Tumor-associated Macrophages (TAM) Research And Lymphoma Cell Interactions

Background:Lymphoma is a group of malignant tumors, deriving from lymph nodes or other lymphoid tissues. Based on the pathological type, lymphoma can be divided into Hodgkin’lymphomas (HLs) and non-Hodgkin’s lymphomas (NHLs). With the development of diagnosis and treatment, the prognosis of lymphoma and other hematological malignancies has been improved. But there are still some paitients who develop PD (progressive disease) during treatment or die due to recurrence. In recent years, many studies have shown tumor-associated macrophages (TAMs) play an important role in promoting disease progression by supporting cancer cell survival, proliferation and invasion. In many subtypes of lymphoma, such as classical type Hodgkin lymphoma (CHL), follicular lymphoma (FL), mucosa associated lymphoid tissue lymphoma and angioimmunoblastic T-cell lymphoma (AITL), high numbers of TAMs have been associated with advanced tumors and poor prognosis. Thus, discussing the transformation between human peripheral blood monocytes and TAMs, considering the interaction of TAMs and tumor cells, can provide an evidence to predict the prognosis and stratify the patients. Also, it can offer a new candidate for future therapeutic strategies.Objective:1. The transformation between peripheral blood monocytes and TAMs. Whether there are differences between the patients of lymphoma or multiple myeloma and their healthy counterparts. Whether there are correlations between TAMs and the patients’ clinical manifestation.2. The effect of TAMs on the proliferation of PTCL (peripheral T-cell lymphoma) tumor cells.Methods:1. Co-culturing THP-1and HUT-78, the markers of TAMs (CD68and CD163) were tested by Flow cytometry. The numbers of tumor cells were counted. These data were analyzed by using SPSS.20and prepared for the follow-up experiments of PBMCs.2. The peripheral blood mononuclear cells (PBMCs) of lymphoma and myeloma patients and their counterparts were obtained by Ficoll-Hypaque gradient centrifugation. The CD14-pos monocytes were purified by using CD14-pos immunomagnetic beads. 3. Co-culture with HUT-78cells (cell line of PTCL) without direct contact in a transwell apparatus.4. Count the numbers of tumor cells. Test the markers of TAMs (CD68and CD163) by Flow cytometry.5. Analyze these data by using SPSS.20.Results:1. After THP-1cells and HUT-78cells co-cultured at a density of1×105cells/ml for72hours, the expression of CD68, CD163and CD68/CD163increased statistically (p=0.00004, p=0.005181, p=0.001804). There was no statistical difference between the MFI (mean fluorescence intensity) of CD68-pos cells, CD163-pos cells and CD68/CD163-pos cells, comparing with the THP-1cultured alone.2. Co-cultured with THP-1cells at a density of1×105cells/ml, the number of HUT-78cells showed an increase at different time point. The increase had statistical significance (p=0.010989).3. Experimental group:After co-cultured with HUT-78cells, the proportion of CD68-pos cells, CD163-pos cells and CD68/CD163-pos cells increased significantly (p=0.000078, p=0.018981, p=0.012987). And the MFI of the CD163-pos cells and CD163-pos part of CD68/CD163-pos cells also showed an increase (p=0.049950, p=0.017414). Control group:There was no statistical significant difference on expression of the markers of TAMs and MFI after co-culture. There was also no statistical significant between the experimental group and control statistically.4. In the experimental group, the number of HUT-78cells after co-cultured was more than that cultured alone in each time point. The data of3patients had statistical significance (p=0.046581, p=0.041563, p=0.007960). Whereas there was no statistical difference in the control group.5. The variation of CD68/163-pos cells after co-culturing was closely associated with marrow infiltration.Conclusions:1. After co-cultured with PTCL tumor cells, THP-1cells can transform into TAMs. These TAMs can promote the proliferation of PTCL tumor cells.2. Co-cultured with PTCL tumor cells in vitro, the peripheral blood monocytes can transform into TAMs. TAMs transformed from peripheral blood monocytes can promote the proliferation of PTCL tumor cells in some extend.3. The monocytes from the patients of lymphoma and multiple myeloma are much easier transformation into TAMs than those from their healthy counterparts. 4. The variation of CD68/163-pos cells after co-culturing was closely associated with marrow infiltration.