CEACAM1Overexpression On Tongue Aquamous Cell Carcinoma Regulates Neutrophils Infiltration And Function,and Effects Its Malignant Phenotype And Prognosis

BankgroundOral squamous cell carcinoma (OSCC) is one of the ten most frequently diagnosed cancers in the world, and is the most common malignant tumor in head and neck. The tongue squamous cells carcinoma (TSCC) is the most common type in OSCC. The prognosis of TSCC is not well due to the relatively limited scope of surgery and the invasive, metastatic and recurrent biological characters of tumor. So it is particularly important to explore the mechanisms of malignant phenotype, and to find the effective blocking target for clinical therapy.Accumulating studies have suggested that inflammation was involved in cancer progression and was the seventh hallmark of cancer. Inflammatory cells include a great variety of cells. Among them, neutrophils represent the maximum fraction of total circulating leukocytes. Neutrophils are traditionally considered antitumoral in the context of their anti-bacterial functions. However, in a series of tumors (renal cell carcinoma, hepatocellular carcinoma, gastric adenocarcinoma etc), there were abundant neutrophils infiltration and the presence of intratumoral neutrophils was associated with poorer clinical outcomes and cancer-related survival. Some researches demonstrated that neutrophils play important roles in cancer biology. Neutrophils have been reported to be closely associated with tumor progression and tumor vasculature. So they were called tumor-associated neutrophils (TANs) by some researchers. Whereas, the research of neutrophils infiltration in TSCC tissues has rarely been reported. Further more, the mechanism research of neutroophils infiltration and its protumoral effects were relatively rare.Carcinoembryonic antigen-related cell adhesion molecule1(CEACAM1) belongs to the immunoglobulin superfamily and is expressed on a variety of epithelial, endothelial and hematopoietic cells. CEACAM1protein has multiple functions, such as regulating cell proliferation, angiogenesis, immunoreaction, tumor invasion and infection of microorganisms etc. A recent study showed that cytokine-induced CEACAM1expression on keratinocytes contributes to a prolonged lifespan of neutrophils. Markel G et al. has also demonstrated that CEACAM1from melanoma cells can inhibit cytotoxicity of NK cells in a class I MHC-independent way. The above studies implied that CEACAM1protein in cancerous tissues may have pivotal roles in regulating neutrophils’ infiltration and functions, and so as to influence the cancer biology. However, the research of the effect of CEACAM1from tumor cells on neutrophils are limited. In this study, we investigated neutrophils infiltration and CEACAM1expression in TSCC tissues using IHC, analyzed the relationship of them with clinical pathological features and cancer-related survival of patients, and explored the association between them.Section INeutrophils infiltration in tongue squamous cell carcinoma and its relationship with CEACAM1expression on tumor cellsObjectives1. To explore neutrophils infiltration in TSCC tissues and its clinical significance.2. To explore CEACAM1expression on TSCC tissues and its clinical significance.3. To probe the association between neutrophils infiltration and CEACAM1expression on tumor cells.Materials and methods1. Patients and specimens. Between2005and2010, a total of74patients underwent primary and curative resection for TSCC at the Affiliated Hospital of Qingdao University were selected for the study population and reviewed retrospectively, after obtaining written informed consent from74patients. All the74patients underwent neck dissection. All the diagnoses were made following the Pathology and Genetics of Head and Neck Tumors of World Health Organization Classification of Tumors. The patients had no treatment of radiotherapy, chemical therapy or other intervention before operation.2. Tissue Microarray and Immunohistochemistry Tow representative areas (away from necrotic and hemorrhagic materials) in each specimen were premarked and selected for tissue microarray. Immunohistochemistry was used to detect CD15+ neutrophils infiltration and CEACAM1expression on tumor cells. The association of neutrophils infiltration and CEACAM1expression with clinical pathological parameters and cancer-related survival was analysed. The correlation between neutrophils infiltration and CEACAM1expression was also analysed.Results1. CEACAM1expression on TSCC and its correlation with clinicopathologic parameters and patients’ survival.IHC results showed that CEACAM1protein expressed mainly on cytoplasm of tumor cells. Some expressed on the cell membrane. The expression of CEACAM1was