Detection of CD39 and a Highly Glycosylated Isoform of Soluble CD73 in the Plasma of Patients with Cervical Cancer: Correlation with Disease Progression.

Persistent infection with high-risk human papillomavirus (HR-HPV) is the main factor in the development of cervical cancer (CC). The presence of immunosuppressive factors plays an important role in the development of this type of cancer. To determine whether CD39 and CD73, which participate in the production of immunosuppressive adenosine (Ado), are involved in the progression of CC, we compared the concentrations and hydrolytic activity of these ectonucleotidases in platelet-free plasma (PFP) samples between patients with low-grade squamous intraepithelial lesions (LSILs) (n = 18), high-grade squamous intraepithelial lesions (HSILs) (n = 12), and CC (n = 19) and normal donors (NDs) (n = 15). The concentrations of CD39 and CD73 in PFP increased with disease progression (r = 0.5929, p < 0.001). The PFP of patients with HSILs or CC showed the highest concentrations of CD39 (2.3 and 2.2 times that of the NDs, respectively) and CD73 (1.7 and 2.68 times that of the NDs, respectively), which were associated with a high capacity to generate Ado from the hydrolysis of adenosine diphosphate (ADP) and adenosine monophosphate (AMP). The addition of POM-1 and APCP, specific inhibitors of CD39 and CD73, respectively, inhibited the ADPase and AMPase activity of PFP by more than 90%. A high level of the 90 kD isoform of CD73 was detected in the PFP of patients with HSILs or CC. Digestion with endoglycosidase H and N-glycanase generated CD73 with weights of approximately 90 kD, 85 kD, 80 kD, and 70 kD. In addition, the levels of transforming grow factor-ß (TGF-ß) in the PFPs of patients with LSIL, HSIL and CC positively correlated with those of CD39 (r = 0.4432, p < 0.001) and CD73 (r = 0.5786, p < 0.001). These results suggest that persistent infection by HR-HPV and the concomitant production of TGF-ß promote the expression of CD39 and CD73 to favor CC progression through Ado generation.

HPV-16 Infection Is Associated with a High Content of CD39 and CD73 Ectonucleotidases in Cervical Samples from Patients with CIN-1.

The development of cervical cancer (CeCa) is associated with high-risk human papilloma virus (HR-HPV) infections, mainly HPV-16, which is present in more than 50% of cases. The presence of immunosuppressive factors in the early stages of the disease is also strongly linked to CeCa progression. In this context, it is unknown whether ectonucleotidases CD39 and CD73, which are involved in the production of adenosine (Ado) that suppresses the specific antitumor immune response, are present in precursor lesions of CeCa. In this pilot study, we analyzed the presence of CD39 and CD73 and their capacity to generate Ado in 25 cervical samples from patients with grade 1 cervical intraepithelial neoplasms (CIN-1) and 25 samples from normal donors (NDs) free of HPV infection. Cells obtained from cervical samples of CIN-1 patients positive for HPV-16 showed higher CD39 and CD73 contents compared to samples obtained from CIN-1 patients negative for HPV-16 and NDs. Interestingly, solubilized cervical mucus from these patients also showed higher contents of soluble CD39 and CD73, which were associated with a greater capacity to produce Ado from the hydrolysis of adenosine triphosphate (ATP) and adenosine monophosphate (AMP). In addition, serum samples of these patients showed higher levels of TGF-ß than those of CIN-1 patients negative for HPV-16 and ND. These results suggest that persistent infection with HR-HPV, mostly HPV-16, in CIN-1 patients may promote the expression of CD39 and CD73 through the production of TGF-ß in precursor lesions to generate an immunosuppressive microenvironment and allow its progression to CeCa.

Mesenchymal Stromal Cells Derived from Normal Cervix and Cervical Cancer Tumors Increase CD73 Expression in Cervical Cancer Cells Through TGF-ß1 Production.

