-sensitive oncolytic Zika virus (ZIKV) for virotherapy application against central nervous system (CNS) tumors. The described methodology produced two synthetic modified ZIKV strains that are safe in normal cells, including neural stem cells, while preserving brain tropism and oncolytic effects in tumor cells. The microRNA-sensitive ZIKV introduces genetic modifications in two different virus sites: first, in the established 3'UTR region, and secondly, in the ZIKV protein coding sequence, demonstrating for the first time that the miRNA inhibition systems can be functional outside the UTR RNA sites. The total tumor remission in mice bearing human CNS tumors, including metastatic tumor growth, after intraventricular and systemic modified ZIKV administration, confirms the promise of this virotherapy as a novel agent against brain tumors-highly deadly diseases in urgent need of effective advanced therapies.

WNT Signaling in Stem Cells: A Look into the Non-Canonical Pathway.

Tissue homeostasis is crucial for multicellular organisms, wherein the loss of cells is compensated by generating new cells with the capacity for proliferation and differentiation. At the origin of these populations are the stem cells, which have the potential to give rise to cells with both capabilities, and persevere for a long time through the self-renewal and quiescence. Since the discovery of stem cells, an enormous effort has been focused on learning about their functions and the molecular regulation behind them. Wnt signaling is widely recognized as essential for normal and cancer stem cell. Moreover, ß-catenin-dependent Wnt pathway, referred to as canonical, has gained attention, while ß-catenin-independent Wnt pathways, known as non-canonical, have remained conspicuously less explored. However, recent evidence about non-canonical Wnt pathways in stem cells begins to lay the foundations of a conceivably vast field, and on which we aim to explain this in the present review. In this regard, we addressed the different aspects in which non-canonical Wnt pathways impact the properties of stem cells, both under normal conditions and also under disease, specifically in cancer.

Functional Roles of CD133: More than Stemness Associated Factor Regulated by the Microenvironment.

CD133 protein has been one of the most used surface markers to select and identify cancer cells with stem-like features. However, its expression is not restricted to tumoral cells; it is also expressed in differentiated cells and stem/progenitor cells in various normal tissues. CD133 participates in several cellular processes, in part orchestrating signal transduction of essential pathways that frequently are dysregulated in cancer, such as PI3K/Akt signaling and the Wnt/ß-catenin pathway. CD133 expression correlates with enhanced cell self-renewal, migration, invasion, and survival under stress conditions in cancer. Aside from the intrinsic cell mechanisms that regulate CD133 expression in each cellular type, extrinsic factors from the surrounding niche can also impact CD33 levels. The enhanced CD133 expression in cells can confer adaptive advantages by amplifying the activation of a specific signaling pathway in a context-dependent manner. In this review, we do not only describe the CD133 physiological functions known so far, but importantly, we analyze how the microenvironment changes impact the regulation of CD133 functions emphasizing its value as a marker of cell adaptability beyond a cancer-stem cell marker.

Genetically modified ZIKA virus as a microRNA-sensitive oncolytic virus against central nervous system tumors

Here we introduce a first-in-class microRNA-sensitive oncolytic Zika virus (ZIKV) for virotherapy application against central nervous system (CNS) tumors. The described methodology produced two synthetic modified ZIKV strains that are safe in normal cells, including neural stem cells, while preserving brain tropism and oncolytic effects in tumor cells. The microRNA-sensitive ZIKV introduces genetic modifications in two different virus sites: first, in the established 3′UTR region, and secondly, in the ZIKV protein coding sequence, demonstrating for the first time that the miRNA inhibition systems can be functional outside the UTR RNA sites. The total tumor remission in mice bearing human CNS tumors, including metastatic tumor growth, after intraventricular and systemic modified ZIKV administration, confirms the promise of this virotherapy as a novel agent against brain tumors—highly deadly diseases in urgent need of effective advanced therapies.

Cancer Stem Cells and Androgen Receptor Signaling: Partners in Disease Progression.

