Analysis of the predictive value of expression ectonucleotidase CD39 peripheral Tregs in patients with colorectal cancer

G.A. Zhulai¹, E.K. Oleinik¹, M.I. Shibaev², P.N. Semakova¹, V.M. Oleinik¹

 

¹Institute of Biology of the Karelian Research Centre of the Russian Academy of Sciences, Petrozavodsk, Republic of Karelia, Russian Federation;

²Republic Oncology Dispensary, Petrozavodsk, Republic of Karelia, Russian Federation

 

Abstract. Regulatory T-lymphocytes (Treg) are considered as potential targets in cancer immunotherapy and as prognostic biomarkers of the course of the disease. In some types of cancer, including colorectal cancer (CRC) the role of Treg cells is not clearly defined and requires further research. Recently, special attention has been paid to the ability of Tregs to express CD39 ectonucleotidase due to its participation in the cleavage of adenosine triphosphate to extracellular adenosine, which inhibits the antitumor immune response. An increased number of CD39⁺ Treg cells is observed both in the tumor tissue and in the peripheral blood of cancer patients. Therefore, the main goal of this research was to assess the prognostic significance of the level of expression (low / high) by Treg cells of CD39 ectonucleotidase in the peripheral blood of CRC patients. Material and methods. We analyzed blood lymphocytes of 32 patients with CRC, the comparison group consisted of 30 healthy donors. The relative content of Treg cells was assessed by multicolor flow cytometry on a Cytomics FC500 (Beckman Coulter, USA) using monoclonal antibodies CD4-FITC, CD25-PC5, CD127-PC7, CD39-PE. The expression of mRNA of the CD39 gene was determined by real-time polymerase chain reaction. Survival curves were constructed using the Kaplan-Meier procedure using application log rank criterion. Results. Previously, we found that in patients with CRC, the content of CD39⁺ Treg-cells is increased both in the peripheral blood in comparison with healthy donors and among tumor-infiltrating cells. Moreover, the share of CD39-positive Treg cells increased with the development of the disease. In this research, the three-year overall survival of patients with CRC was analyzed taking into account the content of CD39⁺ Treg-cells (CD4⁺CD25⁺CD127lo/-) and depending on the level mRNA gene CD39 in the peripheral blood. There were no statistically significant differences in patient survival depending on CD39⁺ Treg cell content and level mRNA gene CD39. Conclusion. Further learning adenosine mechanism of immune suppression with the participation of regulatory T cells, will allow to new therapeutic and prognostic opportunities in the management of patients with CRC, which are represent of great scientific and practical interest.

 

Keywords: colorectal cancer, regulatory T cells, ectonucleotidase, CD39, overall survival

 

There is no conflict of interest.

Contact details of the corresponding author:

Galina A. Zhulai

zhgali-111@yandex.ru

Received 16.11.2020

For citation:

Zhulai G.A., Oleinik E.K., Shibaev M.I., Semakova P.N., Oleinik V.M. Analysis of the predictive value of expression ectonucleotidase CD39 peripheral Tregs in patients with colorectal cance. http://vestnikural.ru/article/1154 [Online] Vestn. Ural. Med. Akad. Nauki. = Journal of Ural Medical Academic Science. 2020, Vol. 17, no. 4, pp. 323–332. DOI: 10.22138/2500-0918-2020-17-4-323-332 (In Russ)

 

Introduction

Colorectal cancer (CRC) is one of the leading localizations among malignant neoplasms in the overall structure of cancer incidence in Russia and in the world [1]. Timely detection of this disease, as well as identification of molecular targets, allowing to predict the course of the disease and the effectiveness of therapy, is currently one of the urgent medical and social problems. 

