Immune Cell Infiltration in Bladder Cancer CURRENT STATUS: POSTED

Background Bladder cancer is one of the most common malignant diseases with high recurrence rates worldwide. Although immunotherapy has been applied in bladder cancer for a long period of time, the tumor-infiltrating immune cells (TIICs) in bladder cancer has not been systematical investigated. Methods CIBERSORT, a versatile computational method for quantifying cell fractions from bulk tissue gene expression profiles (GEPs), was applied to calculate the TIICs fraction proportion in normal bladder tissues and in bladder cancer tissues with the TCGA data. Results compared to normal bladder tissue, B cells naïve, T cells CD4 memory resting and Mast cells resting fractions proportion decreased, and NK resting, macrophages M0 and macrophages M1 increased. In BLCAs tissue, pro-tumorigenic related immune cells were negatively correlated with anti-tumor immune cells. New tumor with locoregional had high fraction proportion of macrophages M0 and macrophages M1. Dendritic cells activated, Monocytes, macrophages M1, Tregs and T cells follicular helper significantly increased in low grade BLCAs. Tregs had a high proportion in BLCAs patients accepted low radiation dose. Conclusions This study indicates that macrophages M2 and Tregs could be the promising immunotherapy targets combined radiotherapy in BLCAs. The result provides valuable information to understand the immunity status in BLCAs. BLCA negative correlated with numerous anti-tumor immunity cells, including NK restingT cells CD8, T cells CD4 naivemast cells activated, plasma cellsB cells naïve, B cells memory, T cells follicular helper and Dendritic cells activated. Tregsa subpopulation of T cells that are immunosuppressive, negative correlated with Macrophages M1 cellsr=-0.21and T cells CD4 memory activated T cells CD8 with T cells CD4 memory activatedr=0.46and Tfh negative Macrophage Macrophage M0 T cells CD4 memory resting T cells CD4 memory activated positively correlated with macrophages T cells CD8 NK restingr=0.29, negative T cells CD4 memory T cells

LM22 signature matrix, which contains 547 genes expression information from 22 human leukocytes, including seven T-cell types, three macrophage types, three B cell types, natural killer (NK) cells, and myeloid subsets, was downloaded from the CIBERSORT web portal (https://cibersort.stanford.edu/index.php). The gene expression matrix with annotation was used to calculate the LM22 fractions by the algorithm method with 100 permutations. The inferred fractions of immune cell populations produced by CIBERSORT were summed up to one matrix and be interpreted for comparison.
Statistical analysis R software (version 3.5.3) and origin 8.0 were applied for all statistical analyses. LM22 correlation index was calculated with corrplot Package. Two-tailed Student t tests or One-way-ANOVA was performed for statistical analysis. The p values less than 0.05 were considered as statistically significant.

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][16][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

Composition of immune cells in new tumor
The new tumor event type and site had important prognostic value in BLCA and other cancers [18]. To

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.

Discussion
Tumor microenvironment acts as an important role in the development and progression of cancer [23,24]. In this work, we aim to clarify the immune microenvironment in BLCA. As far as we know, this is the first time to comprehensively assess the immune infiltration in BLCA based on the deconvolution of bulk gene expression data.
Human T cells and B cells could regulate acquired immune system by secreting cytokines, creating antibodies, etc. The anti-tumor ability was impaired in tumor microenvironment of T cells and B cells [25]. Our results showed that the fraction of B cells and T cells CD4 memory resting decreased in BLCA. It may mean B cells and T cells origination exhaustion in BLCA. This could contribute to the impaired anti-tumor activity of B cells and T cells. Furthermore, we observed that effecter T cells and

