INTERNATIONAL JOURNAL OF IMMUNOLOGY AND IMMUNOBIOLOGY (ISSN:2631-6706)

Background: Some chemokines in tumour microenvironment play an important role in the development and behaviour of many solid tumours. Among all chemokines, CXCL12/ CXCR4 axis is the most studied in endometrial cancer. Physiologically, CXCR4–CXCL12 axis and CXCR7 has been shown to be involved in tumour progression and metastasis. In breast cancer and melanoma cells CXCL8 expression reflects increased invasiveness and angiogenesis, while CXCL11 seems inhibits neo vascularization. In this study we compared CXCL12 with CXCR4, CXCR7, CXCL8 and CXCL11 mRNA expression in endometrial cancer tissue (EC) versus normal endometrial counterparts (NE).

Materials and Methods: Fresh samples of EC tissue and NE were extracted from 15 patients with FIGO stage I–III undergoing primary surgery. Some of the tissue was sent for histology and part of it was treated with RNA later and stored at -80°C in 11 patients RNA was reverse-transcribed, treated with DNase I, quantified and reverse–transcribed into cDNA using random primers. A real-time quantitative PCR determined the relative cDNA levels of genes in each sample.

Results: We collected tissue samples from EC and NE in 15 patients with endometrial cancer FIGO stage I-IIIC. Four patients dropped out. A total of 11 samples were analysed. In the comparison between NE versus EC we found: down-regulation of CXCL12 mRNA in 91% of samples (p<.01), down regulation of CXCR4 mRNA and up-regulation of CXCL8 mRNA in 64% of samples (p=NS), down-regulation of CXCR7 mRNA in 91% of samples (p<.01), overexpression of CXCL11 mRNA in 54% of samples (p=NS). Among all chemokines analysed, expression of CXCL12 was directly correlated only with the expression of CXCR7 (p<.01). CXCL8 mRNA expression was indirectly correlated with CXCR7 (p=0.04). 

Conclusion: We might deduce that CXCL12 is down-regulated in type 1 endometrial histotypes. A down-regulation of CXCL12, being significantly directly correlated with expressions of CXCR7, might express a low tendency of these endometrial cancer cells towards lymph-nodes dissemination, as expected due the low risk histotype (type I). We will expect from a further analysis on a greater sample size the definitive and more significant results. Understanding the relationship in the tumour microenvironment could represent a very attractive therapeutic target. 

Type I endometrial cancer; Tumoral microenvironment; CXCL8; CXCL11; CXCL12; CXCR7; CXCR4

Tumour microenvironment plays an important role in the development and behaviour of solid tumours and some chemokines have been found fundamental mediators of this process. Among all chemokines, CXCL12/CXCR4 axis is the most studied in endometrial cancer, being expressed constitutively in many normal tissues (liver, lung, lymph nodes, adrenal glands and bone marrow) [1]. In addition, physiologically, CXCR4–CXCL12 axis has been shown to be involved in the migration of a subset of embryonic cells implicated in the development of the central nervous system, bone marrow and heart [2,3]. The migration cues employed during embryo genesis, maybe, could be re-established during tumour progression and used by cancer cells in the metastasis process explaining the reason why some tumours prefer particular sites for secondary implants (e.g. brain, bones and no other tissues). In particular, CXCL12–CXCR4 axis and CXCR7 interaction plays a critical role in migration of tumour cells into metastatic sites in many cancers and in particular mediates lymph-nodal meta statisation [1,4-12]. It has been reported that CXCL12 can bind to CXCR7 which is expressed in endometrial stromal cells [13]. Another chemokine expression, CXCL8, in breast cancer and melanoma cells has been supposed to reflect increased invasiveness. Conditioned media from such tumour cells with increased expression of CXCL8 also promoted angiogenesis in vivo when injected subcutaneously into nude mice [14]. CXCL8 is mitogenic for melanoma cells as indicated by the decrease of melanoma cell proliferation by neutralizing monoclonal antibodies against CXCL8 and by decreasing CXCL8 expression by transfection of melanoma cells with anti-sense oligonucleotides [15]. Another chemokine, CXCL11, stimulates metalloproteinase 2 and 9 secretion which is involved in tumour progression, invasion and metastasis in advance stages in multiple myeloma [16]. On another study, CXCL11 has been shown to inhibit angiogenesis in a CXCR3-dependent manner in a murine model of pulmonary fibrosis [17].

This preliminary study represents the first step of a larger research protocol, designed to select the most relevant chemokines, cytokines or growth factors to be analysed in each subpopulation of endometrial tumoral microenvironment inflammatory cells (granulocytes, macrophages, natural killer, fibroblasts, tumoral cells). Therefore, we compared CXCL12 with CXCR4, CXCR7, CXCL8 and CXCL11 mRNA gene expression in endometrial cancer tissue versus normal endometrial counterparts. 