obviously stronger in TSCC tissues than in peritumoral tissues (P<0.05). In lymph node metastasis group, CEACAM1expression was higher than in without LN metastasis group (P<0.05). Likewise, its expressiom was higher in stage Ⅲ/Ⅳ groups than in stage Ⅰ/Ⅱ groups (P<0.05). While there were no correlation between CEACAM1expression with patients’gender, age, grade, tumor size and recurrence. Univariant and multivariant survival analysis revealed that overexpression of CEACAM1was associated with shorter cancer-related survival, but not an independent prognostic factor.2. CD15+neutrophils infiltration in TSCC and its clinical significance. There were abundant neutrophils infiltration in TSCC. The density of neutrophils was higher in TSCC than in peritumoral tissues. Abundant neutrophils infiltration was associated with higher clinical stage, LN metastasis and tumor recurrence. While there was no correlation between neutrophils infiltration with patients’gender, age, grade and tumor size. Univariant and multivariant survival analysis revealed that abuandant neutrophils infiltration was associated with shorter cancer-related survival, and was an independent prognostic factor.3. Correlation of neutrophils infiltration with CEACAM1expression on tumor cells. Using Spearman’s rho coefficient test, we found that there was a positive correlation between CEACAM1expression and neutrophils infiltration.ConclusionsThere were overexpression of CEACAM1protein and abuandant neutrophils infiltration in TSCC tissues. Both of them was associated with poor clinical outcomes and shorter cancer-related survival. What’s more, abundant neutrophils infiltration was an independent prognostic factor. There was a positive correlation between CEACAM1expression and neutrophils infiltration in TSCC tissues. Section ⅡThe effect of CEACAMl overexpression on the biological behavior of the tongue squamous cell carcinoma Cal-27cellsObjective1. To detect the effect of CEACAM1overexpression on proliferation of Cal-27cells;2. To detect the effect of CEACAM1overexpression on invasion and migration of Cal-27cells;Materials and methods1.Cell culture:human tongue squamous cell carcinoma cell lines (Cal-27cells) were routinely cultured in DMEM containing10%fetal bovine serum, at37℃in a humidified air atmosphere containing5%carbon dioxide.2. Construction of CEACAM1-4L and CEACAM1-4S overexpression Lenti-virus vectors. The cDNA sequence of CEACAM1-4L and CEACAM1-4S was a kind gift from an American researcher. The vector construction and virus packaging was finished by Shanghai Genechem Company. The three kinds of Lenti-virus vector were named as CC1-4L-LV,CC1-4S-LV and V-LV respectively.3. Transfection of the three kinds of Lenti-virus into Cal-27cells. Use untrasfected cells cells as the blank control. After3or4day, using semiquantitative RT-PCR and Western blot to testify the transfection efficiency.4. Cell proliferation assay MTT was used to detect cell proliferation in CC1-4L-LV,CC1-4S-LV, V-LV and Blank groups. After digestion and counting, the cells in four groups were inoculated into a96-well plate. Then the growth curve was draw after1,3,5,7days to assess Tca8113cell proliferation activity.5. Wound healing assay The Cal-27cells in4groups were inoculated into a6-well plate. When the cells got90%confluence, made a linear scratch in each well with a tip. After24h, took photos in each well and analysed the healing length.6. Transwell invasion assay The Cal-27cells in4groups were added into the transwell inserts and were incubated for20hours. After incubation, the invaded cells were counted in five fields for each filter under a light microscope at400x magnification.Results1. Transfection of CEACAM1-4L and CEACAM1-4S overexpression Lenti-virus vector could enhance the expression level of CEACAM1-4L and CEACAM1-4S both in mRNA and protein level, using RT-PCR and Western blot analysis.2. MTT result showed that proliferative activity of the four Cal-27cells had no obvious difference in the firstday. However, CC1-4L-LV and CC1-4S-LV cells grew more slowly than V-LV and Blank group after3,5,7days (P<0.05). There was no significant difference between the V-LV and Blank group.3. Wound healing assay result showed that the scratch distance was obviously shorter in CC1-4L-LV group than V-LV and Blank groups (P<0.05) after24h. There was no significant difference between CC1-4S-LV and V-LV, Blank group.4. Transwell invasion assay results showed that the count of the invaded cells was obviously higher in CC1-4L-LV group than in V-LV and Blank group (P<0.05). There was no significant difference between CC1-4S-LV and V-LV, Blank group.Conclusion1. Transfection of CEACAM1-4L and CEACAM1-4S overexpression Lenti-virus vector could obviously enhance the