Distribution, Regulation And Immunosuppressive Function Of Neutrophils In Gastric Cancer

Neutrophils play an important role in host immune responses against infection. Whenlocal tissues are infected, neutrophils extravasate from the blood and reach the sites ofinfection.Then they phagocytize and kill the invading pathogenic microorganisms and causetissue damages and inflammation at the same time. Both clinical trials and experimentalresults suggest that increased number of neutrophils is closely realted to tumor prognosis.Thus, the role of neutrophils in tumors has attracted more and more attention.Two types of tumor-associated neutrophils exist in the tumor microenvironment, N1phenotype (anti-tumor) and N2phenotype (pro-tumor). There are lots of differencesbetween the two tumor-associated neutrophils in induction, regulation, phenotype andfuction. N1phenotype tumor-associated neutrophils have similar phenotype and function toanti-infection neutrophils. They express more immunoactivating cytokines and chemokines,lower levels of immunosuppressing factors, thus exhibit stronger tumor-killing capacity. N2phenotype tumor-associated neutrophils secrete angiogenic factors and matrix degradationenzymes to suppress anti-tumor immune responses, and promote tumor growth.Gastric cancer (GC) is the fourth most common cancer and the second most commoncause of cancer death in the world with very poor prognosis. The types, numbers andlocations of immune cells infiltrating in the tumor microenvironment are closely associatedwith the prognosis of a variety of tumors. Neutrophils are found to infiltrate in many tumorsincluding GC. Current researches on neutrophils in GC mainly focus on the relationshipbetween neutrophils and the prognosis of GC. However, the regulation, function andmechanisims of neutrophils in GC still remain unknown.Objectives:1. To characterize the distribution of neutrophils in peripheral blood and differenttypes of gastric tissues from GC patients2. To investigate the regulation of neutrophils by gastric tumor microenviroment 3. To elucidate the immunosuppressive function of tumor culture supernatant(TCS)-conditioned neutrophilsMethods:1. The distribution of neutrophils in peripheral blood of GC patients and healthydonorsThe fresh blood of GC patients and healthy donors were collected and stained withanti-human CD45and anti-human CD66b antibodies. Flow cytometry (FCM) wasemployed to analyze the ratio of CD45+CD66b+neutrophils in CD45+leukocytes.2. The distribution of neutrophils in different types of gastric tissues from GC patientsDifferent types of gastric tissues (tumor, peritumor and non-tumor) from GC patientswere collected and stained with anti-human CD45and anti-human CD66b antibodies. FCMwas employed to analyze the ratio of CD45+CD66b+neutrophils in CD45+leukocytes.3. The infiltration of neutrophils in situ in different types of gastric tissues from GCpatientsDifferent types of gastric tissues (tumor, peritumor and non-tumor) from GC patientswere paraffin embedded and cut into slices. The infiltration of neutrophils in situ wasexamined by immunohistochemistry staining with mouse anti-human CD15antibody.4. The expression of chemokine receptors on neutrophils in peripheral blood of GCpatients and healthy donorsThe fresh blood of GC patients and healthy donors were collected and stained withanti-human CD45, anti-human CD66b, anti-human CXCR2and anti-human CXCR4antibodies. Flow cytometry (FCM) was employed to analyze the expression of chemokinereceptors CXCR2and CXCR4on neutrophils.5. The expression of chemokine receptors on neutrophils in different types of gastrictissues from GC patientsThe single cell suspension of different types of gastric tissues (tumor, peritumor andnon-tumor) from GC patients were collected and stained with anti-human CD45,anti-human CD66b, anti-human CXCR2and anti-human CXCR4antibodies. Theexpression of chemokine receptors CXCR2and CXCR4on neutrophils was analyzed byFCM.6. Longevity of tumor culture supernatant (TCS)-and non-tumor culture supernatant (NTCS)-conditioned neutrophilsNeutrophils isolated from peripheral blood of healthy donors were cultured with30%tumor culture supernatant (TCS),30%non-tumor culture supernatant (NTCS) andcompletely1640medium for12h, respectively. The survival of conditioned neutrophilswas analyzed by FCM with anti-human7-AAD antibody.7. The immunosuppressive function of TCS-and NTCS-conditioned neutrophilsNeutrophils isolated from peripheral blood of healthy donors were cultured with30%TCS and30%NTCS for12h, respectively. Then, bead-purified peripheral CD3+T cellswere labeled with CFSE and co-cultured with conditioned neutrophils for5d. Theproliferation of CD3+T cells was analyzed by FCM.Results:1. Fifty-three samples of GC patients and fifty peripheral blood samples of healthydonors were collected and the percentage of CD45+CD66b+neutrophils in CD45+leukocytes were analyzed by FCM. Statistical analysis showed that patients with GC had ahigher neutrophil percentage in peripheral blood than healthy donors (78.72%±1.19%vs66.09%±1.17%, P<0.0001).2. Different types of gastric tissues (tumor, peritumor and non-tumor) from fifty-threeGC patients were collected and the percentage of CD45+CD66b+neutrophils in CD45+leukocytes were analyzed by FCM.It showed that tumoral tissues contained a significantlyhigher percentage of neutrophils than peri-tumoral (6.81%±0.85%vs4.09%±0.62%,P<0.01)or non-tumoral tissues(6.81%±0.85%vs3.84%±0.35%, P<0.01).3. Different types of gastric tissues (tumor, peritumor and non-tumor) from GCpatients were paraffin embedded and cut into slices. The infiltration of neutrophils in situwas examined by immunohistochemistry staining and the results revealed that theinfiltration of neutrophils was higher in tumoral tissues than in peri-tumoral or non-tumoraltissues.4. Twenty-four fresh blood samples of GC patients and fifteen samples of healthydonors were collected and the expression of chemokine receptors CXCR2and CXCR4onneutrophils were analyzed by FCM. Statistical analysis showed that the percentage ofCXCR2-expressing neutrophils in peripheral blood of GC patients was less than that inhealthy donors （49.32%±4.27%vs97.80%±0.97%，P<0.01）while the percentage of CXCR4-expressing neutrophils was more in peripheral blood of GC patients than healthydonors (30.53%±3.20%vs9.91%±1.35%, P<0.05).5. Different types of gastric tissues (tumor, peritumor and non-tumor) fromtwenty-four GC patients were collected and the expression of chemokine receptors CXCR2and CXCR4on neutrophils was analyzed by FCM. It showed that neutrophils in gastrictissues mainily expressed chemokine receptors CXCR4and the percentage ofCXCR4-expressing neutrophils in tumoral tissues was higher than that in peritumoral(24.32%±5.84%vs21.77%±7.45%, P>0.05) and non-tumoral tissues (24.32%±5.84%vs21.86%±7.08%, P>0.05).6. Neutrophils isolated from peripheral blood of healthy donors were cultured with30%tumor culture supernatant (TCS),30%non-tumor culture supernatant (NTCS) andcompletely1640medium, respectively. The survival of conditioned neutrophils wasanalyzed by FCM with anti-human7-AAD antibody. FCM results showed that thepercentage of7-AAD+dead cells in TCS-conditioned neutrophils was lower than that ofNTCS-conditioned neutrophils(18.85%±2.35%vs32.45%±1.85%, P<0.05) while thepercentage of7-AAD+dead cells in NTCS-conditioned neutrophils was lower than that of1640-conditioned neutrophils(32.45%±1.85%vs74.3%±2.20%, P<0.01).7. Neutrophils isolated from peripheral blood of healthy donors were cultured with30%TCS and30%NTCS, respectively. Then, bead-purified peripheral CD3+T cells wereco-cultured with conditioned neutrophils. The proliferation of CD3+T cells was analyzedby FCM. The results of FCM showed that compared to NTCS-conditioned neutrophils, theproliferation of CD3+T cell was significantly suppressed by TCS-conditioned neutrophils(9.10%±1.24%vs5.99%±1.01%, P<0.01).Conclusions:1. Neutrophils were higher in peripheral blood of GC patients than healthy donors andaccumulated in tumoral tissues of GC patients.2. The percentage of CXCR2-expressing neutrophils was decreased and the percentageof CXCR4-expressing neutrophils was increased in peripheral blood of GC patients, whileneutrophils in gastric tumor microenviroment mainly expressed chemokine receptorCXCR4.3. TCS-conditioned neutrophils had a prolonged longevity. 4. TCS-conditioned neutrophils could inhibit proliferation of CD3+T cells.