Study On The Factors Influencing Tumor Progression In Tumor Microenvironment Through Tumor Model With Inconsistent Progression Rate

Background: Tumor is a great threat to human,and the tumor microenvironment supports the proliferation,invasion,metastasis and immune escape of tumor cells.At present,it is not clear which factors in tumor microenvironment promote the tumor progression.Hence,to investigate the factors in tumor microenvironment that can promote the tumor progression,and to explore the difference of immune microenvironment of tumor with inconsistent progression rate,is very meaningful and necessary.Here,we try to study the difference of cell components in tumor microenvironment using 4T1 and BALB/c.According to the results we select some related markers and discuss their gene expressions in HNSCC using the GEO dataset,and try to provide a theoretical basis for the further study of tumor microenvironment and immunotherapy,especially in HNSCC.Methods: Firstly,to establish the mouse tumor models with inconsistent tumor progression.Using the mouse breast cancer cell line 4T1 to do the subcutaneous injection on the back of the first batch of BALB/c mice.After sacrifice,using the tumor tissue to make the single cell suspension,and divide it into five.Take one for primary tumor cell culture,carefully remove the possible existing fibroblasts during passage.Then,using the cells from the fastest growing and slowest growing tumor to do the subcutaneous injection on the back of the second batch of BALB/c mice.By the same method making the single cell suspension and get the primary tumor cells from the second batch of mice.Secondly,to detect the tumor cell proliferation using MTS assay,Ki67 immunohistochemical staining,CFSE flow cytometry,to detect the glucose uptake using the 2-NBDG glucose uptake cell-based assay,to detect the cell migration ability using the scratch test,to detect the CD44+CD24-/low tumor stem cell proportion and immune checkpoint associated proteins using the flow cytometry.Thirdly,in terms of the single cell suspension to detect the lymphocyte and subtypes,myeloid cells,cancer associated fibroblasts and tumor stem cells,and immune checkpoint associated proteins PD-L1,MHC1.The cells and microenvironment of tumor with different progression were compared and analyzed,and to discuss the role of infiltrating immune cells in tumor.Lastly,based on the results of the detection of single cell suspension,the conventional dendritic cell related marker CD11 c,fibroblast related markers FSP1,VIM,α-SMA,Hsp47,immune checkpoint related markers MHC1,PD-L1,CTLA-4,PD-1,TIM-3,LAG-3 were selected to study the gene expression in HNSCC through the GEO dataset.Results: Firstly,we successfully establish the mouse tumor models with inconsistent tumor progression.The tumor cell derived from the fastest growing tumor of the first batch can induce the higher rate of tumor progression in the second batch of mice.Secondly,in terms of the tumor derived cells,no significant difference was found in the first batch.And,in the second batch we show that there was no significant difference about the cell proliferation,migration ability,tumor stem cell proportion or PD-L1 protein expression,however,in the faster tumor progression group the glucose absorptivity is higher,CD44+/CD24+ cell proportion lower,MHC1 positive cell proportion lower,MHC1 mean value lower than in the slower tumor progression group,the difference was statistically significant.Thirdly,in terms of the tumor single cell suspension,no significant difference was found in the first batch.And,in the second batch we show that in the faster tumor progression group the tumor stem cell proportion is higher,cancer associated fibroblasts proportion lower,conventional dendritic cell proportion lower than in the slower tumor progression group,the difference was statistically significant.Lastly,in terms of HNSCC,the gene expressions of fibroblast related markers FSP1,VIM,Hsp47 are consistent with the fibroblast proportion in tumor microenvironment.Conclusions: Firstly,the tumor model with inconsistent progression was successfully established through two batches of mice.Secondly,in terms of the tumor derived cells no significant difference was found about the tumor cell proliferation,however,there was significant difference about the glucose absorptivity,CD44+/CD24+ cell proportion and MHC1 expression level.Thirdly,in terms of the tumor microenvironment,there were significant difference about the tumor stem cell proportion,cancer associated fibroblasts proportion and conventional dendritic cell proportion between the faster and slower tumor progression groups.These factors may be related to the tumor progression.Lastly,the gene expressions of fibroblast related markers FSP1,VIM,Hsp47 in HNSCC are consistent with the fibroblast proportion in tumor microenvironment,which provides a theoretical basis for the further study of HNSCC.