Gastric cancer increases transmigratory potential of peripheral blood monocytes by upregulation of β1- and β2-integrins

Introduction

Macrophages represent predominant component of leukocyte infiltrate in many tumors, including gastric cancer [1, 2]. Due to their pleiotropic biological activities they act as orchestrators of immune response within tumor. They may play pivotal role in tumor development acting as tumor suppressors (M1 phenotype – classically activated cells) or tumor supporters (M2 phenotype – alternatively activated cells) [1, 3]. Unfortunately, majority of macrophages within tumor microenvironment acquire the latter phenotype and are referred to as tumor associated macrophages (TAMs). Macrophage polarization depends on the immune modulatory properties of tumor and stroma cells, that can interact locally within tumor tissue or affect peripheral precursors (monocytes) [4].
Recruitment of monocytes into tumor microenvironment is a hallmark of cancer development and progression [5, 6]. Notably, migration of peripheral blood cells to the side of tumor growth is controlled by different soluble factors, namely cytokines, chemokines, growth factors and metabolites [7, 8]. On the other hand, vascular and epithelial junctions represent a barrier for leucocyte migration [9]. Interestingly, monocyte transmigration through vessel wall is possible due to the presence of membrane adhesion molecules, including proteins belonging to 1 and 2-integrin family [10]. Leukocyte adhesion molecules interact with their ligands expressed on cytokine activated endothelium and allows monocytes to avoid forces exerted by the rapid blood flow in vasculature. Consequently, monocytes start to roll along apical endothelial surface until complete immobilization and transmigration [11]. Unfortunately, to date the molecular, humoral and cellular mechanisms that control monocyte trafficking in cancer are not fully elucidated. Therefore, here we aimed to evaluate whether systemic activation of peripheral blood monocytes observed in gastric cancer patients increases transmigratory potential of these cells.

Material and methods

Patients

15 normal donors and 40 gastric cancer patients, succsesive qualified to stomach resection at Chair of Surgical Oncology, Prof. F. Lukaszczyk Memorial Centre of Oncology in Bydgoszcz (Poland), were enrolled to the study (Table 1). None of the patients received chemotherapy and radiotherapy before or was subjected to surgery or blood transfusion for at least six month before blood acquisition. Furthermore, none of the patients showed any clinical or cellular sings on ongoing infection. Peripheral blood was collected upon the approval of the Bioethical Committee of the Collegium Medicum in Bydgoszcz. Each participant was familiarized with the objectives of the study and expressed written consent.

Flow cytometry

100 µl of fresh heparin-anticoagulated blood was stained with panel of mouse anti-human monoclonal antibodies (Table 2). Stain-then-lyse protocol was used as previously described [12]. Appropriate fluorescence-minus-one (FMO) and isotype controls were used for every staining for setting compensation and to assure correct gating. Samples were analyzed by using FACScan flow cytometer (Becton Dickinson) and at least 40 000 events were collected. Next, collected data were analyzed by using FlowJo version 7.6.1. (TreeStar). Used gating strategy is presented on Fig. 1.

Statistics

Statistical analysis was carried out using GraphPad Prism 6 software (GraphPad Software). U Mann-Whitney test was used. The differences were considered statistically significant at p < 0.05. The results are presented as median (interquartile range).

Results

Due to the constitutive expression of analyzed 2-integrins on the surface of monocytes we analyzed their expression levels. First we found significant increase of CD11a and CD11b expression in gastric cancer patients when compared to normal donors. Additionally, we found no differences in CD11c and CD18 expression level. Next, we found that gastric cancer increase frequencies of CD49d (4 subunit of VLA-4 integrin) and CD49f (6 subunit of VLA-6 integrin) expressing monocytes. Interestingly, we did not observed any differences in expression level of above mentioned molecules.

Discussion

Integrins are membrane glycoproteins controlling numerous physiological processes, including cell adhesion, chemotaxis, and phagocytosis [13]. 2-integrins are heterodimeric receptor proteins consisting of  and subunits linked by sulphide bridges. All receptors shear common 2-chain (CD18) and differ in  chain variants, namely L – CD11a; M – CD11b; X – CD11c. They are involved in direct adhesion of leukocytes to endothelial cells [14, 15]. Similarly, 1-integrins shear common 1 chain (CD29) and differ in  subunits. To date, at least 10 different chains were discovered including CD49d and CD49f expressed on monocytes. In contrast to 2-integrins, the latter represent a group of protein receptor responsible for cell interactions with extracellular matrix [13]. Here, we found that gastric cancer increase frequencies of both VLA-4 and VLA-6 expressing monocytes. Interestingly, Jin et al. showed that VLA-4 is playing leading role in monocyte transmigration to tumor microenvironment [16]. However, in some contrast to previous studies by Zhang et al., they found that this process occurred in the M2 (CD11b/CD18) integrin independent manner [16, 17]. It seems, that 2-integrins may play supportive role in monocyte transmigration process and observed upregulation of CD11a and CD11b expression is a consequence of monocyte activation and their inflammatory phenotype [18]. Notably, in our previous report we found that gastric cancer patients showed increased frequencies of inflammatory (activated) monocytes, namely intermediate (CD14++CD16+) and non-classical (CD14+CD16++) cells [12]. Interestingly, VLA-4 support not only transmigration of inflammatory monocytes but also the transition of macrophages from classically activated (M1) to pro-tumoral (M2) cells, by Rac2 activation [19]. Therefore, increased frequencies of VLA-4 expressing monocytes may be a consequence of their non-classical activation. Furthermore, VLA-6 was shown to support tumor growth and angiogenesis by promoting tumor infiltration of Tie-2 expressing monocytes and macrophages (TEMs) [20]. In summary, we showed here that peripheral blood monocytes from gastric cancer patients possess high transmigratory potential and are sensitive for non-classical polarization.

