The Function And Mechanism Of TLR3 Agonist And Tumor Stroma Lnc-CAF During The Treatment Of Oral Squamous Cell Carcinoma

Oral squamous cell carcinoma is an increasingly serious problem that threatens human health.Currently,the survival rate and prognosis of patients have not been significantly improved.Therefore,the etiology,the choice of treatment methods,and the evaluation of prognosis of oral squamous cell carcinoma need to be continuously investigated.Toll-like receptors(TLRs),as a pattern recognition receptor(PRR)of pathogen-associated molecular patterns(PAMPs),are widely investigated in host cell recognition and responses to microbial pathogens and initiate innate immune responses such as inflammation.Although TLRs expression is prevalent in immune cells,TLRs is also found to be expressed in non-malignant or malignant epithelial cells and fibroblasts.It has been found that TLRs are activated by PAMP or DAMP(Damage-associated molecular patterns)and are involved in tumorigenesis during inflammation-cancer transformation.The expression patterns of different TLRs in tumors can be used as risk factors for the diagnosis and prognosis of patients.Additionaly,TLR agonists are currently being developed and used as adjuvants in anti-cancer immunotherapy.TLR agonists promote the maturation and activation of DCs(Dendritic cells)as well as other APCs(Antigen-presenting cells).They also enhance phagocytosis and migration of APCs to draining lymph nodes,leading to the induction of optimized presentation of tumor antigens to T cells,inducing pro-inflammatory cytokines(IL-12 and TNF-?)and type ? IFNs,enhancing anti-tumor activity of various effector cells.Therefore,investigations should focus on the expression patterns of TLRs in different tumors,the development of TLR agonists and their application in anti-cancer immunotherapy.The application of TLR agonists in clinical can enhance the anti-cancer effect of clinical treatment.Although TLR3 agonists are safe and well-tolerated as cancer vaccine adjuvants,its treatment alone has no or only low antitumor activity and is associated with significant human toxicity.In addition,different routes of poly(I:C)administration(including intravenous,intratumoral,peritumor injection,intramuscular injection or transfection)lead to different therapeutic effects.In glioblastoma clinical trials,glioblastoma patients first received immunotherapy,followed by chemotherapy,the clinical response rate was significantly higher than the chemotherapy alone.It is noteworthy that once the initial tumor control has been achieved by conventional doses of chemotherapy,drug doses could be reduced and treatment strategies using only low doses of chemotherapy drugs can inhibit tumor progression and prolong the survival of breast cancer mice.Therefore,we need to explore optimal dosages and combination strategies for TLR3 agonists and chemotherapeutics for the effective treatment of tumors and reduction of patient suffering.It has been reported that oral squamous cell carcinoma expresses certain types of TLRs,but the expression pattern of TLR3 and its agonists in oral squamous cell carcinoma is unclear.The establishment of tumor microenvironment can promote tumor development and chemoresistance,and the tumor microenvironment mainly consists of tumor cells,infiltrating immune cells and fibroblasts.Among them,dysfunctional immune cells are mainly regulatory T cells,tumor-associated macrophages,dendritic cells and myeloid suppressor cells.Cancer-associated fibroblasts(CAFs)are the most important non-immune cell types in TME.They maintain the immunosuppressive microenvironment through autocrine or paracrine actions and promote tumor growth and chemoresistance.Many studies have shown that effective inhibition of CAF formation helps target therapy of oral squamous cell carcinoma.Long-chain non-coding RNAs(LncRNAs)are involved in the development of tumors,but the role of LncRNAs in chemotherapy tolerance is still very limited.Moreover,the research of LncRNA mainly focuses on tumor cells,but the LncRNA profiles of LncRNAs in stroma microenvironment,especially in tumor-associated fibroblasts are completely unknown.We hypothesized