Cancer stem cells exhibit self-renewal, tumorigenesis, and a high differentiation potential. These cells have been detected in every type of cancer, and different signaling pathways can regulate their maintenance and proliferation. Androgen receptor signaling plays a relevant role in the pathophysiology of prostate cancer, promoting cell growth and differentiation processes. However, in the case of prostate cancer stem cells, the androgen receptor negatively regulates their maintenance and self-renewal. On the other hand, there is evidence that androgen receptor activity positively regulates the generation of cancer stem cells in other types of neoplasia, such as breast cancer or glioblastoma. Thus, the androgen receptor role in cancer stem cells depends on the cellular context. We aimed to analyze androgen receptor signaling in the maintenance and self-renewal of different types of cancer stem cells and its action on the expression of transcription factors and surface markers associated with stemness.

Mechanisms involved in cancer stem cell resistance in head and neck squamous cell carcinoma.

Despite scientific advances in the Oncology field, cancer remains a leading cause of death worldwide. Molecular and cellular heterogeneity of head and neck squamous cell carcinoma (HNSCC) is a significant contributor to the unpredictability of the clinical response and failure in cancer treatment. Cancer stem cells (CSCs) are recognized as a subpopulation of tumor cells that can drive and maintain tumorigenesis and metastasis, leading to poor prognosis in different types of cancer. CSCs exhibit a high level of plasticity, quickly adapting to the tumor microenvironment changes, and are intrinsically resistant to current chemo and radiotherapies. The mechanisms of CSC-mediated therapy resistance are not fully understood. However, they include different strategies used by CSCs to overcome challenges imposed by treatment, such as activation of DNA repair system, anti-apoptotic mechanisms, acquisition of quiescent state and Epithelial-mesenchymal transition, increased drug efflux capacity, hypoxic environment, protection by the CSC niche, overexpression of stemness related genes, and immune surveillance. Complete elimination of CSCs seems to be the main target for achieving tumor control and improving overall survival for cancer patients. This review will focus on the multi-factorial mechanisms by which CSCs are resistant to radiotherapy and chemotherapy in HNSCC, supporting the use of possible strategies to overcome therapy failure.

Non-canonical Wnt/Ca2+ signaling is essential to promote self-renewal and proliferation in colon cancer stem cells.

Introduction: Cancer Stem Cells (CSC) are responsible for maintaining tumor growth, chemoresistance, and metastasis. Therefore, understanding their characteristics is critical to progress in cancer therapy. While the contribution of the canonical Wnt/b-catenin signaling in both normal and CSCs had been well established, the function of non-canonical Wnt signaling cascades in stem cells is unclear. Recently, we reported that Wnt ligands trigger complex signaling in which the canonical and non-canonical responses can be simultaneously activated by one ligand in colon cancer cells, suggesting, therefore, that noncanonical Wnt pathways may also be important in CSCs. Methods: The present work aimed to know the role of the Wnt/Ca2+ pathway in colon CSCs. We used tumorspheres as a model of CSCs enrichment of CRC cell lines with different Wnt/b-catenin contexts. Results: Using Wnt3a and Wnt5a as prototype ligands to activate the canonical or the non-canonical pathways, respectively, we found that both Wnt3a and Wnt5a promote sphere-formation capacity and proliferation without stimulating b-catenin-dependent transcription. Upregulation of sphere formation by Wnt5a or Wnt3a requires the downstream activation of Phospholipase C and transcriptional factor NFAT. Moreover, the single specific inhibition of PLC or NFAT, using U73122 and 11R-VIVIT, respectively, leads to impaired sphere formation. Discussion: Our results indicate that both types of ligands activate the Wnt/Ca2+ signaling axis to induce/maintain the self-renewal efficiency of CSCs, demonstrating to be essential for the functions of CSC in colon cancer.

Effect of ZEB1 Associated with microRNAs on Tumor Stem Cells in Head and Neck Cancer.