This study was partially supported by the grant for young scientists (CM UMK no. 7/WF/2013) from the Nicolaus Copernicus University; AE, MM are supported by funds from Leading National Scientific Center in Bialystok of Medical University of Bialystok.

References

1. Eljaszewicz A, Wiese M, Helmin-Basa A, et al. Collaborating with the enemy: function of macrophages in the development of neoplastic disease. Mediators Inflamm 2013; 2013: 831387.
2. Eljaszewicz A, Gackowska L, Kubiszewska I, et al. Macrophage activity in tumour development. Contemp Oncol (Pozn) 2010; 14: 1-6.
3. Solinas G, Germano G, Mantovani A, Allavena P. Tumor-associated macrophages (TAM) as major players of the cancer-related inflammation. J Leukoc Biol 2009; 86: 1065-73.
4. Mantovani A, Sozzani S, Locati M, et al. Macrophage polarization: tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes. Trends Immunol 2002; 23: 549-55.
5. Baggiolini M. Chemokines and leukocyte traffic. Nature 1998; 392: 565-8.
6. Schmid MC, Varner JA. Myeloid cell trafficking and tumor angiogenesis. Cancer Lett 2007; 250: 1-8.
7. Nagarsheth N, Wicha MS, Zou W. Chemokines in the cancer microenvironment and their relevance in cancer immunotherapy. Nat Rev Immunol 2017; 17: 559-72.
8. Ingersoll MA, Platt AM, Potteaux S, Randolph GJ. Monocyte trafficking in acute and chronic inflammation. Trends Immunol 2011; 32: 470-7.
9. Bazzoni G, Dejana E. Endothelial cell-to-cell junctions: molecular organization and role in vascular homeostasis. Physiol Rev 2004; 84: 869-901.
10. Vestweber D. How leukocytes cross the vascular endothelium. Nat Rev Immunol 2015; 15: 692-704.
11. Gerhardt T, Ley K. Monocyte trafficking across the vessel wall. Cardiovasc Res 2015; 107: 321-30.
12. Eljaszewicz A, Jankowski M, Gackowska L, et al. Gastric cancer increase the percentage of intermediate (CD14++CD16+) and nonclassical (CD14+CD16+) monocytes. Centr Eur J Immunol 2012; 37: 355-61.
13. Hynes RO. Integrins: versatility, modulation, and signaling in cell adhesion. Cell 1992; 69: 11-25.
14. Walzog B, Weinmann P, Jeblonski F, et al. A role for beta(2) integrins (CD11/CD18) in the regulation of cytokine gene expression of polymorphonuclear neutrophils during the inflammatory response. FASEB J 1999; 13: 1855-65.
15. Kourtzelis I, Mitroulis I, von Renesse J, et al. From leukocyte recruitment to resolution of inflammation: the cardinal role of integrins. J Leukoc Biol 2017; 102: 677-83.
16. Jin H, Su J, Garmy-Susini B, et al. Integrin alpha4beta1 promotes monocyte trafficking and angiogenesis in tumors. Cancer Res 2006; 66: 2146-52.
17. Zhang L, Yoshimura T, Graves DT. Antibody to Mac-1 or monocyte chemoattractant protein-1 inhibits monocyte recruitment and promotes tumor growth. J Immunol 1997; 158: 4855-61.
18. Chuluyan HE, Issekutz AC. VLA-4 integrin can mediate CD11/CD18-independent transendothelial migration of human monocytes. J Clin Invest 1993; 92: 2768-77.
19. Joshi S, Singh AR, Zulcic M, et al. Rac2 controls tumor growth, metastasis and M1-M2 macrophage differentiation in vivo. PLoS One 2014; 9: e95893.
20. Bouvard C, Segaoula Z, De Arcangelis A, et al. Tie2-dependent deletion of 6 integrin subunit in mice reduces tumor growth and angiogenesis. Int J Oncol 2014; 45: 2058-64.

Address for correspondence

Andrzej Eljaszewicz
Department of Regenerative Medicine and Immune Regulation
Medical University of Bialystok
Waszyngtona 13
15-269 Bialystok, Poland
e-mail: andrzej.eljaszewicz@umb.edu.pl

Marcin Moniuszko
Department of Regenerative Medicine and Immune Regulation
Medical University of Bialystok
Waszyngtona 13
15-269 Bialystok, Poland
e-mail: Marcin.Moniuszko@umb.edu.pl