Toll-like receptor 3 is a potential prognosis marker and associated with immune infiltration in stomach adenocarcinoma

BACKGROUND: Toll-like receptors participate in various biological mechanisms, mainly including the immune response and inflammatory response. Nevertheless, the role of TLRs in STAD remains unclear. OBJECTIVE: We aimed to explore the expression, prognosis performance of TLRs in STAD and their relationship with immune infiltration. METHODS: Student’s t-test was used to evaluate the expression of TLRs between STAD tissues and normal tissues. Kaplan-Meier method was applied to explored the prognosis value of TLRs in STAD. And qRT-PCR validated their expression and prognosis value. Spearman’s correlation analysis and Wilcoxon rank-sum test were used to assess the association between TLRs and immune infiltration in STAD. RESULTS: The mRNA level of TLR3 was downregulated in STAD. We summarized genetic mutations and CNV alteration of TLRs in STAD cohort. Prognosis analysis revealed that STAD patients with high TLR3 expression showed better prognosis in OS, FP and PPS. The result of qRT-PCR suggested that TLR3 expression was decreased in STAD tissues and STAD patients with high TLR3 mRNA level had a better OS. Univariate and multivariate cox regression analysis suggested TLR3 expression and clinical stage as independent factors affecting STAD patients’ prognosis. A positive association existed between TLR3 expression and the abundance of immune cells and the expression of various immune biomarkers. Furthermore, key targets related to TLR3 were identified in STAD, mainly including MIR-129 (GCAAAAA), PLK1, and V$IRF1_01. CONCLUSIONS: Our result demonstrated TLR3 as a prognosis marker and associated with immune infiltration in STAD.

TLR3 Serves as a Prognostic Biomarker and Associates with Immune Infiltration in the Renal Clear Cell Carcinoma Microenvironment

Background. Clear cell renal cancer (KIRC) is one of the most common cancers globally, with a poor prognosis. TLRs play a vital role in anticancer immunity and the regulation of the biological progress of tumour cells. However, the precise role of TLRs in KIRC is still ambiguous. Methods. Various bioinformatics analysis and clinical validation of tissues were performed to evaluate the prognostic value of TLRs and their correlation with immune infiltration in KIRC. Results. The expression of TLR2/3/7/8 was increased at both mRNA and protein levels in KIRC. TLRs in KIRC were involved in the activation of apoptosis, EMT, RAS/MAPK, and RTK pathways, as well as the inhibition of the cell cycle and the hormone AR pathway. Drug sensitivity analysis revealed that high expression of TLR3 and low expression of TLR7/9/10 were resistant to most of the small molecules or drugs from CTRP. Enrichment analyses showed that TLRs were mainly involved in innate immune response, toll-like receptor signalling pathway, NF-kappa B signalling pathway, and TNF signalling pathway. Furthermore, a high-level TLR3 expression was associated with a favourable prognosis in KIRC. Validation research further confirmed that TLR3 expression was increased in KIRC tissues, and high TLR3 levels were associated with poor overall survival. Moreover, TLR3 in KIRC showed a positive association with an abundance of immune cells, including B-cells, CD4+ T-cells, CD8+ T-cells, macrophage, neutrophils, and dendritic cells, and the expression of the immune biomarker sets. Several TLR3-associated kinase, miRNA, or transcription factor targets were also identified in KIRC. Conclusion. Our results indicate that TLR3 serves as a prognostic biomarker and associated with immune infiltration in KIRC. This work lays a foundation for further studies on the role of TLR3 in the carcinogenesis and progression of KIRC.