Cancer biologists have focused on studying cancer stem cells (CSCs) because of their ability to self-renew and recapitulate tumor heterogeneity, which increases their resistance to chemotherapy and is associated with cancer relapse. Here, we used two approaches to isolate CSCs: the first involved the metabolic enzyme aldehyde dehydrogenase ALDH, and the second involved the three cell surface markers CD44, CD117, and CD133. ALDH cells showed a higher zinc finger E-box binding homeobox 1 (ZEB1) microRNA (miRNA) expression than CD44/CD117/133 triple-positive cells, which overexpressed miRNA 200c-3p: a well-known microRNA ZEB1 inhibitor. We found that ZEB1 inhibition was driven by miR-101-3p, miR-139-5p, miR-144-3p, miR-199b-5p, and miR-200c-3p and that the FaDu Cell Line inhibition occurred at the mRNA level, whereas HN13 did not affect mRNA expression but decreased protein levels. Furthermore, we demonstrated the ability of the ZEB1 inhibitor miRNAs to modulate CSC-related genes, such as TrkB, ALDH, NANOG, and HIF1A, using transfection technology. We showed that ALDH was upregulated upon ZEB1-suppressed miRNA transfection (Mann-Whitney ** p101 = 0.009, t-test ** p139 = 0.009, t-test ** p144 = 0.002, and t-test *** p199 = 0.0006). Overall, our study enabled an improved understanding of the role of ZEB1-suppressed miRNAs in CSC biology.

Cancer Stem Cells and Chemoresistance in Ewing Sarcoma.

Resistance to chemotherapy poses a major challenge for cancer treatment. Reactivating a stem cell program resembling that seen in embryonic development can lead cancer cells to acquire a stem-cell phenotype characterized by expression of stemness genes, pluripotency, high self-renewal ability, and tumor-initiating capability. These cancer stem cells (CSCs) are usually resistant to anticancer drugs and are likely involved in treatment failure in many cancer types. Ewing sarcoma (ES) is a pediatric cancer type typically resulting from a typical genetic alteration affecting bone or soft tissues. Despite advances in treatment, survival prognostic remains poor for patients with refractory or recurrent disease. Here, we review the increasing evidence indicating that ES tumors contain a CSC subpopulation expressing stem cell genes, including BM1, OCT3/4, NANOG, and SOX2, that plays a role in resistance to drug treatment, and current experimental strategies that successfully counteract chemoresistance mediated by CSCs in ES.

Oral squamous cell carcinoma cancer stem cells have different drug sensitive to pharmacological NFκB and histone deacetylation inhibition.

Despite many progresses in the development of new systemic therapies for oral squamous cell carcinoma (OSCC), the five-year survival rate of OSCC is low. The traditional chemotherapies approach (cisplatin - CDDP) shows some limitations like drug toxicity, limited efficacy, and drug resistance. Promising studies suggested OSCC cancer stem cells (CSC) presented resistance to CDDP. We have previously studied many targets, and we extensively showed the efficacy of the NFκB signaling and the role of histones acetylation, on different malignant tumors, including adenoid cystic carcinoma and mucoepidermoid carcinoma, but until then the effects of the NFkB inhibitor and histone deacetylase (HDAC) inhibitor on the biology of OSCC were not evaluated. Here we assessed the pharmacological inhibitor of NFκB emetine and HDAC inhibitor SAHA on the behavior of CSC derived from OSCC. Our data suggested that CDDP administration resulted in reduced viability of bulk OSCC cells and increased CSC. A single and isolated shot of emetine and SAHA were able to disrupt CSC by inhibiting the NFκB pathway and increasing the histone acetylation levels, respectively. Further, the combined administration of emetine and SAHA presented the same CSC disruption as seen in emetine alone.

Cancer Stemness/Epithelial-Mesenchymal Transition Axis Influences Metastasis and Castration Resistance in Prostate Cancer: Potential Therapeutic Target.

Prostate cancer (PCa) is a leading cause of cancer death in men, worldwide. Mortality is highly related to metastasis and hormone resistance, but the molecular underlying mechanisms are poorly understood. We have studied the presence and role of cancer stem cells (CSCs) and the Epithelial-Mesenchymal transition (EMT) in PCa, using both in vitro and in vivo models, thereby providing evidence that the stemness-mesenchymal axis seems to be a critical process related to relapse, metastasis and resistance. These are complex and related processes that involve a cooperative action of different cancer cell subpopulations, in which CSCs and mesenchymal cancer cells (MCCs) would be responsible for invading, colonizing pre-metastatic niches, initiating metastasis and an evading treatments response. Manipulating the stemness-EMT axis genes on the androgen receptor (AR) may shed some light on the effect of this axis on metastasis and castration resistance in PCa. It is suggested that the EMT gene SNAI2/Slug up regulates the stemness gene Sox2, and vice versa, inducing AR expression, promoting metastasis and castration resistance. This approach will provide new sight about the role of the stemness-mesenchymal axis in the metastasis and resistance mechanisms in PCa and their potential control, contributing to develop new therapeutic strategies for patients with metastatic and castration-resistant PCa.

