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(A) Wound healing assay, showing the migratory abilities of untreated and EDA knockout cells. (original magnification, 100×; scale bar: 100 μm). (B) Transwell invasion assay results, showing the invasive rate of untreated and EDA knockout cells (original magnification, 200×; scale bar: 50 μm). (C) The speed of wound edge in the untreated and EDA knockout cells, including CNE-2Z and SW480. (D) The invasive rate of EDA knockout cells, compared with untreated CNE2Z or SW480 cells, respectively.
Source publication
Background
The low efficiency of clustered, regularly interspaced, palindromic repeats-associated Cas (CRISPR/Cas) system editing genes in vivo limits the application. A components of the extracellular matrix (ECM), the extra domain A positive fibronectin (EDA+FN), may be a target for CRISPR/Cas system for the pro-oncogenic effects. The exclusion o...
Contexts in source publication
Context 1
... to the EDA over-expressing cells, which illustrated the pro-oncogenic effect (Supplementary Figures 1 to 3
Figure 2C&2D), while the levels of Ki-67 decreased as well in each EDA knockout cell line ( Figure ...
Context 2
... the wound healing assay, the speed of EDA knockout cells was shown to be 9.15±0.84 μm/h in CNE- 2Z and 8.76±0.66 μm/h in SW480 cells, which was significantly lower than untreated controls (CNE-2Z: 17.84±2.4μm/h, P=0.005 & SW480: 13.91±0.86μm/h, P=0.001) ( Figure 3A&3C). Similarly, the invasion rate of EDA knockout CNE-2Z (45.67±14.01 cells per field) and SW480 cells (45.00±10.44 cells per field) were significantly decreased, in comparison with that of untreated controls respectively (CNE-2Z: 135.00±11.79 cells per field, P=0.001 & SW480: 68.67±5.51 cells per field, P=0.003) ( Figure ...
Context 3
... the wound healing assay, the speed of EDA knockout cells was shown to be 9.15±0.84 μm/h in CNE- 2Z and 8.76±0.66 μm/h in SW480 cells, which was significantly lower than untreated controls (CNE-2Z: 17.84±2.4μm/h, P=0.005 & SW480: 13.91±0.86μm/h, P=0.001) ( Figure 3A&3C). Similarly, the invasion rate of EDA knockout CNE-2Z (45.67±14.01 cells per field) and SW480 cells (45.00±10.44 cells per field) were significantly decreased, in comparison with that of untreated controls respectively (CNE-2Z: 135.00±11.79 cells per field, P=0.001 & SW480: 68.67±5.51 cells per field, P=0.003) ( Figure ...
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Citations
... According to the optical density, EDA +FN staining was demarcated as groups of high or low expression as in our previous studies. 15,22 Cell Culture and Colony Forming Unit (CFU) Assay ...
... To explore the role of EDA+FN in the interaction between clusters, the EDA exon was knocked out in the Cluster 4 fibroblasts. According to our previous studies, 15,22,31 the width of the band lacking the EDA exon (415 bp) exceeded the band containing the EDA exon (675 bp), which resulted in decreased EDA+FN protein levels, suggesting that the EDA exon was excluded from the majority of cells in which the total FN level remained stable ( Figure 4A and B). Cluster 1 coculture with EDA knockout Cluster 4 induced significantly less Trap+MNCs (n = 6, 7.5000±2.8247/well) ...
... This study has identified 4 subtypes (Cluster 1, 2 3, 4) of fibroblasts, and the Cluster 4 expressing relatively high level of EDA+FN stimulated inflammatory factor expression in other clusters, leading to reinforced osteoclastogenesis and bone destruction. As the main component of the ECM, abnormal alternative splicing of the fibronectin (FN) gene occurs during inflammation, wound healing or tumors, 22,35 generating abnormal FN isoforms. 36 Our previous studies have demonstrated that one of the abnormal isoforms, EDA+FN, is not only increased in the inflammatory stroma of radicular cysts, but also participates in jawbone destruction by stimulating the expression of a series of osteoclastogenesis-related genes in fibroblasts. ...