Mesenchymal stromal cells (MSCs) in the tumor microenvironment (TME) participate together with tumor cells to suppress antitumor effector cells through the production of immunosuppressive factors, such as transforming growth factor-beta 1 (TGF-ß1). Furthermore, TGF-ß1 can induce 5'-nucleotidase (CD73) expression in various cell types; this functional activity is associated with the production of adenosine (Ado), which is an immunosuppressive nucleoside. In this study, we provide evidence that coculture of MSCs derived from cervical tumors (CeCa-MSC) with CeCa tumor cells increases CD73 expression in tumor cells and the capacity of these cells to generate Ado in a MSC ratio-dependent manner. Interestingly, the increase in CD73 in the CeCa cell membrane corresponded to an increase in the TGF-ß1 expression level in the tumor cells and the TGF-ß1 content in the supernatants of the CeCa/CeCa-MSC cocultures. The addition of anti-hTGF-ß neutralizing antibodies strongly reversed CD73 expression in the tumor cells. This phenomenon was not exclusive to CeCa-MSCs; coculture of MSCs derived from the normal cervix with CeCa cells produced similar results. These results suggest that the interaction of MSCs with CeCa tumor cells in the TME may condition higher TGF-ß1 production to maintain an immunosuppressive status not only through the activity of this cytokine per se but also through its ability to induce CD73 expression in tumor cells and generate an immunosuppressive microenvironment rich in Ado.

Cervical cancer cells produce TGF-ß1 through the CD73-adenosine pathway and maintain CD73 expression through the autocrine activity of TGF-ß1.

In cancer, the adenosinergic pathway participates in the generation of an immunosuppressive microenvironment and in the promotion of tumor growth through the generation of adenosine (Ado). The present study analyzed the participation of Ado, generated through the functional activity of the cervical cancer (CeCa) pathway in CeCa cells, to induce the expression and secretion of TGF-ß1, as well as the participation of this factor to maintain CD73 expression. Ado concentrations greater than 10 µM were necessary to induce an increase of over 50% in the production and expression of TGF-ß1 in CeCa tumor cells. Blockade of A2AR and A2BR with the specific antagonists, ZM241385 and MRS1754, respectively, strongly reversed the production of TGF-ß1. TGF-ß1 produced by CeCa cells was necessary to maintain CD73 expression because the addition of anti-TGF-ß neutralizing antibodies or the inhibition of TGF-ßRI strongly reversed the expression of CD73 in the CeCa cells. These results suggested a feedback loop in CeCa cells that favors immunosuppressive activity through the production of TGF-ß1 and Ado as well as the autocrine activity of TGF-ß1 and expression of CD73.

Mesenchymal stromal cells derived from cervical cancer produce high amounts of adenosine to suppress cytotoxic T lymphocyte functions.

BACKGROUND: In recent years, immunomodulatory mechanisms of mesenchymal stem/stromal cells (MSCs) from bone marrow and other "classic" sources have been described. However, the phenotypic and functional properties of tumor MSCs are poorly understood. The aim of this study was to analyze the immunosuppressive capacity of cervical cancer-derived MSCs (CeCa-MSCs) on effector T lymphocytes through the purinergic pathway. METHODS: We determined the expression and functional activity of the membrane-associated ectonucleotidases CD39 and CD73 on CeCa-MSCs and normal cervical tissue-derived MSCs (NCx-MSCs). We also analyzed their immunosuppressive capacity to decrease proliferation, activation and effector cytotoxic T (CD8+) lymphocyte function through the generation of adenosine (Ado). RESULTS: We detected that CeCa-MSCs express higher levels of CD39 and CD73 ectonucleotidases in cell membranes compared to NCx-MSCs, and that this feature was associated with the ability to strongly suppress the proliferation, activation and effector functions of cytotoxic T-cells through the generation of large amounts of Ado from the hydrolysis of ATP, ADP and AMP nucleotides. CONCLUSIONS: This study suggests that CeCa-MSCs play an important role in the suppression of the anti-tumor immune response in CeCa through the purinergic pathway.