A growing tumor uses various mechanisms to evade immunological surveillance. One of these methods is the induction of immune suppression for suppression antitumor immune response. The direct participants in this process are regulatory T cells (Treg). These cells normally suppress an excessive immune response, maintaining immunological tolerance and inhibiting the development of autoimmune responses. In some cases, suppressor Tregs can promote tumor development. Treg cells are present in high numbers in peripheral blood and tumor tissue of various types and sites of cancer. Increased CD4⁺ count Treg cells among tumor-infiltrating lymphocytes are often accompanied by a decrease in the ratio CD8+ T-cells to CD4⁺CD25⁺FOXP3⁺ Treg cells and is associated with a poor prognosis for most patients with malignant neoplasms [2]. It is noted that Tregs can interfere with successful anticancer therapy. A detailed study of the relationship between immune cells and tumors in colorectal cancer will reveal immunological targets that can be used in the clinic.

CD39 (ectonucleoside triphosphate diphosphohydrolase 1, E-NTPDase1) is a membrane protein that binds and converts extracellular adenosine triphosphate with CD73 in extracellular adenosine possessing immunosuppressive effect and promoting carcinogenesis [3, 4]. CD39 is expressed various immune and non-immune cells (endothelial, fibroblasts), as well as some tumor cells. It is noted that tumor-infiltrating immune cells show enhanced expression of CD39. Currently the blockade of adenosine mediated suppressor mechanism is considered as a potential direction of immunotherapy for cancer patients.

It has been established that Treg cells can participate in the formation of adenosine from extracellular nucleotides, which is considered as one of the main mechanisms of their suppressor activity [5]. Under the conditions of cancer development, Treg cells are characterized by increased expression of CD39 both in the tumor microenvironment and in the periphery [6]. We have previously shown activation adenosine-mediated suppression in patients with CRC [7], in which the population of CD39⁺ Treg cells can play an important role. In this regard, the aim of this work was to assess the prognostic significance of the expression level (low/high) by Treg cells ectonucleotidase CD39 in peripheral blood in CRC patients.

 

Materials and methods

In the study 32 patients with CRC were examined, the average age of which was 64±11.5 years. The study included patients who met the criteria: age 18–70 years, the presence of a confirmed diagnosis “colorectal cancer». The diagnosis of colorectal cancer was established on the basis of clinical, laboratory, endoscopic and morphological research methods. The exclusion criteria were: the presence of other forms of neoplasms in the anamnesis, as well as immunoinflammatory diseases. The patients were divided into two groups: the first group consisted of patients at stages I and II of CRC, the second — at stages III and IV. To conduct the study, permission was obtained from the Medical Ethics Committee under the Ministry of Health and Social Development of the Republic of Karelia and Petrozavodsk State University (protocol No. 25 of 12.02.2013). Informed consent was obtained from the examined patients for the study. Lymphocytes from 30 healthy donors aged 54.4±20.6 years were analyzed as a comparison group. 

In the study the immunophenotype lymphocytes of patients with CRC was researched by the method of multicolor flow cytometry on the device Cytomics FC500 (Beckman Coulter, USA). Monoclonal antibodies CD4-FITC, CD25-PC5, CD127-PC7 (Beckman Coulter, France), CD39-PE (R&D Systems, USA) and corresponding isotypic controls were used. Expression mRNA gene CD39 was determined by real-time PCR. Isolation and purification of nucleic acids was carried out using the kit «AxyPrep Blood Total RNA Miniprep Kit» (Axygen, USA). For synthesis cDNA random hexaprimers and MMLV reverse transcriptase (Sileks, Russia) were used. Amplification cDNA and analysis of amplification products in real time was performed using a reaction mixture with intercalating dye SYBR Green I (Evrogen, Russia) on the device «iCycler Thermal Cycler» (Bio-Rad, USA). The analysis of the obtained data was carried out by the method 2–∆∆Ct where Ct is the threshold cycle, and ∆Сt — the difference between the values of the reference (GAPDH) and targeted threshold cycles. The final expression level of the studied gene was calculated relative to the control (healthy donors), taking the value of the expression of the studied gene in the control as a unit.