Background
To analyze the fibroblasts subtypes in the gingival tissues of healthy controls, gingivitis and periodontitis patients, as well as the effects of interaction between subtypes on alveolar bone destruction.
Methods
Gingival tissues were divided into three groups according to clinical and radiographic examination, and the immunostaining of EDA+FN was assessed. Fibroblasts from gingiva developed colony formation units (CFUs) and induced Trap+MNCs. The expression of osteoclastogenesis-related genes was assessed by real-time PCR. Variances in the gene profiles of CFUs were identified by principal component analysis, and cluster analysis divided CFUs into subtypes. The induction of Trap+MNCs and gene expression were compared among individual or cocultured subtypes. The fibroblast subtypes exerted critical effect on Trap+MNCs formation were selected and edited by CRISPR/Cas to investigate the influence on osteoclastogenesis in the periodontitis in mice.
Results
Most periodontitis samples exhibited intensive EDA+FN staining (P < 0.05), and these fibroblasts also induced most Trap+MNCs among three groups; consistently, fibroblasts from periodontitis highly expressed genes facilitating osteoclastogenesis. According to gene profiles and osteoclastogenic induction, four clusters of CFUs were identified. The proportion of clusters was significantly different (P < 0.05) among three groups, and their interaction influenced osteoclastogenic induction. Although Cluster 4 induced less osteoclasts, it enhanced the effects of Clusters 1 and 3 on Trap+MNCs formation (P < 0.05). EDA knockout in Cluster 4 abrogated this promotion (P < 0.05), and decreased osteoclasts and alveolar bone destruction in experimental periodontitis (P < 0.05).
Conclusion
Heterogeneous fibroblast subtypes affect the switch or development of periodontitis. A subtype (Cluster 4) played important role during alveolar bone destruction, by regulating other subtypes via EDA+FN paracrine.
... This knockout decreased cancer cell growth and motility and inhibited epithelial-mesenchymal transition (EMT). These findings could be a result of decreased EDA+FN protein levels, modified ECM composition, and disrupted interaction of tumor cells and their surroundings (Lv et al., 2017b). ...
Drug resistance is a serious challenge in cancer treatment that can render chemotherapy a failure. Understanding the mechanisms behind drug resistance and developing novel therapeutic approaches are cardinal steps in overcoming this issue. Clustered regularly interspaced short palindrome repeats (CRISPR) gene-editing technology has proven to be a useful tool to study cancer drug resistance mechanisms and target the responsible genes. In this review, we evaluated original research studies that used the CRISPR tool in three areas related to drug resistance, namely screening resistance-related genes, generating modified models of resistant cells and animals, and removing resistance by genetic manipulation. We reported the targeted genes, study models, and drug groups in these studies. In addition to discussing different applications of CRISPR technology in cancer drug resistance, we analyzed drug resistance mechanisms and provided examples of CRISPR's role in studying them. Although CRISPR is a powerful tool for examining drug resistance and sensitizing resistant cells to chemotherapy, more studies are required to overcome its disadvantages, such as off-target effects, immunotoxicity, and inefficient delivery of CRISPR/cas9 into the cells.
... In fact, it has been demonstrated that FN-EDA plays an important role in the development of pathological neoangiogenesis 41 42 promoting intratumoral microvessel formation and tumor progression. 43 We have demonstrated that EDA CAR-T cells can recognize EDA-expressing tumor cells and prevent tumor growth in vivo. But notably, EDA CAR-T cell infusion showed antitumor therapeutic efficacy against the challenge with tumor cells not expressing EDA. ...