Immediately after surgery, fresh samples of endometrial cancer (EC) and their normal endometrial counterpart (NE) were obtained from patients submitted to primary surgery for endometrial cancer at Humanitas Clinical Institute in Milan (Italy). Parts of the samples were used for the histologic diagnosis and other parts were immediately treated with RNA later (Ambion) for 24-36 h at 4°C, and subsequently dried and stored at -80°C.

The study was approved by the Ethical Committee of Humanitas Research Institute and informed, written consent was obtained for all patients. All the clinical and surgical data were recorded on a database. The total RNA was isolated both from endometrial cancer and normal endometrial specimen using TRI Reagent (Ambion). RNA was quantified by Nanodrop spectrophotometer ND-1000 and its quality was examined by 1.5% agarose gel electrophoresis.

According to the manufacturer’s instructions, 1 mg of total RNA was reverse-transcribed using the High-Capacity cDNA Archive kit (Applied Biosystems), treated with DNase I, quantified and reverse-transcribed into cDNA using random primers. A real-time quantitative polymerase chain reaction, using Syber Green I (Applied Biosystem) as detection dye, was used to determine the relative cDNA levels of genes in each sample. The amplification protocol was used as following: 2 min at 50°C to activate uracil-DNA glycosylase, 10 min at 94.5°C (activation), 40 cycles of denaturation at 97°C for 30 s and annealing and extension at 59.7°C for 1 min. The relative amount of each target gene mRNA to the housekeeping gene (18S) was calculated as 2^(-DDCt), where DCt=Ct gene -Ct housekeeping gene. The fold-change of each target gene mRNA to the corresponding normal tissue was calculated as 2^(-DDCt), where DDCt=DCt target gene in tumour tissue –DCt target gene in normal tissue. The threshold cycle Ct was automatically given by the SDS2.2 software package (Applied Biosystems). For preliminary results we analysed some genes CXCL12, CXCR4, CXCL11, CXCR7 and CXCL8.

Statistical analysis

Statistical significance of proportion of samples with was determined by t -test and considered significant at a p value of <0.05.

We collected tissue samples from endometrial cancer (EC) and from normal corresponding endometrium (NE) in 15 patients with endometrial cancer FIGO stage I-IIIC. All patients were submitted to primary laparoscopic total hysterectomy and bilateral salpingooophorectomy with pelvic lymphadenectomy. Four patients dropped out from the study: two because the endometrial sample was damaged during the storage, making them impossible to process, and two because no residual tumour was found in the samples, despite an initial histologic diagnosis by endometrial biopsy. A total of 11 samples were treated and analysed. Tables 1 and 2 are described the patient’s clinical characteristics and histology characteristics of each sample. In the comparison between normal endometrium versus endometrial cancer, we found CXCL12 gene mRNA downregulation in 10/11 (91%) samples (Figure 1, p<.01), CXCR4 mRNA gene expression down-regulation and CXCL8 mRNA gene expression up-regulation in 7/11 (64%) samples (Figure 2 & 3, p=NS in both cases), CXCR7 mRNA gene expression down-regulation in 10/11 (91%) samples (Figure 4, p<.01) and CXCL11 mRNA gene expression up-regulation in 6/11 (54%) samples (Figure 5, p=NS).

Among all chemokines analysed, CXCL12 mRNA gene expression was significantly directly correlated only with CXCR7 mRNA gene expression in 11/11 samples (100%, p<.01). CXCL8 mRNA gene expression was significantly indirectly correlated with CXCR7 in 7/11 (63%, p=0.04). 

In this study, we analysed the correlation between CXCL12 mRNA gene expression and other chemokines mRNA gene expression less investigated in endometrial cancer. Among 11 samples, 11 were stage IA (according FIGO classification) and 9/11 samples were type I endometrial cancer, therefore globally a study population with a low risk behaviour.

Considering CXCL12 as a chemokine related to invasiveness and tumour growth, unexpectedly we found it down-regulated in almost all endometrial cancer tissues (10/11 samples) in comparison with the normal tissue counterpart. We observed that the only one sample in which CXCL12 was up-regulated was a type II endometrial cancer (clear cell histyotype, grade 3 even if stage IA) while all the others samples were type I endometrial cancer and the only one patient with type II endometrial cancer had a clear cell histotype mixed with adenocarcinoma and it was grade 2. The deduction is that CXCL12 might be over-expressed only in type 2 high risk endometrial histotypes.

The expression of CXCL12 in our study, not always directly correlated to the expression of CXCR4, suggests that may be other mediators are implicated in the CXCL12/CXCR4 axis involved in tumour progression and metastases or can be the expression of a particular trend of some tumoral cell to metastasise towards particular targeted organs, like in other kind of tumour.

CXCR7 is a chemokine mostly implied in lymph nodal metastases. A down-regulation of CXCL12, being significantly directly correlated with expressions of CXCR7 might express a low tendency of these endometrial cancer cells towards lymph-nodal spread, as expected due to the prevalence of this low risk histotype (type I) in the study population: the only case in which CXCR7 was over-expressed in a stage I endometrial cancer was a Clear Cell grade 3 (type II) histotype.