Statistical processing of the results obtained during the study was carried out using the GRAPH Pad Prism 6.0 software. Survival curves were constructed using the Kaplan – Meier procedure. When comparing survival curves in groups log rank criterion was applied. The censored data included data from patients who were alive at the end of the study. Uncensored data refere to the data of deceased patients. The level of statistical significance was taken equal to 0.05. Survival analysis and imaging were performed using IBM SPSS software Statistics Subscription. The study was carried out on the equipment of the Shared Facility of the Federal Research Center Karelian Research Center of the Russian Academy of Sciences.

 

Results and discussion

We have previously shown that in colorectal cancer patients, the frequency of CD39⁺ Treg cells is increased as in the peripheral blood compared with healthy donors and among tumor-infiltrating cells. Moreover, the proportion of CD39⁺ Treg cells increased with the development of the disease [7]. 

In this research, the three-year overall survival (OS) of 32 patients with CRC was analyzed taking into account the content of CD39⁺ Treg cells (CD4⁺CD25⁺CD127lo/-) in the peripheral blood. The patients were divided into two groups: depending on the value of M+2SD frequency (more or less) CD39⁺ Treg cells in healthy individuals, which accounted for 65.36%. It was noted that OS in the group with low CD39⁺ Treg cells is equal to 64.3% with an average survival of 26.3±3.5 months (95% CI 19.3-33.3 months) and did not differ from the group with a high frequency of CD39⁺ Treg cells, where it was 61.1% at average survival 25.9±3.7 months (95% CI 18.5-33.3 months, p=0.79) (figure). The analysis of patient survival, taking this factor into account, did not show statistically significant differences and when considering data depending on the stage of the disease (table 1).

In a previous study [7], it was found that the patients with CRC had an increased level of expression CD39 mRNA in blood leukocytes. In this regard, the work analyzed the three-year OS of patients with CRC, depending on the level of mRNA geneCD39. High and low CD39 mRNA expression was determined by the median. In the group of patients with a low level of CD39 expression mRNA OS was 69.2% with an average survival rate of 14.1±6.7 months (95% CI 1.0-27.3) versus an OS of 54.5% with an average survival rate of 16.7±6.2 months (95% CI 4.4-30.0) in the group with a high level of CD39 expression mRNA. No significant differences were found between these groups, p=0.47 (Figure). OS in groups with high and low CD39 expression mRNA depending on the stage of the disease also did not differ statistically (Table 2).

It has now been shown that in many malignant diseases, the accumulation of Treg cells in tumor tissue, and a subsequent decrease in the ratio of CD8+ cells to Treg cells correlates with a poor prognosis for patients [2]. However, for cancer types such as colorectal cancer and head and neck cancer, there is conflicting information about the prognostic role of Treg cells. Some studies show that increasing frequency Treg cells associated with positive dynamics of the disease. It is assumed that the duration of survival is influenced restriction by infiltrating FOXP3⁺ Treg cells of the inflammatory response caused by intestinal microbiota in the early stages of CRC [8]. 

Recent studies have found that extracellular adenosine actively accumulates in the tumor microenvironment, which provides immunosuppressive action and promoting carcinogenesis. The formation of adenosine is due to well-coordinated action ectonucleotidase CD39 and CD73, which degrade adenosine triphosphate released in condition of intratumoral hypoxia. High CD39/CD73 expression genes and their products are shown by many tumor cells and cells in the tumor microenvironment and has potential value as a prognostic factor. It is noteworthy that the prognostic role of expression in tumor CD73 is better studied and considered as an unfavorable factor in many oncological diseases [9]. The significance of CD39 expression in tumor tissue has been less studied. So, with head and neck cancer intratumoral expression of the CD39 molecule was an unfavorable factor in patient survival [10], and in pancreatic cancer it turned out to be a favorable factor of postoperative survival [11]. There are a number of studies devoted to determining the role of CD39⁺ Treg lymphocytes as a potential marker of the course of the disease and/or the effect of anticancer therapy, most often with the use of blockers immune checkpoints. So, Cai et al. showed that at hepatocellular carcinoma increased content of CD39⁺FOXP3⁺ cells were independent an unfavorable factor of OS in patients after radical tumor resection [12]. In melanoma, accumulation CD39⁺ Treg cells in peripheral blood are also an unfavorable factor in OS and relapse-free survival [13, 14]. It was noted that with head and neck cancer, an increase in the number of CD39⁺ Treg cells in the blood are associated with a short OS in patients receiving nivolumab [15].