Background:
One of the main difficulties of adoptive cell therapies with chimeric antigen receptor (CAR)-T cells in solid tumors is the identification of specific target antigens. The tumor microenvironment can present suitable antigens for CAR design, even though they are not expressed by the tumor cells. We have generated a CAR specific for the splice variant extra domain A (EDA) of fibronectin, which is highly expressed in the tumor stroma of many types of tumors but not in healthy tissues.
Methods:
EDA expression was explored in RNA-seq data from different human tumor types and by immunohistochemistry in paraffin-embedded tumor biopsies. Murine and human anti-EDA CAR-T cells were prepared using recombinant retro/lentiviruses, respectively. The functionality of EDA CAR-T cells was measured in vitro in response to antigen stimulation. The antitumor activity of EDA CAR-T cells was measured in vivo in C57BL/6 mice challenged with PM299L-EDA hepatocarcinoma cell line, in 129Sv mice-bearing F9 teratocarcinoma and in NSG mice injected with the human hepatocarcinoma cell line PLC.
Results:
EDA CAR-T cells recognized and killed EDA-expressing tumor cell lines in vitro and rejected EDA-expressing tumors in immunocompetent mice. Notably, EDA CAR-T cells showed an antitumor effect in mice injected with EDA-negative tumor cells lines when the tumor stroma or the basement membrane of tumor endothelial cells express EDA. Thus, EDA CAR-T administration delayed tumor growth in immunocompetent 129Sv mice challenged with teratocarcinoma cell line F9. EDA CAR-T treatment exerted an antiangiogenic effect and significantly reduced gene signatures associated with epithelial-mesenchymal transition, collagen synthesis, extracellular matrix organization as well as IL-6-STAT5 and KRAS pathways. Importantly, the human version of EDA CAR, that includes the human 41BB and CD3ζ endodomains, exerted strong antitumor activity in NSG mice challenged with the human hepatocarcinoma cell line PLC, which expresses EDA in the tumor stroma and the endothelial vasculature. EDA CAR-T cells exhibited a tropism for EDA-expressing tumor tissue and no toxicity was observed in tumor bearing or in healthy mice.
Conclusions:
These results suggest that targeting the tumor-specific fibronectin splice variant EDA with CAR-T cells is feasible and offers a therapeutic option that is applicable to different types of cancer.
... Overexpression of lncRNA CRNDE facilitates EMT phenotype in intrahepatic cholangiocarcinoma, bladder cancer, thyroid carcinoma and modulates PI3K/AKT signal and Notch1 pathways to enhance EMT in gastric cancer and osteosarcoma, respectively [125,126,128]. Cancer stemness is principally responsible for EMT related tumor reoccurrence and therapeutic resistance and is promoted by several of the lncRNAs such as ROR, OIP5-AS1, SOX2OT, NEAT1, HOTAIR and DANCR [108,125,129]. ...
... XIST accounts for EMT in non-small cell lung carcinoma by activating TGF-β pathway through sponging miR367/141 [131]. Likewise, overexpressed CRNDE and DANCR promote cell proliferation, invasion and metastasis by competing to bind with the miR384 in papillary thyroid cancer and miR33a-5p in osteosarcoma, respectively [125,129]. In addition, adding another layer of complexity to the ceRNA regulatory network, miR203 was found to inhibit HOTAIR in renal cell carcinoma and regulates tumorigenesis through EMT [132]. ...
... For example, knockdown of EDA + FN by siRNA actually decreases the total amount of FN [155], while FN is vital to normal tissues. CRISPR/Cas9 EDA editing, allowed to abrogate the pro-oncogenic effects of EDA + FN and to induce inhibition of tumor progression both in vitro and in vivo [129]. ...
Epithelial to mesenchymal transition (EMT) is a complex plastic and reversible cellular process that has critical roles in diverse physiological and pathological phenomena. EMT is involved in embryonic development, organogenesis and tissue repair, as well as in fibrosis, cancer metastasis and drug resistance. In recent years, the ability to edit the genome using the clustered regularly interspaced palindromic repeats (CRISPR) and associated protein (Cas) system has greatly contributed to identify or validate critical genes in pathway signaling. This review delineates the complex EMT networks and discusses recent studies that have used CRISPR/Cas technology to further advance our understanding of the EMT process.