Effects of 5-ALA mediated photodynamic therapy in oral cancer stem cells.

The aim of the present study was to investigate the effects of PDT using the photosensitizer 5-aminoulevulinic acid (5-ALA) in oral squamous cell carcinoma (OSCC) behavior, mainly regarding its role on the cancer stem cell (CSC) phenotypes and in maintenance of the stem cell properties. Two OSCC cell lines were used and divided in the groups: Control, 5-ALA, LED 6 J/cm2 and PDT. MTT and Neutral red assays were used to access cellular viability, cell migration was evaluated by the wound healing assay. The stem cell phenotype was analyzed by flow cytometry to evaluate the CD44high/ESAhigh, CD44high/ESAlow and CD44low populations, by the clonogenic and tumor sphere formation assays as well as by RT-qPCR. The presence of Protoporphyrin IX in each CSC fraction was evaluated by flow cytometry. The OSCC cell lines showed a significant decrease in cell viability and migration after PDT. The percentage of CD44high/ESAhigh cells decreased after PDT, which was associated with an increase in the CD44low cells and with a functional decrease in the colony and sphere formation capacity. CD44high/ESAhigh cells showed increased PpIX, which contributed for their greater sensitivity to PDT. INV gene increased significantly after PDT, indicating cellular differentiation. Altogether, our results demonstrate that 5-ALA mediated PDT decreases not only the fraction of oral CSC but also their functional capabilities, inducing their differentiation.

Mechanisms of immune evasion by head and neck cancer stem cells.

Different mechanisms are involved in immune escape surveillance driven by Oral and Head and Neck Cancer Stem Cells (HNCSCs). The purpose of this review is to show the most current knowledge regarding the main impact of HNCSCs on tumor evasion through immunosuppression, CSCs phenotypes and environmental signals, highlighting strategies to overcome immune evasion. The main results drive the participation of cell surface receptors and secreted products and ligands, the crosstalk between cells, and genetic regulation. The reduction in CD8+ T cell recruitment and decreased effector of anti-PD-1 therapy by cells expressing BMI1 is a key event; Natural Killer cell ligands and cytokines needed for its activation and expansion are crucial to control tumor growth and to target CSCs by immunotherapy; CSCs expressing ALDH1 are related to increased expression of PD-L1, with a positive link between DNMT3b expression; CD276 expression in CSCs can act as a checkpoint inhibitor and together with Activator Protein 1 (AP-1) activation, they create continuous positive feedback that enables immune evasion by suppressing CD8+ T cells and prevent immune cell infiltration in head and neck cancer. These data demonstrate the relevance of the better understanding of the interaction between HNCSCs and immune cells in the tumor microenvironment. The ultimate clinical implication is to ground the choice of optimized targets and improve immune recognition for ongoing treatments as well as the response to approved immunotherapies.

AMPK's double-faced role in advanced stages of prostate cancer.