Increased expression ectonucleotidase CD39 has also been demonstrated in CRC, both by tumor cells and blood cells in the periphery [16, 17]. In mouse models, CD39 has been shown to stimulate the growth and metastasis of colon cancer. In addition, the results in vitro studies have shown that the use of inhibitors activity of CD39 or blocking antibodies weakened tumor-induced suppression CD4⁺ and CD8+ T cell proliferation and increased the cytotoxic activity of CTL and NK cells [16]. It is noteworthy that along with this information Zhang et al. showed that the expression of CD39 in epithelial cells of human rectal carcinoma correlates with a favorable clinical outcome of the disease [17]. Despite the significant increased expression CD39 ectonucleotidase by peripheral lymphocytes, we did not reveal a significant relationship between high/low levels of CD39⁺ Treg cells and the survival of the examined patients. The level of CD39 mRNA was also not very informative in this regard. Apparently, further study is needed of adenosine-mediated immune suppression, activation of which occurs in colorectal cancer patients, in order to determine the characteristics of the functioning of cells both inside the tumor and among circulating lymphocytes to develop and improve the effectiveness of approaches in antitumor immunotherapy.   

 

Conclusion 

A dual role is currently confirmed for tumor-infiltrating lymphocytes in the tumor microenvironment. Some cells are capable of recognizing and eliminating neoplastic cells in the course of an antitumor immune response, while others, on the contrary, promote tumor growth and metastasis, creating conditions for evading transformed cells from immune surveillance. The changing molecular profile immunocytes is promising in the use of cell therapy and the search of biomarkers for prognosis of the course of the disease and/or its treatment. Currently, the prognostic value of Treg cells in the development of cancer remains controversial for many types of malignant neoplasms, in particular in colorectal cancer. Therefore, further research is needed to identify predictive significance of immune cells in the development of cancer and the development of effective ways to use them.

 

The research was financed from the federal budget for the fulfillment of the state assignment of the KarRC RAS, budget theme No. 0218-2019-0083

 