... Though the authors did not directly assess the anti-apoptotic role of EDA per se, they hypothesized that it is the EDA-mediated strengthening of FN-α5β1 interactions (151) that results in the protective effect of FN against cell death. Finally, in two human tumor cell lines it was shown utilizing CRISPR/Cas9 technology that exclusion of EDA resulted in a pronounced decrease in cell proliferation (152). ...
Normal tissue homeostasis and architecture restrain tumor growth. Thus, for a tumor to develop and spread, malignant cells must overcome growth-repressive inputs from surrounding tissue and escape immune surveillance mechanisms that curb cancer progression. This is achieved by promoting the conversion of a physiological microenvironment to a pro-tumoral state and it requires a constant dialog between malignant cells and ostensibly normal cells of adjacent tissue. Pro-tumoral reprogramming of the stroma is accompanied by an upregulation of certain extracellular matrix (ECM) proteins and their cognate receptors. Fibronectin (FN) is one such component of the tumor matrisome. This large multidomain glycoprotein dimer expressed over a wide range of human cancers is assembled by cell-driven forces into a fibrillar array that provides an obligate scaffold for the deposition of other matrix proteins and binding sites for functionalization by soluble factors in the tumor microenvironment. Encoded by a single gene, FN regulates the proliferation, motile behavior and fate of multiple cell types, largely through mechanisms that involve integrin-mediated signaling. These processes are coordinated by distinct isoforms of FN, collectively known as cellular FN (as opposed to circulating plasma FN) that arise through alternative splicing of the FN1 gene. Cellular FN isoforms differ in their solubility, receptor binding ability and spatiotemporal expression, and functions that have yet to be fully defined. FN induction at tumor sites constitutes an important step in the acquisition of biological capabilities required for several cancer hallmarks such as sustaining proliferative signaling, promoting angiogenesis, facilitating invasion and metastasis, modulating growth suppressor activity and regulating anti-tumoral immunity. In this review, we will first provide an overview of ECM reprogramming through tumor-stroma crosstalk, then focus on the role of cellular FN in tumor progression with respect to these hallmarks. Last, we will discuss the impact of dysregulated ECM on clinical efficacy of classical (radio-/chemo-) therapies and emerging treatments that target immune checkpoints and explore how our expanding knowledge of the tumor ECM and the central role of FN can be leveraged for therapeutic benefit.
... As the main component of the extracellular matrix (ECM), fibronectin (FN) is important and is widely distributed in various tissues. Despite its absence in most adult tissues, the alternatively spliced exon extra domain A (EDA) is included in the FN protein (EDA + FN) under pathological conditions, particularly inflammation, wound healing and tumors [1,2]. This protein facilitates cellular proliferation, motility, and aggressiveness in different lesions [3,4]. ...
... In a previous study, the EDA exon maintained VEGF expression and in turn ensured the vascularization and dissemination of tumors, thereby contributing to tumor aggressiveness [2]. We have previously investigated hyperplastic vessels in odontogenic cysts [8] and the osteoclastogenesis induced by VEGF [9], since this growth factor sometimes induces osteoclastogenesis as a substitute for macrophage colony-stimulating factor (M-CSF) [10,11]. ...
... Therefore, in the present study, the associations among EDA + FN, vessels, and bone destruction in radicular cysts were investigated. Bevacizumab [15] or the type II bacterial clustered, regularly interspaced, palindromic repeats (CRISPR)-associated (Cas) system [2,4] was used to block the effects of either VEGF or EDA + FN [16], respectively, and inhibit osteoclastogenesis. ...
