Nicotinamide N’-methyltransferase-mediated Type Ⅰ Collagen Deposition Promotes The Self-renewal Of Cancer Stem Cells In Oral Squamous Cells Carcinoma

Oral squamous cell carcinoma(OSCC)is commonly infiltrated by cancerassociated fibroblasts(CAFs),which give rise to highly dynamic extracellular matrix(ECM)in tumor microenvironment(TME).Specifically,studies have reported that the expression of type Ⅰ collagen(Col1),which represent a major component of ECM,is always up-regulated in TME.However,the role of cancer-related ECM in OSCC development remains unclear.In the present study,we generated primary fibroblasts and tumor cells,using fresh tumor and paracancerous specimens from patients with OSCC,to allow for the establishment of patient-derived organoids(PDOs).Results of the immunohistochemical/immunofluorescence staining confirmed that the expression pattern of OSCC tumor nest were highly recapitulated in PDO model.Results of the serial passage assay demonstrated that the purified cancer stem cells(CSCs)population(i.e.CD44+ cells or sphere forming cells)was able to stably propagate PDOs in three dimensional(3D)culture system.Results of the pharmacological intervention and coculture assay showed that the CAF-derived lactate can promote the organoid forming ability of CSCs.Furthermore,RNA-seq analysis disclosed that the ECM-receptor interaction-and Focal adhesion kinase(FAK)-related genes were up-regulated in CSCs compared to parent OSCC cells,and the titration of Coll can aggravate the CSCs properties in OSCC cells,such as the expression of CD44 protein and sustaining of Wnt activity.Using a fibroblast-attached organoids(FAOs)model,which mimic the symbiotic environment of CAFs and OSCC cells,the results showed that CSCs phenotypes were impaired when the expression of Collal in CAFs or FAK signal in OSCC cells was silenced or inhibited.Mechanistically,we found that the expression of nicotinamide N-methyltransferase(NNMT)was elevated in stromal compartment of OSCC when compare to that in paracancerous specimens,and contributed to the trigger and maintenance of CAFs phenotypes.In tumor regeneration assay with co-implanted CAFs/PFs and SFCs,the tumor-initiating ability of SFCs was reduced when the expression of NNMT in CAFs/PFs was silenced/overexpressed.Moreover,knockout of NNMT in CAFs decreased the expression of COL1A1 in FAOs,while the overexpression of NNMT promoted the transcription of lysyloxidase(LOX)in PFs.Overall,we clarify that NNMT-mediate Col1 deposition may promote the self-renewal of CSCs in patients with OSCC.Part 1.Establishment of patient-derived oral carcinoma organoids1.Objective:To generate a panel of patient-derived organoids using tumor specimens from patients with oral squamous cell carcinoma(OSCC),and simultaneously,to explore whether co-culture with cancer-associated fibroblasts(CAFs)promote the organoid forming efficiency of OSCC cells.2.Methods:Fresh tumor and paracancerous specimens of patients with OSCC were collected to generate tumor organoids and fibroblasts.The Hematoxylin and Eosin(HE)staining assay was utilized to clarify the structure of patient-derived tumor organoids.The immunofluorescence staining assay was performed to detect the expression of epithelial lineage marker(i.e.keratin 19,CK19),cancer stem cells(CSCs)marker(i.e.CD44 and CD 133),mesenchyme lineage marker(i.e.Vimentin,VIM),and CAFs activation markers(i.e.α-SMA and FAP)in tumor organoids as well as fibroblasts for their authentication.CD44-based Flow cytometry cell sorting or sphere forming assay was employed to enrich for OSCC cells subset with enhanced stem-like properties(i.e.CSCs population)for further serial propagation.Organoid reconstitution assay was employed for CSCs administrated with glucose,galactose,and lactate.Three dimension(3D)co-culture assay was arranged for CSCs with paralleled patient-derived CAFs,in which the uptake or production of lactate in CSCs or CAFs was inhibited by pharmacological treatment3.Results:Results of the HE staining and immunofluorescence staining showed that the tumor nest structure and cellular heterogeneity of OSCC specimens were highly recapitulated in corresponding tumor organoids.The CSCs were able to serially propagate tumor organoids in virto.Treatment with lactate promoted the organoid forming efficiency of CSCs and increased the expression of CD44 and OCT-4 in CSCderived OSCC organoids.In 3D co-culture system of tumor organoid with corresponding CAFs,the organoid forming efficiency was decreased when the uptake or production of lactate in CSCs or CAFs was inhibited.4.Conclusion:A panel of patient-derived oral carcinoma organoids was established for further study;in 3D co-culture system,the CAF-derived lactate