Regarding CXCL8 mRNA gene expression, a chemokine related to angioinvasiveness, it was significantly indirectly related with CXCR7 mRNA gene expression, a chemokine related to lymph nodal metastatisation: this aspect might express a trend of endometrial cancer to spread preferably only in one of these ways.

This study is ongoing and we will expect from a further analysis on a greater sample size the definitive results. Moreover, we will pass to the second step of our research, trying to identify in which inflammatory cells subpopulation of the endometrial cancer tumoral microenvironment these chemokines are over or under expressed. Understanding the relationship in the tumour microenvironment could represent a very attractive therapeutic target. 

The authors declare no conflicts of Interest.

1. Luker KE, Luker GD. Functions of CXCL12 and CXCR4 in breastcancer. Cancer Lett. 2006 Jul;238(1):30-41.
2. Nagasawa T, Hirota S, Tachibana K, Takakura N, Nishikawa S, et al.Defects of B-cell lymphopoiesis and bone-marrow myelopoiesisin mice lacking the CXC chemokine PBSF/SDF-1. Nature. 1996Aug;382(6592):635-638.
3. Ma Q, Jones D, Borghesani PR, Segal RA, Nagasawa T, et al.Impaired B-lymphopoiesis, myelopoiesis, and derailed cerebellarneuron migration in CXCR4- and SDF-1-deficient mice. Proc NatlAcad Sci U S A. 1998 Aug;95(16):9448-9453.
4. Scotton CJ, Wilson JL, Milliken D, Stamp G, Balkwill FR. Epithelialcancer cell migration: a role for chemokine receptors? CancerRes. 2001 Jul;61(13):4961-4965.
5. Phillips RJ, Burdick MD, Lutz M, Belperio JA, Keane MP, et al.The stromal derived factor-1/CXCL12–CXC chemokine receptor4 biological axis in non-small cell lung cancer metastases. Am JRespir Crit Care Med. 2003 Jun;167(12):1676-1686.
6. Ding Y, Shimada Y, Maeda M, Kawabe A, Kaganoi J, et al. Associationof CC chemokine receptor 7 with lymph node metastasis ofesophageal squamous cell carcinoma. Clin Cancer Res. 2003Aug;9(9):3406-3412.
7. Schimanski CC, Schwald S, Simiantonaki N, Jayasinghe C, GönnerU, et al. Effect of chemokine receptors CXCR4 and CCR7 on themetastatic behavior of human colorectal cancer. Clin Cancer Res.2005 Mar;11(5):1743-1750.
8. Schimanski CC, Bahre R, Gockel I, Müller A, Frerichs K, et al.Dissemination of hepatocellular carcinoma is mediated viachemokine receptor CXCR4. Br J Cancer. 2006 Jul;95(2):210-217.
9. Chen GS, Yu HS, Lan CC, Chow KC, Lin TY, et al. CXC chemokinereceptor CXCR4 expression enhances tumorigenesis andangiogenesis of basal cell carcinoma. Br J Dermatol. 2006May;154(5):910-918.
10. Wald O, Izhar U, Amir G, Avniel S, Bar-Shavit Y, et al.CD4+CXCR4highCD69+ T cells accumulate in lungadenocarcinoma. J Immunol. 2006 Nov;177(10):6983-6990.
11. Balabanian K, Lagane B, Infantino S, Chow KY, Harriague J, etal. The chemokine SDF-1/CXCL12 binds to and signals throughthe orphan receptor RDC1 in T lymphocytes. J Biol Chem. 2005Oct;280(42):35760-35766.
12. Bleul CC, Farzan M, Choe H, Parolin C, Clark-Lewis I, et al. Thelymphocyte chemoattractant SDF-1 is a ligand for LESTR/fusinand blocks HIV-1 entry. Nature. 1996 Aug;382(6594):829-833.
13. Kodama J, Hasengaowa, Seki N, Kusumoto T, Hiramatsu Y.Expression of the CXCR4 and CCR7 chemokine receptors in humanendometrial cancer. Eur J Gynaecol Oncol. 2007;28(5):370-375.
14. Lin Y, Huang R, Chen L, Li S, Shi Q, et al. Identification ofinterleukin-8 as estrogen receptor regulated factor involved inbreast cancer invasion and angiogenesis by protein arrays. Int JCancer. 2004 Apr;109(4):507-515.
15. Schadendorf D, Möller A, Algermissen B, Worm M, SticherlingM, et al. IL-8 produced by human malignant melanoma cells invitro is an essential autocrine growth factor. J Immunol. 1993Sep;151(5):2667-2675.
16. Pellegrino A, Ria R, Di Pietro G, Cirulli T, Surico G, et al. Bonemarrow endothelial cells in multiple myeloma secrete CXCchemokines that mediate interactions with plasma cells. Br JHaematol. 2005 Apr;129(2):248-256.
17. Burdick MD, Murray LA, Keane MP, Xue YY, Zisman DA, et al.CXCL11 attenuates bleomycin- induced pulmonary fibrosis viainhibition of vascular remodeling. Am J Respir Crit Care Med.2005 Feb;171(3):261-268.