Fibronectin (FN) is a main component of extracellular matrix (ECM) in most adult tissues. Under pathological conditions, particularly inflammation, wound healing and tumors, an alternatively spliced exon extra domain A (EDA) is included in the FN protein (EDA+FN), which facilitates cellular proliferation, motility, and aggressiveness in different lesions. In this study we investigated the effects of EDA+FN on bone destruction in human radicular cysts and explored the possibility of editing FN gene or blocking the related paracrine signaling pathway to inhibit the osteoclastogenesis. The specimens of radicular cysts were obtained from 20 patients. We showed that the vessel density was positively associated with both the lesion size (R = 0.49, P = 0.001) and EDA+FN staining (R = 0.26, P = 0.022) in the specimens. We isolated fibroblasts from surgical specimens, and used the CRISPR/Cas system to knockout the EDA exon, or used IST-9 antibody and bevacizumab to block EDA+FN and VEGF, respectively. Compared to control fibroblasts, the fibroblasts from radicular cysts exhibited significantly more Trap+MNCs, the relative expression level of VEGF was positively associated with both the ratio of EDA+FN/total FN (R = 0.271, P = 0.019) and with the number of Trap+MNCs (R = 0.331, P = 0.008). The knockout of the EDA exon significantly decreased VEGF expression in the fibroblasts derived from radicular cysts, leading to significantly decreased osteoclastogenesis; similar results were observed using bevacizumab to block VEGF, but block of EDA+FN with IST-9 antibody had no effect. Furthermore, the inhibitory effects of gene editing on Trap+MNC development were restored by exogenous VEGF. These results suggest that EDA+FN facilitates osteoclastogenesis in the fibrous capsule of radicular cysts, through a mechanism mediated by VEGF via an autocrine effect on the fibroblasts. Bevacizumab inhibits osteoclastogenesis in radicular cysts as effectively as the exclusion of the EDA exon by gene editing.
Aim:
To analyse the heterogeneity of fibroblasts isolated from the fibrous capsules of radicular cysts and explore the effects of fibroblast subsets on bone destruction.
Methodology:
Radicular cysts were divided into groups according to varying perilesional sclerosis identified by radiograph. Colony forming units (CFUs) were isolated from the fibrous capsules of cysts, by which Trap+MNCs were induced, and the expression of osteoclastogenesis-related genes was compared among groups by real-time PCR. The variances in gene profiles of CFUs were identified by principal component analysis, and then CFUs were divided into subsets using cluster analysis. The induction of Trap+MNCs and related gene expression were compared among subsets, and osteoclastogenic induction was blocked by IST-9 or bevacizumab. The fibroblast subsets in cysts were investigated by retrospective immunostaining with IST-9, VEGF-A, and CD34. A fibroblast subset that underwent gene editing by CRISPR/Cas was injected into the site of bone defects in animal models, and the in vivo effects on osteoclastogenesis were investigated.
Results:
The fibroblast CFUs isolated from radicular cysts with perilesional unsclerotized cysts induced more Trap+MNCs than those with perilesional sclerotic cysts (P<0.05). Most fibroblast CFUs from unsclerotized cysts belonged to Cluster 2, which induced more Trap+MNCs (P<0.05) and highly expressed genes facilitating osteoclastogenesis; these results were different from those of Cluster 1 (P<0.05), in which most CFUs were isolated from perilesional sclerotic cysts or controls (P<0.05). The high expression of EDA+FN and VEGF-A was investigated in both the fibroblasts of Cluster 2 and the fibrous capsules of unsclerotized cysts (P<0.05), and the number of Trap+MNCs induced by Cluster 2 was decreased by treatment with IST-9 and bevacizumab (P<0.05). Consistently, EDA exon exclusion significantly decreased the osteoclastogenic induction of fibroblasts from Cluster 2 in vivo (P<0.05).
Conclusion:
The fibrous capsules of radicular cysts contain heterogeneous fibroblasts that can form subsets exhibiting different effects on osteoclastogenesis. The subset which depending on the autocrine effects of EDA+FN on VEGF-A, mainly contributes to the osteoclastogenesis and bone destruction of radicular cysts. The regulation of the proportion of subsets is a possible strategy for artificially interfering with osteoclastogenesis.