that LncRNA could control tumor growth and chemoresistance by regulating CAF function.Therefore,we first analyzed the clinical basis of TLR3 agonist-based therapy,evaluated the different ways of administration,doses and side effects of chemoimmunotherapy based on TLR3 agonist and cisplatin,analyzed the characteristics of chemoimmunotherapy and clarified that anti-tumor mechanism;focused on the role of LncRNA in strom fibroblasts in drug resistance.This study provides a new instruction and new strategy for the immunochemotherapy of oral squamous cell carcinoma,and found that strom LncRNA can be used as a prognostic indicator of tumor progression and therapeutic effect.1.Functional heterogeneity of TLR3 in oral squamous cell microenvironment:To investigate the potential value of TLR3 agonists in the treatment of oral squamous cell carcinoma,we first analyzed the expression of TLR3 during the development of oral squamous cell carcinoma.The results showed that TLR3 expression patterns in tumor cells and strom fibroblasts are inconsistent.Immunohistochemistry(IHC)results showed the positive TLR3 staining on tumor cells as well as on TILs and tumor-associated fibroblasts(CAFs),but TLR3 expression was very slight in the normal epithelial part.There were 115 of 166(69%)patients with OSCC showed TLR3 positive expression,and 123(74%)of 166 patients had TLR3 positive expression in stroma fibroblasts.In addition,we also analyzed the clinical significance of TLR3 expression in different cellular components in the tumor microenvironment.High expression of TLR3 in tumor cells correlated with poor prognosis of patients,while high expression of TLR3 in fibroblasts correlated with good prognosis.Therefore,the therapeutic efficacy of TLR3 agonists may be related to the different expression patten of TLR3 on different cell types in the tumor microenvironment.2.The chemoimmunotheraputic effect of TLR3 agonist/cisplatin depends on different dosage and combination strategy:In order to explore the combind chemotherapy strategy of TLR3 agonist poly(I:C)and the clinical first line chemotherapy durgs of oral squamous cell carcinoma-cisplatin.We first investigated the combined effect of different doses of cisplatin and TLR3 agonist in vitro.We chose a concentration(20 ?g/ml)of poly(I:C)with low or no cytotoxicity and used it in combination with different doses of cisplatin,including IC25,IC50 and IC75.We found that the combined efficiencies were maximized in the cisplatin-treated group at the IC25 concentration compared to the cisplatin-alone treated group,but not in the cisplatin-treated group at the higher IC50 concentration,whereas the IC75 concentration have no combined effect with poly(I:C).In addition to the dose of cisplatin affects the combination efficiency,we also found that the order of administration is an important factor in combination chemotherapy.After cells were pretreated with 20 ?g/ml poly(I:C)for 12 hours,the cells were co-treated with IC25 concentrations of cisplatin and poly(I:C)for 48 hours.The combined effect of the pretreatment group was significantly higher compared to the group without pretreatment,and the inhibition rate of this combination treatment was equivalent to the cisplatin-induced inhibition rate at IC50 concentration alone.These findings strongly suggest that pre-treatment of poly(I:C)with enhanced chemotherapeutic efficacy at low doses of cisplatin,thus we propose an ordered Poly(I:C)25/DDP2.5 immunotherapy strategy for effective anti-tumor treatment.3.Poly(I:C)/cisplatin-based chemoimmunotherapy can not only inhibit cisplatin efflux in the tumor but overcome the immunosuppressive tumor microenvironment:We further investigated the mechanism and characteristics of the chemoimmunotherapy.We found that pretreatment of poly(I:C)at different times and concentrations significantly inhibited the gene expression of most ABC transporters including P-gp,ABCB4,MRP-1 etc.Down-regulated drug transporter protein can inhibit cisplatin efflux,thereby promoting low-dose cisplatin in the cell residency and improve the efficiency of chemotherapy.In a co-culture of spleen cells and tumor cells,we found that TLR3 agonists can activate