Screening The Granulocytes With Cancer Killing Activity And Observation Their In Vitro Anti-tumor Ability

Granulocytes play important roles in inherent immunity, with high ability to preventthe invasion of microorganism but do not response effectively to cancer cells. Recently, aseries of explorations in mouse models have indicated that granulocytes from some micehave strong antineoplasmic activity, suggesting that granulocytes may serve as a new typeof effetor cell for anticancer immunotherapy. However, it remains a question either aboutthe existence of the granulocytes with cancer killer activity (CKA) in human poluation, orabout the therapeutic effect and mechanism of the CKA granulocytes to cancer cells. In thisstudy, we screened CKA granulocytes from21healthy human samples and identified boththe therapeutic effect and mechanisms of the screened CKA granulocytes. Our resuts mightshed light on the application of CKA granulocytes in anticancer immunotherapy.Objective：To screen CKA granulocytes from healthy human samples, identify thetherapeutic effect and work mechanism of the screened CKA to kill various cancer cells,and unfold the molecular characteristic of CKA granulocytes relative to granulocyteswithout cancer killer activity as well.Methods:1. The CKA granulocytes were screened out by dtetecting their capacity to inhibit thegrowth of A549cancer cells via CCK-8assay.2. Utilizing CCK-8assay, the therapeutic effect of screened CKA granulocytes to ona list of cancer cell lines and normal tissue cells lines were examined at variouseffector-target ratios, respectively.3. The ability of screened CKA granulocytes to induce apotosis of various cancercells were detected by analyse of flow cytometry. By means of three kinds of contactsbetween CKA granulocytes and cancer cells, including direct co-cultivation, transwellcultivation, and contact indirectly throuth the culture of cancer cells, the anticancer abilityof screened CKA granulocytes were examined, respectively. 4. The expression of TGFβ1in CKA granulocytes and granulocytes without cancerkiller activity were detected by realtime PCR and ELISA assay. Then, the anticancer abilitywas examined when used Ly-364947, a TGFβ1inhibitor to block the pathway of TGFβ1.Results1. Among21human samples, granulocytes derived from six samples (28.57%)remarkedly inhibit the growth of A549and the No.3samples showed highest anticancercapacity.2. The No.3CKA granulocytes could distinctly inhibit the growth of humanadenocarcinoma cell A549, human hepatocarcinoma cell HepG2, and human cervicalcarcinoma cells Hela in a dose dependent manner while left human normal myofibroblasticcell MRC-5unharmed.3. When co-culivation with various caner cells, the No.3CKA granulocytes couldeffectively induce cell apoptosis. By means of direct co-cultivation, CKA granulocytesshowed highest ability to kill cancer cell, followed by means of transwell cultivation, andcontact indirectly throuth the culture of cancer cells.4. Realtive to granulocytes without cancer killer activity, CKA granulocytes hadlower expression of TGFβ1both at mRNA level and at protein level. With the use ofLy-364947to block the TGFβ1pathway, the anticancer ability was improved in adose-dependent manner.Conclusion:1. Similar to that of mouse model, CKA granulocytes are existed in humanpopulations.2. To a certain extent, human CKA granulocyte can selectively kill cancer cells.3. Human CKA granulocyte can kill cancer cells by inducation of apoptosis and directcontact may be critical during the process of CKA granulocyte to kill cancer cells.4. The expression of TGFβ1is negative correlation with the anticancer capacity ofCKA granulocytes. It can be a useful way to enhance the therapeutic effect of CKAgarnulocytes by blocking its TGFβ1pathway.