B cells, including T cells CD8/ T cells CD4 memory activated / T cells follicular helper/plasma cells
negatively correlated with pro-tumorigenic related immune cells, such as Tregs/macrophages M2, etc.
Macrophages, which could mediate the antigen presentation process, are major components in cancer immune infiltration [26]. It is also characterized by plasticity and heterogeneity. Macrophages M0 could differentiate into macrophages M1 or macrophages M2 under different cytokines stimulation.
The disturbance of macrophages M1/M2 balance could alter antitumor effects. In human bladder cancer, increased infiltration of tumor associated macrophages has been showed to be associated with the poor clinical prognosis [27]. Some researcher suggested that targeting macrophage may be a promising strategy for anticancer therapy [28,29]. Our analyses revealed t a high level of macrophages M0 and M1 existed in BLCA. Although macrophages M0 and macrophages M1 fraction increased in BLCA, compared to normal, the M1/M2 balance and M1/total macrophages balance did not shift.
Mast cells contain large granules, which could release the immunomodulatory and vasoactive molecules. Mast cells infiltration has been showed involved in several types of cancer physiological and pathological conditions [30]. Previous studied showed that mast cells influence BLCA patient outcomes [31,32]. Mast cells could enhance bladder cancer metastasis by increasing CCL2/CCR2/EMT/MMP9 signals axis. In this study, we discovered that total mast cells and mast cells resting decreased in BLCA. Mast cells negative correlated with several types of immune cells, such as

macrophages M1/T cells CD8/T cells CD4 memory activated/T cells follicular helper, etc. the result
consists with its pro-tumorigenic role in BLCA [31].
More interesting, our data firstly exposed the TIICs fraction in different types of new tumor event type and site. New primary tumor seemed had a T cells pool, T cells CD4 memory resting and T cells naïve, compared to locoregional and distant metastasis BLCA. It suggested that activation of T cells may be a useful strategy to BLCA patients with new primary tumor. Furthermore, TIICs fraction difference also existed in different type of metastasis BLCAs. These results could be helpful to improve BLCAs treatment.
TIICs have been reported in association with tumor grade [10,33]. Herein, the association between TIICs and tumor grade was also investigated. A high estimated Tregs fraction proportion was observed in low grade BLCA. A high estimated Dendritic cells resting fraction proportion was observed in high grade BLCA. It suggested that Regulatory T Cell-Based Immunosuppressive Effect may play an important role in low grade BLCA and inhibition the Tregs function in low grade BLCA may be a useful immunotherapy in treating low grade BLCA. Moreover, Dendritic cells resting activation may be another therapy strategy for high grade BLCA [34].
Radiotherapy is generally applied to the cancerous tumor for its cytotoxicity. However, radiotherapy could not only cause damage to cancer cells, but also leading to immunologic cells death indiscriminately [35,36].
We found TIICs fraction proportion are different in BLCA patients who accepted different radiation dose or accepted with different radiotherapy sites. Interestingly, results showed that Tregs are more sensitive to high radiation dose. It determined that combining immunotherapy by inhibiting of Tregs activity with radiotherapy provided an improved treatment for BLCA rather than single low radiation dose [37]. Our data further showed the TIICs fraction proportion in patients with different radiotherapy sites. Results implied that macrophages M2 may act as a vital role in local recurrence and in local recurrence and radiation therapy combined M2 inhibition may be more useful to these patients. In distant recurrence patients, T cells CD4 memory resting and Macrophages M0 had a high fraction proportion. Activation T cells CD4 memory resting and Macrophages M0 may be a promising strategy to improved radiotherapy effect to distant recurrence patients.

Conclusions
This is the first time to explore the TIICs in BLCA by using the accurately estimate tumor-infiltrating leukocytes analysis tool, CIBERSORT. The complex relationship between different kinds of TIICs was further revealed. Furthermore, we studied the TIICs fraction proportion in BLCAs patients in high or low grade, and the relationship between TIICs fraction proportion and radiotherapy. More importantly, we highlighted the promising immunotherapy value of Tregs, macrophages M2, etc. Our work advances the understanding of TIICs and provides valuable resources for research to improve  [38,39].

Competing interests
The authors declare that they have no competing interests.