REFERENCES

  1.      Fedorov V.E., Podelyakin K.A. Epidemiological aspects of colorectal cancer. Medical Almanac. 2017; 4(49): 145-148 (in Russian).
  2.      Tanaka, A. Sakaguchi, S. Targeting Treg cells in cancer immunotherapy. Eur. J. Immunol. 2019; 49: 1140-1146.
  3.      Moesta, A.K., Li, X., Smyth, M.J. Targeting CD39 in cancer. Nat Rev Immunol. 2020.
  4.      Golovkin A.S., Asadullina I.A., Kudryavtsev I.V. Purinergic regulation of basic physiological and pathological processes. Medical Immunology (Russia)/Meditsinskaya Immunologiya. 2018; 20(4): 463-476 (in Russian).
  5.      Deaglio S., Dwyer K.M., Gao W., et al. Adenosine generation catalyzed by CD39 and CD73 expressed on regulatory T cells mediates immune suppression. J Exp Med. 2007; 204(6): 1257-1265.
  6.      Schuler P.J., Schilling B., Harasymczuk M. et al. Phenotypic and functional characteristics of CD4⁺CD39⁺ FOXP3⁺ and CD4⁺CD39⁺FOXP3neg T‐cell subsets in cancer patients. Eur. J. Immunol. 2012; 42: 1876-1885.
  7.      Zhulai G.А., Churov A.V., Oleinik E.K et al. Activation of СD4+CD39⁺ T cells in colorectal cancer. Bulletin of Russian state medical university. 2018; 3: 49-55
  8.      Saleh R., Elkord E. FOXP3⁺ T regulatory cells in cancer: Prognostic biomarkers and therapeutic targets. Cancer Lett. 2020; 490:174-185.
  9.      Wang R., Zhang Y., Lin X., Gao Y., Zhu Y. Prognostic value of CD73-adenosinergic pathway in solid tumor: A meta-analysis and systematic review. Oncotarget. 2017; 8(34):57327-57336.
  10.    Mandapathil M., Boduc M., Roessler M. et al. Ectonucleotidase CD39 expression in regional metastases in head and neck cancer. Acta Oto-Laryngologica. 2018; 138 (4): 428-432
  11.    Künzli B.M., Berberat P.O., Giese T., et al. Upregulation of CD39/NTPDases and P2 receptors in human pancreatic disease. Am J Physiol Gastrointest Liver Physiol. 2007; 292 (1): G223-G230.
  12.    Cai X.Y., Ni X.C., Yi Y. et al. Overexpression of CD39 in hepatocellular carcinoma is an independent indicator of poor outcome after radical resection. Medicine (Baltimore). 2016; 95(40): e4989.
  13.    Retseck J., Nasr A., Lin Y. et al. Long term impact of CTLA4 blockade immunotherapy on regulatory and effector immune responses in patients with melanoma. J Transl Med. 2018; 16 (1): 184.
  14.    Shevchenko A., Mathes C., Groth et al. Enhanced expression of CD39 and CD73 on T cells in the regulation of anti-tumor immune responses OncoImmunology. 2020; 9 (1): 1744946
  15.    Ottonello S., Genova C., Cossu I., et al. Association between response to nivolumab treatment and peripheral blood lymphocyte subsets in patients with non-small cell lung cancer. Front Immunol. 2020; 7 (11):125.
  16.    Hajizadeh F., Masjedi A., Heydarzedeh Asl. S. et al. Adenosine and adenosine receptors in colorectal cancer. Int Immunopharmacol. 2020; 87:106853.
  17.    Zhang B., Cheng B., Li F.S. et al. High expression of CD39/ENTPD1 in malignant epithelial cells of human rectal adenocarcinoma. Tumour Biol. 2015; 36(12): 9411-9.

 

Authors

 

Galina A. Zhulai

Institute of Biology of the Karelian Research Centre of the Russian Academy of Sciences

Junior Researcher, Immunology Group

11, Pushkinskaya str., Petrozavodsk, Republic of Karelia, Russian Federation, 185014

zhgali-111@yandex.ru

 

Evgeniya K. Oleinik

Institute of Biology of the Karelian Research Centre of the Russian Academy of Sciences

PhD, MD (Biology), Associate Professor, Main Research Associate, Head of Immunology Group

11, Pushkinskaya str., Petrozavodsk, Republic of Karelia, Russian Federation, 185014

ole@krc.karelia.ru

 

Mikhail I. Shibaev

Republic Oncology Dispensary

PhD (Medicine), Endoscopist, Surgical Oncologist

5, Lososinskoye Highway, Petrozavodsk, Republic of Karelia, Russian Federation, 185002

mshib@karelia.ru

 

Polina N. Semakova

Institute of Biology of the Karelian Research Centre of the Russian Academy of Sciences

Junior Researcher, Immunology Group

11, Pushkinskaya str., Petrozavodsk, Republic of Karelia, Russian Federation, 185014

k-polina13@mail.ru

 

Victor M. Oleinik

Institute of Biology of the Karelian Research Centre of the Russian Academy of Sciences

MD (Biology), Leading Research Associate, Immunology Group

11, Pushkinskaya str., Petrozavodsk, Republic of Karelia, Russian Federation, 185014

scigraph@yandex.ru

 
 
 

Authorization