Data Set Containing 414 Bladder Cancer Samples and 19 Normal Bladder Tissue Samples
In this study, a summary of available patient characteristics were presented in Table 1. Bladder cancer samples were classified by age at diagnosis, gender, tumor grade, stage status and metastasis pathologic.
Immune composition differs in Bladder Cancer Tissues and Normal Bladder Tissues
The gene expression matrix was obtained from the TCGA download datasets. Then CIBERSORT algorithm was applied to define the immune cell fraction proportion of BLCA tissues and normal bladder tissues. Figure 1 showed the immune cell fraction proportion in bladder cancer tissues and normal bladder tissues. Compared with normal bladder tissue, the B cells naïve, T cells CD4 memory resting, Mast cells resting fractions in bladder cancer tissue were generally lower, whereas the fractions of NK cells resting, Macrophages M0 and M1 cells were higher in bladder cancer tissue (table 2 and figure 1).
Correlation with 22 immune cell types in bladder cancer tissues
The degree of tumor immune cell infiltration is a crucial factor in cancer development. Interactions between tumor infiltrating leukocytes (TILs) existed in tumor microenvironment [15-17]. However, the TILs interaction in bladder cancer was rarely reported. To further elucidate the interdependence between the degree of immune cell fraction and immune cell composition in bladder cancer tissues，we calculated the coefficient of association of 22 immune cell types in bladder cancer tissues with the corrplot package. Our results showed that macrophages M2, which was much abundant in BLCA，negative correlated with numerous anti-tumor immunity cells, including NK resting，T cells CD8, T cells CD4 naive，mast cells activated, plasma cells，B cells naïve, B cells memory, T cells follicular helper and Dendritic cells activated. Tregs，a subpopulation of T cells that are immunosuppressive, negative correlated with Macrophages M1 cells（r=-0.21）and T cells CD4 memory activated (r=-0.24). T cells CD8 positively correlated with T cells CD4 memory activated（r=0.46）and Tfh (r=0.2), negative correlated with Macrophage M2 (r=-0.17), Macrophage M0 (r=-0.33) and T cells CD4 memory resting (r=-0.4). T cells CD4 memory activated positively correlated with macrophages M1 (r=0.33), T cells CD8 (r=0.46) and NK resting（r=0.29）, negative correlated with T cells CD4 memory resting（r=-0.36）and T cells regulatory（r=-0.24）(figure 2).
Composition of immune cells in new tumor
The new tumor event type and site had important prognostic value in BLCA and other cancers . To investigate the composition of immune cells in new tumor, we analysised the immune cells fraction change in new type or site tumor. Results showed that new Primary Tumor had a high fraction of T cells CD4 memory resting, T cells naïve, T cells follicular helper and had a lower fraction of plasma cells, macrophages M2, macrophages M0, compared to locoregional, distant metastasis type tumor.
When we analyzed the immune cells fraction in different types of bladder cancer tissues, It was found dendritic cells and resting macrophages M0 maybe correlate with tumor metastasis (figure3). compared to metastasized to other sites, when cancer cells metastasized to the lymph node only，macrophages M0 content has a great change，which implied high content of Macrophages M0 may be a risk factor for metastasis. When bladder cancer cell transfer to bone, fraction high content of the dendritic cells resting was found in the bladder cancer tissue.
Immune cells fraction change in high and low grade BLCA
To explore the relationship between tumor immune cell infiltration and the bladder cancer grade, we found T cells CD4 memory activated, Macrophages M0 and macrophages M1 fraction proportion increased in high grade BLCA tissues (fig.4a, d, e). However, regulatory T cell-based immunosuppressive effect was weak in high grade BLCA cancer tissue for Treg is much less abundant in high grade BLAC tissue than in low grade BLCA tissue (fig.4b). Monocytes decreased in high grade BLCA tissues (fig.4d). Dendritic cells activation was inhibited in high grade BLCA tissue for the activated Dendritic cells decreased significantly and Dendritic cells resting fraction increased significantly compared to in low grade BLCA tissue (fig.4f, g).
Tumor immune cell infiltration and radiation in bladder cancer
Radiotherapy was one of the most common treatments in the management of various types of cancer. Over half of people who were treated for cancer had accepted with radiotherapy [19, 20]. However，radiotherapy could contribute to the anti-immunogenic effect by recruiting TAMs and MDSCs in tumor microenvironment[21, 22]. The tumor immune cell infiltration in bladder cancer which accepted with radiotherapy remains unclear. Our work demonstrated that radiation dose could cause the tumor immune cell infiltration change (figure5). In low radiation dose group, Tregs increased. In high radiation dose group, macrophages M2 increased. When studying TIICs fraction proportion in BLCA patient with different radiotherapy sites, we found macrophages M2 had a high fraction proportion in local recurrence, T cells CD4 memory resting and Macrophages M0 had a high fraction proportion in distant recurrence, macrophages M2 and T cells CD8 fraction proportion increased in local recurrence.