824 Modulation of TLR3 protein in response to radiation in squamous cell lung carcinoma

BackgroundSquamous cell lung cancer (SCLC) is the second most common type of lung cancer. Treatment is complicated due to the lack of mutated molecular targets.1 Radiotherapy (RT) is commonly used to treat SCLC, but relapse and tumor progression are common. The combination of immunotherapy (IT) with RT can enhance the effect observed with RT alone.2 Effective combination of IT and RT requires an understanding of the pathways that synergize to enhance tumor cell kill in SCLC. Our lab has identified Toll-like receptor 3 (TLR3) as a molecule that is regulated by RT and can be targeted with IT. Toll-like receptors serve a crucial role against tumor cells by activating innate and adaptive immune responses that boost antitumor immunity.3 4 TLR3 is the only receptor whose molecular mechanism functions independent of MyD88, leading to NF-κB mediated apoptosis.5 We hypothesized that increased TLR3 expression would be associated with improved response to RT. We further hypothesized that RT can downregulate TLR3 and that this effect can be reversed with TLR3 agonists leading to enhanced tumor antigen recognition. We aim to use this data to formulate further studies using combined RT and IT.MethodsMouse (KLN205) and human (SW900) squamous cell carcinoma (SCC) cell lines were used to study the effect of radiation on TLR3 expression. Irradiation was performed using the gammacell 3000 elan irradiator. Cells were irradiated with 0, 5, 10 and 20 Gy. Protein extraction was performed 48 and 72 hours after RT. Protein extracts were analyzed by Western Blot. Further, TLR3 mRNA expression and 5-year overall survival of SCLC patients was obtained from public databases. Kaplan-Meier method was used to correlate between TLR3 mRNA expression and survival.ResultsIn vitro studies and western blot analysis demonstrated a decrease of TLR3 expression in response to increasing doses of radiation. This observation was consistent in mouse and human SCC cell lines. In silico analysis of SCLC patients who received RT showed that increased TLR3 mRNA expression was associated with improved overall survival and disease-free survival.ConclusionsOur findings point to an important role for TLR3 in SCLC. Combining RT with TLR3 agonists may enhance the tumor response to RT. Several complementary experiments are underway in our lab to use the TLR3 agonist, Poly I:C, which will allow a better understanding of the effect of RT on TLR3.ReferencesGeorge J, Lim SJ, Jang SJ, et al. Comprehensive genomic profiles of small cell lung cancer. Nature 2015;524:47–53.Darragh L, Oweida A, Karam SD. Overcoming resistance to combination radiation-immunotherapy: a focus on contributing pathways withing the tumor microenvironment. Frontiers in Immunology 2019;9:3154.Shcheblyakov D, Logunov DY, Tukhvatulin AI, et al. Toll-Like Receptors (TLRs): The Role in Tumor Progression. Acta Naturae 2010;2(3):21-9.Kawai T, Akira S. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat Immunol 2010;11:373–384.Bianchi F, Alexiadis S, Camiasaschi C, et al. TLR3 expression induces apoptosis in Human Non-Small-Cell Lung Cancer. Int J Mol Sci 2020 Feb;21(4):1440.

P09.11 TLR3 suppresses colorectal carcinogenesis, presumably through up-regulation of T-cell attracting CXC chemokines

