Many studies have demonstrated that several cellular and molecular mechanisms can help tumors escape the body’s own natural immune response [21, 22]. The importance of immune regulation to cancer progression can be explained by the increase in the number of immunosuppressive factors and cells and the lack of immune-system–activating signals in tumor microenvironment. TLRs is one of the important receptors that activate immune cells on the surface of immune cells. Hence, it is worthwhile to explore TLRs role in tumor development. TLRs can up-regulate the expression of costimulatory molecules such as CD40, CD80, CD86 and cytokines such as IL-12, thus stimulating other immune cells such as T lymphocytes. [23, 24]. Meanwhile, TLR expression can lead to tumor growth by triggering on other cells including cancer cells . In this study, we are committed to explore the relationship between TLRs’ transcriptional expression and TCGA tumor characteristics, including TME, clinical significance, immune subtypes, stem cells and drug response. TLRs isotype has a significant effect on tumorigenesis. First, we analyzed the differential expression of 33 TCGA tumor types in 11057 samples (including 10327 tumor samples and 730 paracancerous samples). Through multidimensional analysis, we found that there were significant differences in TLRs expression levels among different tumor types. Moreover, survival analysis and Cox proportional hazard regression were also performed. In some types of cancers, we found that there was a statistically significant difference in survival between patients with high and low expression of TLRs, suggesting that TLRs may be a potential prognostic indicator for clinical application. Furthermore, we carried out the drug response analysis to explore the relationship between drug sensitivity and TLRs. This is expected to provide some help for anticancer therapies.
In our study, it was obvious that TLR2 was high expressed in most type of tumors. This result is similar to that of most previous studies [26–28]. Gergen et al.  reported TLR2 activation induced proliferation of lung adenocarcinoma cells by activating NF-ĸB. As a special medium between lung cancer cells and mesenchymal stem cells in tumor microenvironment, TLR2 promotes crosstalk and ultimately promotes the change of tumor supporting phenotype of mesenchymal cells . Furthermore, the expression of TLR2 protein has been confirmed to be up regulated in colon cancer, so the high expression of TLR2 is significantly correlated with the low overall survival rate of patients with colon cancer [31, 32]. This means the TLR2 signaling pathway may be an important potential therapeutic target in tumors. In addition, we found that TLR9 was hardly expressed compared to other TLR genes, which lead to TLR9 showed less correlation with both immune scores and stromal scores. But several studies have reported TLR9 were associated with the development of cancer, such as gynecologic cancer [33, 34]. The activation of TLR9 on DC and pDC promotes the secretion of a large amount of type I IFN, which has direct (tumor cell inhibitory effect) and indirect (antitumor immune responses) effects on cancer cells, and is most obvious in the early stage of anti-tumor immune response .
Thorsson et al.  identified the immune landscape of cancer into C1-C6 immune subtypes. In our study, we classified tumor samples by representative immune signatures and detected the RNA-seq level of TLR 1-10 from C1 to C6. Interestingly, all the TLRs were related with immune subtype of tumor samples that all of them were differentially expressed in differential immune subtype samples. Tumor microenvironment (TME) is closely related to immune functions such as extracellular matrix, tumor vascular system and tumor cells, and has an important impact on treatment response and clinical prognosis . TLRs are expressed in TME . Our study further confirmed this point, we extracted the fractions of stromal cells and immune cells in tumor samples of 33 TCGA cancer types by calculating stromal scores, immune scores, and ESTIMATE scores. Expression of TLRs were positively related with immune scores and stromal scores in almost all types of tumor. On the one hand, TLRs is expressed in programmed cell death induced by TME, on the other hand, it releases cytokines and chemokines in the tumor environment, and recruits immune cells to further release pro-inflammatory cytokines, angiogenic factors and growth factors, such as TGF β, IL-8, CXCR4, ICAM-1 and VEGF,. It can repair the anti-tumor function and apoptosis response of antigen-presenting cells (APCs) and effector T cells [39, 40]. TLRs signaling pathway plays an essential role in controlling tumor progression, metastasis, recurrence and chemotherapy tolerance by inappropriate immune enhancement and anti-tumor immunity .
Stemness was applied to distinguish the stem cell-like characteristics of the tumor, such as self-renewal and dedifferentiation . Two types of stemness indices were accessed including DNAss and RNAss . We found that expression of TLRs were negatively correlated with RNAss in nearly almost type of tumors except KIRC, and also negatively correlated with DNAss in many types of tumors except ACC, CHOL, KIRC, LAML, LGG, TGCT, THCA, THYM, and UVM. TLR3 activation facilitated the expression of stemness-associated genes, including OCT3/4, NANOG, and SOX2 . TLR4 expression in HCC were associated with increased stem-like properties . NF-𝜅B, activated by TLR signaling, was closely bound up with the proliferation, invasion and tumorigenesis, invasion, and tumorigenesis of tumor stem cells . Meanwhile, our study also found that the transcriptional expression level of TLR7 and TLR9 was associated with drug responses. Among them, the expression of TLR9 was significant positively correlated with the drug sensitivity of Fluphenazine, Alectinib, Carmustine, and 7−Hydroxystaurosporine. There was a significant positive correlation between TLR7 and the drug sensitivity of Alectinib. These results have clinical relevance for guiding selection of antitumor therapies.
Finally, we explored the relationship between TLRs and KIRC. TLR2, TLR3, TLR4, TLR10 were significantly differentially expressed between Stage I to Stage IV. TLR1, TLR3, TLR4, TLR7, TLR8, TLR10 were significantly differential expressed between C1 to C6 immune subtype. All the TLRs were positively correlated with the immune scores, stromal scores, and estimate scores. Morikawa et al.  reported that TLR3 is overexpressed in KIRC, which suggested TLR3 pathway may be a novel therapeutic target in KIRC. Moreover, the expression of TLR9 is an independent prognostic marker of KIRC, and the loss of TLR9 expression is related to the poor prognosis of KIRC . Our results provide guidance for further exploration of the role of TLR in KIRC.
Although this is the first study to multidimensionally analyze TLRs in pan-cancer, it still has some limitations. First, our results have not been verified by other independent databases, and thus, it is necessary to validate the conclusions by our own data and other public database in the future. Second, we have not explored the underlying mechanisms behind bioinformatics analysis through molecular and animal experiments. Finally, we studied the relationship between the TLRs family and a variety of combinatorial data. However, biometric correlation may not directly clarify the mechanisms of interaction and regulation Thus, further studies need to be carried to verify these potential mechanisms via laboratory molecular experiments. Besides, in order to find the potential of TLRs and its coactivators as tumor targets, further investigation is needed.