spleen DCs and cytotoxic T cells in spite of low doses of cisplatin,which promoted their killing of tumor cells.In addition,as another important tumor therapeutic resistance factor,CAF secret immunosuppressive cytokines or form a drug defense barrier by directly contacting with tumor cells,participating in tumor microenvironment angiogenesis and promoting tumor cell growth,metastasis and drug resistance.We found that poly(I:C)25/DDP2.5 combination was able to induce significant apoptosis in tumor cells,even in the presence of CAF that supported tumor growth,indicating that this combined strategy could overcome the CAF-supported immunosuppressive environment.4.Lnc-CAF regulates stromal fibroblast activation for tumor growth and therapeutic resistance:Stromal fibroblasts consists of normal fibroblasts(NFs)and CAF.Targeting the abnormally expressed LncRNAs in CAF can promote tumor regression,improve chemotherapeutic efficiency and reduce therapeutic resistance of oral squamous cell carcinoma.To determine the LncRNA profile during malignant transformation of stromal fibroblasts,we first collected 10 matched normal and tumor tissues from patients with oral squamous cell carcinoma to isolate and culture fibroblasts.After phenotypic and functional validation of the paired NFs/CAFs,we performed a screening of patient clinical data,and these 5 paired NFs/CAFs were subjected to further transcriptome RNA sequencing.Based on their levels in fibroblasts and their fold changes,we found that an uncharacterized IncRNA LOC400221(also known as FLJ22447(NCBI Gene ID:400221))which was found to be significantly up-regulated in CAF,namely Lnc-CAF.Lnc-CAF is highly expressed in fibroblasts.Further,we analyzed the expression of Lnc-CAF in NFs,CAFs and oral squamous cell carcinoma tissues by fluorescence in situ hybridization.The results showed that Lnc-CAF was expressed in tumor cells and interstitial fibroblasts,but the expression of Lnc-CAF in cancer nests seemed to be higher than that in CAF.Interestingly,as an important cytokine involved in inflammation and cancer,IL-3 3 is significantly associated with Lnc-CAF in the co-expression network and is also significantly up-regulated in CAF.Furthermore,we found that IL-33 is mainly expressed in tumor stromal cells in clinical tumor samples,especially stromal fibroblasts,but is rarely expressed in cancer nests,thus the Lnc-CAF/IL-3 3 signaling may be involved in the malignant transformation of tumor-associated fibroblasts.Further analysis of its function found that overexpression of Lnc-CAF in NFs could promote the expressions of ?-SMA,vimentin and N-cadherin for fibroblasts activation,which induced tumor proliferation.Knockdown of IL-33 expression in CAF inhibited the activation phenotype of CAF and attenuated the growth of CAF-supported oral cancer cells.Subsequently,we analyzed the molecular mechanism by which Lnc-CAF regulates IL-33.We found that upregulated Lnc-CAF inhibited the p62-dependent autophagic lysosome degradation pathway,preventing the protein degradation of IL-33 and promoting the functional stability of IL-33,which was not associated with the IncRNA scaffold function.Interestingly,Lnc-CAF expression is not restricted to stroma fibroblasts but also expressed in tumor cells.We found that oral squamous cell carcinoma cells can excrete exo somes containing Lnc-CAF which could be uptake by stromal fibroblasts and up-regulate of Lnc-CAF levels in CAF to activate Lnc-CAF/IL-33 signaling and induce CAF Activation,forming a positive regulatory loop to induce tumor proliferation and therapeutic resistance.These findings have also been validated in mouse xenograft modelsIn summary,this study proposed a new chemoimmunotherapy strategy:an ordered combination therapy based on TLR3 agonist and low-dose cisplatin.We found that TLR3 agonists promoted cisplatin in-tumor residence,decreased cisplatin dosage,increased cytotoxicity of tumor cells,and activated the immune system to overcome CAF-dependent suppressive tumor microenvironment.In addition,highly expressed Lnc-CAF/IL33 signaling in CAF may serve as an important molecular target for inhibiting tumor growth and overcoming therapeutic resistance.