Prostate cancer (PCa) is the second leading cause of cancer deaths in men. Unfortunately, a very limited number of drugs are available for the relapsed and advanced stages of PCa, adding only a few months to survival; therefore, it is vital to develop new drugs. 5´ AMP-activated protein kinase (AMPK) is a master regulator of cell metabolism. It plays a significant role in the metabolism of PCa; hence, it can serve well as a treatment option for the advanced stages of PCa. However, whether this pathway contributes to cancer cell survival or death remains unknown. The present study reviews the possible pathways by which AMPK plays role in the advanced stages of PCa, drug resistance, and metastasis: (1) AMPK has a contradictory role in promoting glycolysis and the Warburg effect which are correlated with cancer stem cells (CSCs) survival and advanced PCa. It exerts its effect by interacting with hypoxia-induced factor 1 (HIF1) α, pyruvate kinase 2 (PKM2), glucose transporter (GLUT) 1 and pyruvate dehydrogenase complex (PDHC), which are key regulators of glycolysis; however, whether it promotes or discourage glycolysis is not conclusive. It can also exert an anti-CSC effect by negative regulation of NANOG and epithelial-mesenchymal transition (EMT) transcription factors, which are the major drivers of CSC maintenance; (2) the regulatory effect of AMPK on autophagy is also noticeable. Androgen receptors' expression increases AMPK activation through Calcium/calmodulin-dependent protein kinase 2 (CaMKK2) and induces autophagy. In addition, AMPK itself increases autophagy by downregulating the mammalian target of rapamycin complex (mTORC). However, whether increased autophagy inhibits or promotes cell death and drug resistance is contradictory. This study reveals that there are numerous pathways other than cell metabolism by which AMPK exerts its effects in the advanced stages of PCa, making it a priceless treatment target. Finally, we mention some drugs developed to treat the advanced stages of PCa by acting on AMPK.

The inverse paradigm and the ancestral cell of IDH-wildtype glioblastoma.

Rethinking IDH-wildtype glioblastoma through its unique features can help researchers find innovative and effective treatments. It is currently emerging that, after decades of therapeutic impasse, some traditional concepts regarding IDH-wildtype glioblastoma need to be supplemented and updated to overcome therapeutic resistance. Indeed, multiple clinical aspects and recent indirect and direct experimental data are providing evidence that the supratentorial brain parenchyma becomes entirely and quiescently micro-infiltrated long before primary tumor bulk growth. Furthermore, they are indicating that the known micro-infiltration that occurs during the IDH-wildtype glioblastoma growth and evolution is not at the origin of distant relapses. It follows that the ubiquitous supratentorial brain parenchyma micro-infiltration as a source for the development of widespread distant recurrences is actually due to the silent stage that precedes tumor growth rather than to the latter. All this implies that, in addition to the heterogeneity of the primary bulk, there is a second crucial cause of therapeutic resistance that has never hitherto been identified and challenged. In this regard, the ancestral founder cancer stem cell (CSC) appears as the key cell that can link the two causes of resistance.

Isolated cancer stem cells from human liver cancer: morphological and functional characteristics in primary culture.

BACKGROUND: Primary liver cancer cells (PLCs) could more directly simulate the human tumor microenvironment. Compared with liver cancer cell lines, PLCs could reflect the human situation. As in previous studies, tumor stem cells were a small number of cancer cells in the microenvironment and considered to be one of the origins of liver cancer. This study aimed to screen stem cells in PLCs, analyze their biological characteristics, propose the possibility that liver cancer originated from stem cells. METHODS: Liver cancer tissues of 17 patients were taken from the Affiliated Hospital of Guangdong Medical College, and PLCs were isolated by tissue slice method. The proliferation, tumor formation in nude mice, stem protein expression of PLCs were observed. C-kit+ liver cancer cells were screened and their biological characteristics were analyzed. RESULTS: PLCs could be stably passaged. Transmission electron microscopy indicated that the nucleus was irregular, there were many mitochondria, and the endoplasmic reticulum was irregularly distributed. PLCs could express E-Cadherin, Oct-4, ß-Catenin, Sox2, CD326, C-kit, GPC3, Nanog. The proliferation curve of PLCs and Hep3B cells were similar, and they all could form tumors in nude mice. Flow-sorted C-kit+ PLCs, as well as C-kit+ Hep3B cells could highly express Bmi1, Sox2, Oct4, Notch1, Nanog, C-kit, ß-Catenin, Smo, Nestin, ABCG2, ABCB1. And they also could clone and form tumors in vivo. But C-kit+ PLCs were more sensitive to chemotherapy drugs than C-kit+ liver cancer cell lines. CONCLUSION: C-kit+ PLCs had the characteristics of tumor stem cells and were more sensitive to chemotherapy drugs.

Glucose and Amino Acid Metabolic Dependencies Linked to Stemness and Metastasis in Different Aggressive Cancer Types.

