A three‐dimensional microenvironment alters CD73 expression in cervical cancer

Abstract

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.

Evidence that cervical cancer cells cultured as tumorspheres maintain high CD73 expression and increase their protumor characteristics through TGF‐β production

“…The expression of CD73 and the capacity to generate Ado were significantly higher in CaSki-T cells than in CaSki-M cells. As reported in previous studies, [43][44][45] CaSki-T cells were enriched in CSC-like characteristics due to increases in the expression of stem cell markers (CD49f, CK17 and P63, OCT4 and SOX2), greater SFE, and an increase in the percentage of PS cells.…”

“…The expression of CD73 and the capacity to generate Ado were significantly higher in CaSki‐T cells than in CaSki‐M cells. As reported in previous studies, 43 , 44 , 45 CaSki‐T cells were enriched in CSC‐like characteristics due to increases in the expression of stem cell markers (CD49f, CK17 and P63, OCT4 and SOX2), greater SFE, and an increase in the percentage of PS cells. CaSki‐T cells also produced higher amounts of TGF‐β1 than CaSki‐M cells and exhibited a markedly protumoral phenotype characterized by a significant increase in the expression levels of proteins related to the EMT state, such as vimentin, Snail, and Twist, and a reduced expression level of N‐cadherin.…”

“…In the context of tumor development and progression, at least two important functions of CD73 have been described: (a) the catalytic activity of this enzyme to generate Ado from AMP hydrolysis and (b) the ability of CD73 to promote adhesion and modulate cell migration. Accordingly, the low CD73 expression found in CC spheres has been hypothesized to be related to tumor progression, leading to tumor migration and invasiveness 43 . Interestingly, our results provide evidence that high CD73 expression in CC spheres, which is related to high TGF‐β1 production, also results in the promotion of protumoral characteristics related to tumor progression, such as migration and invasiveness, immune evasion, immunosuppression, and chemoresistance, suggesting that both functions of CD73 can be relevant in CC progression.…”

“…4,6 On the other hand, in nonadherent and serum-free conditions, the in vitro tumorsphere formation assay has been considered a functional method to produce tumor cultures enriched in subpopulations of CSC-like cells and a microenvironment where cell behavior can be studied under different experimental conditions. [21][22][23][24][25] However, a recent in silico analysis carried out by Iser et al 43 Representative data from 3 independent experiments ±SEM. * , **Significant differences at p < .05 and p < .01, respectively.…”

“…On the other hand, in nonadherent and serum‐free conditions, the in vitro tumorsphere formation assay has been considered a functional method to produce tumor cultures enriched in subpopulations of CSC‐like cells and a microenvironment where cell behavior can be studied under different experimental conditions. 21 , 22 , 23 , 24 , 25 However, a recent in silico analysis carried out by Iser et al 43 related to CD73 expression on cells derived from CSC‐enriched tumorspheres using microarray gene expression profiles of 7 GSE datasets from the Expression Omnibus Database (GEO) reported that protocols for CSC induction or isolation, as well as the maintenance conditions or exposure to treatments, presented significant differences among the studies analyzed. In addition, similar to their results obtained from SiHa cells, they reported that in human primary cervical cells isolated from healthy tissue, the human anaplastic thyroid cancer cell line (THJ‐11 T cells), 53 and breast cancer cell lines from human primary tumors, 54 a decrease in the CD73 expression of CSC‐enriched tumorspheres compared to monolayer cells was evident.…”