may promote the efficiency of CSCs to expand organoids outgrowth.Part 2.CAF-derived Type Ⅰ Collagen promotes the stem-like properties of OSCC cells1.Objective:To construct a fibroblast-attached organoid model and investigate whether CAF-derived type Ⅰ collagen promote the stem-like properties of OSCC cells.2.Methods:The sphere forming assay was employed to enrich for the OSCC cells with enhanced stem-like properties.The RNA-sequencing analysis was utilized to dissect the transcription prolife of sphere forming cancer cells(SFCs).A lentivirus harboring Wnt-reporter genes was constructed and transfected into OSCC cells for visualization of Wnt activity in SFCs,which were expanded in matrigel mixed with various dosage of type Ⅰ collagen.The SFCs and corresponding CAFs were mixed and suspended in ultra-low adsorption plate to generate CAF-OSCC cells clusters,which were then embedded in matrigel for construction of Fibroblast-attached organoids(FAOs).The HE and immunofluorescence staining assay was performed to test the pathological structure as well as the expression pattern of epithelial lineage marker(i.e.β-catenin),and fibroblasts markers,such as COL1A1 and FSP-1,in OSCC specimens and corresponding FAOs.The expression of cancer stem cells marker(i.e.CD44),proliferative cells marker(i.e.Ki67),and apoptosis cells marker(i.e.caspase3)was evaluated in FAO administrated with losartan,a specific inhibitor of type Ⅰ collagen synthesis.The conditional FAO reconstitution assay was employed for SFCs,in which the expression of collal in CAFs or focal adhesion kinase(FAK)signal in OSCC cells was silenced or inhibited.3.Results:Results of the RNA-sequencing showed that the expression of ECM-receptor interaction-and FAK signal-related genes was elevated in SFCs when compared to parent OSCC cells.Results of the matrigel-based culture assay demonstrated that the titration of type Ⅰ collagen promoted the expression of CD44 and activation of Wnt signaling in SFC-derived organoids.Results of the HE and immunofluorescence staining assays confirmed that the spatial distribution pattern of fibroblasts with tumor nest in OSCC specimens was highly recapitulated in corresponding FAOs.Treatment with losartan reduced the expression of CD44 and Ki67,and increased the expression of caspase3 in FAOs.In conditional reconstitution assay,the FAOs propagation was impaired when the expression of collal in CAFs or FAK signal in SFCs was silenced or inhibited.4.Conclusion:In a patient-derived FAO model,the CAF-derived type Ⅰ collagen promote the stem-like properties of OSCC cells.Part 3.NNMT-mediated collagen deposition promotes the selfrenewal of CSCs in patients with OSCC1.Objective:To investigate the expression pattern and the biological functions of NNMT in patients with OSCC.2.Methods:The immunohistochemical(IHC)and immunocytochemical(ICC)staining was employed to determine the expression pattern of NNMT in OSCC specimens,as well as that in primary CAFs and PFs.The CCK8 assay,transwell migration assay,and collagen contraction assay were employed to test the phenotype transition of CAFs that were manipulated with either knockdown of NNMT(CAF-sh-NNMT)or knockout of NNMT in genome(CAF-sg-NNMT),as well as the PFs that were manipulated with over-expression of NNMT(PF-oe-NNMT).The in vitro FAO and in vivo co-implanted xenograft model were constructed using SFCs and corresponding CAFs or PFs.The immunofluorescence assay was utilized to interrogate the expression of COL1A1 in transgenic FAOs,in which the NNMTin CAFs genome was knockout.The real time quantitative PCR(RT-qPCR)assay was performed to evaluate the expression of collagen deposition-related genes in PF-oeNNMT.3.Results:Results of the IHC and ICC staining showed that the expression of NNMT was significantly elevated in CAFs when compared to PFs.Knockdown of NNMT or knockout of NNMT in CAFs reduced their proliferation,migration,and collagen contraction abilities.On the contrary,the above phenotypes in PFs were triggered by over-expression of NNMT.In CAF-SFC derived FAO and co-implanted xenograft model,the self-renewal of SFCs was impaired when the expression of NNMT in CAFs was silenced.Likewise,over-expression of NNMT in PFs promoted the organoid and xenograft formation abilities of SFCs.Results of the immunofluorescence assay confirmed that the expression of COL1A1 in FAOs was reduced when the NNMT in CAFs was knockout.Results of the RT-qPCR assay indicated that the expression of LOX in PFs was significantly increased upon over-expression of NNMT.4.Conclusion:The enhanced expression of NNMT in CAFs is associated with the progression of patients with OSCC,and the NNMT-mediated collagen deposition promotes the selfrenewal of CSCs in OSCC.