Malignant cells are commonly characterised by being capable of invading tissue, growing self-sufficiently and uncontrollably, being insensitive to apoptosis induction and controlling their environment, for example inducing angiogenesis. Amongst them, a subpopulation of cancer cells, called cancer stem cells (CSCs) shows sustained replicative potential, tumor-initiating properties and chemoresistance. These characteristics make CSCs responsible for therapy resistance, tumor relapse and growth in distant organs, causing metastatic dissemination. For these reasons, eliminating CSCs is necessary in order to achieve long-term survival of cancer patients. New insights in cancer metabolism have revealed that cellular metabolism in tumors is highly heterogeneous and that CSCs show specific metabolic traits supporting their unique functionality. Indeed, CSCs adapt differently to the deprivation of specific nutrients that represent potentially targetable vulnerabilities. This review focuses on three of the most aggressive tumor types: pancreatic ductal adenocarcinoma (PDAC), hepatocellular carcinoma (HCC) and glioblastoma (GBM). The aim is to prove whether CSCs from different tumour types share common metabolic requirements and responses to nutrient starvation, by outlining the diverse roles of glucose and amino acids within tumour cells and in the tumour microenvironment, as well as the consequences of their deprivation. Beyond their role in biosynthesis, they serve as energy sources and help maintain redox balance. In addition, glucose and amino acid derivatives contribute to immune responses linked to tumourigenesis and metastasis. Furthermore, potential metabolic liabilities are identified and discussed as targets for therapeutic intervention.

ALCAM/CD166: A pleiotropic mediator of cell adhesion, stemness and cancer progression.

Activated Leukocyte Cell Adhesion Molecule (ALCAM/CD166) is a glycoprotein involved in homotypic and heterotypic cell adhesion. ALCAM can be proteolytically cleaved at the cell surface by metalloproteases, which generate shedding of its ectodomain. In various tumors, ALCAM is overexpressed and serves as a valuable prognostic marker of disease progression. Moreover, CD166 has been identified as a putative cancer stem cell marker in particular cancers. Herein, we summarize biochemical aspects of ALCAM, including structure, proteolytic shedding, alternative splicing, and specific ligands, and integrate this information with biological functions of this glycoprotein including cell adhesion, migration and invasion. In addition, we discuss different patterns of ALCAM expression in distinct tumor types and its contribution to tumor progression. Finally, we highlight the role of ALCAM as a cancer stem cell marker and introduce current clinical trials associated with this molecule. Future studies are needed to define the value of shed ALCAM in biofluids or ALCAM isoform expression as prognostic biomarkers in tumor progression.

A three-dimensional microenvironment alters CD73 expression in cervical cancer.

Stem-like cells (CSCs) have a tumour-initiating capacity and play critical role in tumour metastasis, relapse and resistance to therapy. The ectoenzyme CD73, encoded by the NT5E gene, which catalyses the hydrolysis of AMP into adenosine, has been associated to an immunosuppressive tumour microenvironment, tumour cell adhesion and migration. Therefore, we investigated the expression and activity of CD73 in sphere-forming cells from cervical cancer in comparison to monolayer cells in vitro. In addition, in silico analysis was performed to determine the expression of CD73 and other members of purinergic signalling in CSC-like population derived from different tumour types in comparison to monolayer cells. CD73 protein expression levels and functionality in SiHa cells were analysed by flow cytometry and enzymatic assay, respectively. In silico investigation was performed through the analysis of seven datasets from different tumour types using GEO database. In vitro analysis showed a decreased CD73 protein expression and enzymatic activity in cervical spheres, when compared to monolayers. In addition, when sphere-derived cells are re-plated as monolayer culture, the CD73 expression and activity are restored. Supporting the in vitro results, in silico analysis showed that three-dimensional spheres derived from cervical, thyroid and breast cancer presented decreased expression of CD73, when compared to their adherent counterparts. The decreased expression of CD73 in sphere-derived cells or CSC-enriched population reinforce its important role in cell adhesion, tumour spreading ability and metastasis, suggesting CD73 as potential target to be further investigated in cervical cancer.