Aim:
To analyse the effects of the alternatively spliced fibronectin (FN) gene and its isoforms on osteoclastogenesis in radicular cysts.
Methodology:
Specimens of radicular cysts were collected surgically from 22 patients whose radiolucent periapical areas were measured on digital panoramic radiographs before surgery. The associations between the radiolucent areas and FN isoforms, Vascular Endothelial Growth Factor (VEGF) expression or micro-vessel density, as well as the relationships among them, were analysed by immunohistochemical staining using the antibodies IST-9, BC-1, P1F11, VEGF, and CD34. Fibroblasts isolated from those specimens were used to induce Trap+MNCs, and the effects of induction were assessed by blocking FN containing extra domain A (EDA+FN), COX-2, or VEGF in vitro. The effects of EDA exon knockout using CRISPR/Cas system was also assessed. Quantitative PCR was used to analyse relative expression of FN isoforms and osteoclastogenic genes. Data were analysed using Linear regression, Spearman's rank correlation analysis, Chi-square test and Student's t-test; P < 0.05 was considered significant.
Results:
Micro-vessel density and EDA+FN staining were positively associated with the size of radiolucent periapical areas (mm2 ) (P<0.05), consistent with a positive association between Trap+MNCs and VEGF expression in fibroblasts (P<0.05). Blocking the interaction between EDA+FN and fibroblasts inhibited Trap+MNC formation. In addition, EDA exon knockout decreased VEGF expression, and inhibited Trap+MNC formation to the extent of blocking VEGF by bevacizumab, but osteoclastogenic induction was restored by recombinant VEGF. Using retrospective clinicopathological data, VEGF staining was shown to be positively associated with EDA+FN staining, micro-vessel density, and the size of radiolucent areas (P<0.05).
Conclusion:
In fibrous capsules of radicular cysts, the alternatively spliced isoform EDA+FN generated by fibroblasts stimulated VEGF expression via an autocrine effect, and then facilitated osteoclastogenesis. Both blockage of VEGF and EDA exon knockout could be used to inhibit bone destruction.
Objective
To investigate the alternative spliced isoforms of Fibronectin (FN) in the stroma of radicular cysts; and analyze the associations between these isoforms and the osteoclastogenic effects of fibroblasts.
Methods and materials
The specimens of radicular cysts were stained with immunohistochemistry, and the associations between each FN isoform and clinical parameters were assessed. The fibroblasts isolated from cysts or jaw bone were cultured to induce the Trap+MNCs. In the conditioned medium, the Fibronectin containing extra domain A (EDA+FN) were neutralized by antibody IST‐9, and the EDA exon of fibroblasts were knockout by CRISPR/Cas system, for assessing the osteolastogenic effects. The mRNA level of FN isoforms and the osteoclastogenesis related genes were analyzed by quantitive PCR.
Results
EDA+FN staining was positively associate with the size of the lesions (P<0.05). In contrast with the controls, the ratio of EDA+FN/total FN in the fibroblasts from radicular cysts was significantly higher (P<0.05), and positively associate with Trap+MNCs counting, it was consistent with increased expression of COX‐2, IL‐6, IL‐17 and the RANKL/OPG (P<0.05). The Trap+MNCs counting and osteoclastogenesis related genes were decreased by IST‐9 blocking and EDA exon knockout in fibroblasts, but the blockage of the interaction between EDA+FN and pre‐osteoclasts exhibited little effects on Trap+MNCs formation.
Conclusion
The microenvironment of the fibrous capsule of radicular cysts facilitate the splicing of EDA exon, it endues EDA+FN with autocrine effects on fibroblast itself, and increases the expression of osteoclastogenesis related genes, by which the osteoclastogenesis in radicular cysts could be initiated.
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