BackgroundToll-like-receptors (TLRs) are main components of the innate immune system which recognize endogenous or pathogen-associated molecular ‘danger patterns’. Previous findings from us and others highlighted a role of TLRs in the formation of tumors. However, TLRs may have differing roles in immune and cancer cells, and the underlying mechanisms are still unclear. TLR downstream signaling is mediated by two adapter proteins; MyD88 (myeloid differentiation primary response gene 88) and TRIF (TIR-domain-containing adapter-inducing interferon-β). The MyD88-mediated signaling pathway is activated by all TLRs, except TLR3. We could show previously that it leads to the initiation of oncogenic, proliferative and pro-inflammatory responses in colorectal cancer. The endosomal receptor TLR3, in contrast, solely depends on TRIF. It recognizes viral, microbial and endogenous dsRNA leading to production of type-I interferon and chemokines, and induces apoptosis. The role of TRIF dependent TLR3 signaling in colorectal cancer is still disputed. Within this study, we show tumor-suppressive TLR3 functions prevailing over oncogenic effects in colorectal cancer.Materials and MethodsTLR3-deficient colon cancer cell lines were engineered by CRISPR-Cas9. Genetically modified mouse models were generated, based on a ‘switch-on mutagenesis’ approach, with global inactivation of Tlr3 or TRIF (Ticam1), allowing tissue specific re-expression based on Cre recombination. The mice were interbred with the Apc1638N mouse model for digestive cancer. Furthermore, clinical significance of TLR3 expression levels was assessed in human colorectal cancer tissue samples from our clinic (n=81) and from TCGA datasets. A putative correlation between intratumoral TLR3 expression and T-cell infiltration is currently analyzed.ResultsGlobal deficiency of murine Tlr3, or TRIF, induced significantly increased digestive tumor formation, associated with increased morbidity indicating a tumor suppressive role. Coherently, TLR3 expression is highly significantly decreased in human colorectal cancer compared to normal mucosa, significantly correlated with poor survival. TLR3 deficient cell lines show reduced migration and slightly declined proliferation suggesting an oncogenic role on the cell-autonomous level. Nevertheless, gene expression analysis revealed that the dsRNA induced expression of T-cell attracting cytokines CXCL10 and CXCL11 in colon cancer cell lines is exclusively dependent on TLR3. These chemokines were shown to favor a TH1-type antitumoral response.ConclusionsTLR3 favors tumor suppression in vivo, presumably resulting from non-cell-autonomous factors such as the production of CXCL10 and 11 and resulting T-cell infiltration. This may outweigh the putative cell-autonomous oncogenic functions of TLR3 deficiency.Disclosure InformationA. Sichler: None. M. Frey: None. W. Johannes: None. K. Janssen: None.

Angiogenic and immunomodulatory biomarkers in axitinib-treated patients with advanced renal cell carcinoma

Aim: Immunomodulatory mechanisms contributing to angiogenic inhibition in renal tumors are not well characterized. We report associations between efficacy and tumor-associated immune cells and mRNA/miRNA expression in patients from AXIS. Materials & methods: Immunohistochemistry (n = 52) and mRNA/miRNA expression analyses (n = 72) were performed on tumor samples. Results: In axitinib-treated patients, higher CXCR4 and TLR3 expression, respectively, was associated with longer progression-free survival (hazard ratio; 95% CI: 0.3; 0.1–0.8 and 0.4; 0.2–0.9) and showed interaction with treatment (p = 0.029 and p < 0.001); lower CCR7 expression was associated with objective response (odds ratio: 0.1; 95% CI: 0.01–1.0) and longer overall survival (hazard ratio: 3.9; 95% CI: 1.4–10.3). Conclusion: CCR7, CXCR4 and TLR3 expression levels may be prognostic/predictive of clinical benefit with axitinib. Clinical trial identifier: ClinicalTrials.gov NCT00678392.

TLR3 Expression Induces Apoptosis in Human Non-Small-Cell Lung Cancer

The prognostic value of Toll-like receptor 3 (TLR3) is debated in cancer, differing between tumor types, methods, and cell types. We recently showed for the first time that TLR3 expression on early stage non-small-cell lung cancer (NSCLC) results associated with a good prognosis. Here, we provide experimental evidences explaining the molecular reason behind TLR3’s favorable prognostic role. We demonstrated that TLR3 activation in vitro induces apoptosis in lung cancer cell lines and, accordingly, that TLR3 expression is associated with caspase-3 activation in adenocarcinoma NSCLC specimens, both evaluated by immunohistochemistry. Moreover, we showed that TLR3 expression on cancer cells contributes to activate the CD103+ lung dendritic cell subset, that is specifically associated with processing of antigens derived from apoptotic cells and their presentation to CD8+ T lymphocytes. These findings point to the relevant role of TLR3 expression on lung cancer cells and support the use of TLR3 agonists in NSCLC patients to re-activate local innate immune response.