expression level of CEACAM1-4L and CEACAM1-4S both in mRNA and protein level.2. Overexpression of CEACAM1-4L and CEACAM1-4S in Cal-27cells could inhibit cell proliferation.3. Overexpression of CEACAM1-4L in Cal-27cells could enhance the ability of cell migration and invasion. Section ⅢThe effect of CEACAM1overexpession in Cal-27on neutrophilsand its mechanismsObjective1. To explore the effect of CEACAM1overexpession in Cal-27on neutrophils infiltration;2. to probe the possible effect of CEACAM1overexpession in Cal-27on the function and type change of neutrophils. Materials and methods1.Cell culture:1) Human tongue squamous cell carcinoma cell line (Cal-27cells) were routinely cultured in DMEM containing10%fetal bovine serum, at37℃in a humidified air atmosphere containing5%carbon dioxide.2) Human pro-myeloid cell line (HL-60) were routinely suspended cultured in IMEM containing10%fetal bovine serum, at37℃in a humidified air atmosphere containing5%carbon dioxide.2. Quantitative real-time RT-PCR(qRT-PCR) was used to detect the mRNA expression of IL-8, CXCL-6and MCP-1.3. HL-60cells was induced by all trans retinoid acid for3days. Flow cytometry (FC) was used to detect the total CEACAM expression on HL-60cell surface. HL-60cells could be induced toward neutrophils differentiation, and was named as iHL-60.4. Direct and indirect coculture of4groups of Cal-27and iHL-60cells.(1) Supernatant of the4group cells was collected. After centrifugation, the supernatant was use to coculturwith iHL-60. After24h, the total RNA of iHL-60from4groups and untreated iHL-60was extracted. qRT-PCR was used to detect the mRNA expression of IL-8, MMP-9, VEGF and TNF-αin5groups of iHL-60.(2) Let iHL-60cells directed co-cultured with4groups of Cal-27cells in a ratio of1:5. After24h, MTT was used to detect the490nm absorbance values of Cal-27cells in different groups.5. qRT-PCR was used to detect the mRNA expression of TGF-β and IFN-β.6. Eliasa was used to detect the secretory TGF-βprotein in4group of Cal-27.7. IHC was used to exlore the TGF-β protein expression on TSCC. The correlation of TGF-p and CEACAM1expression was evaluated. Immunofluorescent double-labeling was used to observe the co-localization of TGF-p and CEACAM1.8. Add TGF-P neutrolizing antibodies to the above direct and indirect coculture systems, and redetect the mRNA expression of various factors in iHL-60and its tumor-killing ability in different groups.Results1. qRT-PCR result showed that overexpression of CEACAM1-4L could upregualte mRNA expression of IL-8and CXCL-6, while has no influence to MCP-1.2. FC result showed that after induction of all trans retinoid acid for3days, the CEACAM expression on HL-60cell surface was obviously upregualted, which was a marker of neutrophil differentiation.3. Results of direct and indirect coculture of Cal-27and iHL-60.(1) qRT-PCR result showed that co-culture with tumor cells could upregulate the mRAN expression of IL-8, MMP-9and VEGF and downregulate TNF-ain iHL-60cells.This trend was particularly obvious in CC1-4L-LV and CC1-4S-LV group,and the difference had statistical significane compared with V-LV and Blank group (P<0.05). After adding of TGF-β neutrolizing antibodies, the differences in different groups was obviously reduced, and had no statistical significance.(2) MTT result showed that co-culture with tumor cells from CC1-4L-LV and CC1-4S-LV group could weaken the tumor-killing ability of iHL-60compared with V-LV and Blank group (P<0.05). After adding of TGF-β neutrolizing antibodies, the tumor-killing ability of iHL-60was obviously enhanced in all groups. And the differences in various groups was obviously reduced, and had no statistical significance.4. Correlation of CEACAM1exprssion and TGF-β expression.(1) qRT-PCR and Elisa results showed that overexpression of both CEACAM1-4L and-4S could upregulate mRNA and protein expression of TGF-β.(2) IHC result showed that TGF-βexpression was higher in TSCC than in peritumoral tissues, and was positively related to CEACAM1expression. Immunofluorescent double-labeling result showed that there were co-localization of TGF-β and CEACAM1.Conclusion1. Overexpression of CEACAM1-4L could upregulate mRNA expression of IL-8and CXCL-6.2. In TSCC tissues, there was a positive relationship between CEACAM1and TGF-β expression, and had co-localization of them two. Overexpression of both CEACAM1-4L and CEACAM1-4S could upregulate TGF-β expression.3. Overexpression of CEACAM1in Cal-27cells could weaken the tumor-killing ability of iHL-60that Co-cultured with tumor cells, and can more easily to transform iHL-60cells from antitumoral type (N1) to protumoral type (N2).4. The effect of CE AC AMI overexpression in Cal-27on the type conversion of iHL-60was mainly through upregulating TGF-β.