Effect Of Hyperthermia On Biological Behavior Of Oral Squamous Cell Carcinoma By Regulating Macrophage Polarization

BACKGROUND AND OBJECTTumor-associated macrophages(TAMs)are the largest number of immune cells in tumor microenvironment(TME).Their phenotypes are mainly M2 macrophages,which play a tumor-promoting role.There is a large amount of TAM infiltration in oral squamous cell carcinoma(OSCC),which is closely related to the growth,metastasis and prognosis of OSCC.The phenotype of TAM is affected by TME,suggesting that the potential of antitumor immune response may be changed by regulating the polarization phenotype of TAM,and reversing its immunosuppression is the key to mobilize the autoimmune system to fight,slow down and even cure tumors.Hyperthermia(HT)is called green therapy because of its non-invasive,little side effect and ability to start systemic immunity.With more and more studies on heating technology,temperature measurement and thermobiology,tumor HT has entered a period of rapid development.The effect of HT on anti-tumor immune response has received great attention,but the effect of HT on TAM polarization in TME is rarely reported.In view of the importance of polarization of TAM to the occurrence and development of OSCC,it is necessary to deeply understand the effect of HT on TAM in OSCC immune microenvironment.Therefore,in this study,through the construction of macrophage model in vitro,molecular experiments were used to detect the effect of hyperthermia on the polarization phenotype of macrophages,and to further study the effect of macrophages on the malignant biological behavior of OSCC cells after hyperthermia.The purpose of this study is to study the anti-tumor mechanism of HT from the perspective of immunology,and to provide new therapeutic ideas for the clinical treatment of OSCC.METHODS1.Human leukemic mononuclear cell line THP-1 was induced into M0 macrophages,and then induced to differentiate into M1 and M2 macrophages by adding LPS,INF-γ and IL-4 respectively.Human tongue squamous cell carcinoma cell line CAL-27 and M0 macrophages were used to construct Transwell chamber co-culture system to induce OSCC-TAM model.The morphology of macrophages with different phenotypes was observed under inverted microscope,and the expression of macrophage polarization markers TNF-α,IL-1 β,TGF-β and IL-10 was detected by q RT-PCR,the expression of macrophage surface marker antigens CD86 and CD206 was detected by flow cytometry,and the expression of INOS and Arg-1 was detected by cellular immunofluorescence.2.q RT-PCR,flow cytometry and cellular immunofluorescence assay were used to detect the expression levels of M1 and M2 macrophages polarization markers in M1,M2 and TAM macrophages after heat treatment at 39.5 ℃ and 42 ℃ for 1 h.3.With the normal cultured CAL-27 cells as the control group,M1,M2 and TAM subtypes of macrophages were co-cultured with CAL-27 cells to construct indirect coculture system.The effects of different macrophage phenotypes on the proliferation,migration and invasion of CAL-27 cells were detected by CCK-8 method,cell cloning test,scratch test and Transwell test.4.Indirect co-culture systems of macrophages and CAL-27 cells with different phenotypes were constructed in vitro.with constant temperature culture at 37 ℃ as control group,CCK-8 method,cell cloning test,scratch test and Transwell test were used to detect the effects of macrophages on the proliferation,migration and invasion of CAL-27 cells after hyperthermia at 39.5 ℃ and 42 ℃.RESULTS1.M1,M2 and OSCC-TAM macrophage models were constructed in vitro.The expression levels of macrophage polarization markers were detected by q RT-PCR,flow cytometry and cell immunofluorescence assay.The results showed that M0 macrophages were successfully induced into M1 by LPS+IFN-γ,M2 by IL-4 stimulation,and OSCCTAM by co-culture of M0 macrophages with CAL-27 cells.Among them,the expression of M2 polarization markers is relatively high.2.The expression of macrophage polarization markers in M1,M2 and TAM macrophages at different temperatures(37 ℃,39.5 ℃ and 42 ℃)was detected by the above experimental methods.The results showed that the expression of polarization markers TNF-α,IL-1 β,CD86 and INOS of M1 macrophages in 42 ℃ group was higher than that of 37 ℃ group and 39.5 ℃ group,while the expression levels of polarization markers TGF-β,IL-10,CD206 and Arg-1 of M2 macrophages decreased.The above phenomena were consistent in M1,M2 and TAM macrophages.3.With the normal cultured CAL-27 cells as the control group,the results of CCK-8assay,cell clone test,scratch test and Transwell test showed that the proliferation and migration and invasion ability of CAL-27 cells were decreased in M1+CAL27 group,while the proliferation,migration and invasion ability of CAL-27 cells were enhanced in M2+CAL-27 group and TAM+CAL-27 group,and the proliferation,migration and invasion ability of CAL-27 cells in M2+CAL-27 group was stronger than that in TAM+CAL-27 group.4.The number of proliferation,migration and invasion of CAL-27 cells in the coculture system of M1+CAL-27 group,M2+CAL-27 group and TAM+CAL-27 group after42 ℃ hyperthermia was lower than that of 37 ℃ control group.CONCLUSIONS1.OSCC-TAM was successfully constructed after co-culture of macrophages and oral squamous cell carcinoma,and the main expression of TAM was M2.2.42 ℃ hyperthermia may regulate the phenotype of macrophages by promoting the polarization markers of M1 macrophages and inhibiting the expression of polarization markers of M2 macrophages,and make the macrophages transform from M2 to M1.3.M1 macrophages inhibit the proliferation,migration and invasion of CAL-27 cells,M2 macrophages and TAM promote the proliferation,migration and invasion of CAL-27 cells,and M2 macrophages have stronger ability of promoting proliferation,migration and invasion than TAM.4.42 ℃ hyperthermia may inhibit the proliferation,migration and invasion of CAL-27 cells by inducing macrophages to transform into M1 type.