Submitted Successfully!
Thank you for your contribution! You can also upload a video entry related to this topic through the link below: https://encyclopedia.pub/user/video_add?id=26772
Check Note
2000/2000
Ver. Summary Created by Modification Content Size Created at Operation
1 -- 1693 2022-09-01 07:54:53 |
2 format correction Meta information modification 1693 2022-09-02 03:02:48 |
Roles of CAFs in the TME of PDAC
Edit
Upload a video

Pancreatic ductal adenocarcinoma (PDAC) is one of the most malignant digestive tumors, characterized by a low rate of early diagnosis, strong invasiveness, and early metastasis. The abundant stromal cells, dense extracellular matrix, and lack of blood supply in PDAC limit the penetration of chemotherapeutic drugs, resulting in poor efficacy of the current treatment regimens. Cancer-associated fibroblasts (CAFs) are the major stromal cells in the tumor microenvironment. Tumor cells can secrete exosomes to promote the generation of activated CAFs, meanwhile exosomes secreted by CAFs help promote tumor progression. 

PDAC exosomal miRNAs cancer-associated fibroblasts
Information
Subjects: Surgery
Contributors : , ,
View Times: 71
Revisions: 2 times (View History)
Update Time: 02 Sep 2022
Table of Contents

    1. Identification, Origin, and Classification of CAFs in PDAC

    In PDAC, 60–70% of the tumor tissue is composed of CAF-dominant stromal cells and the deposition of extracellular matrix (ECM) components such as collagen [1]. CAFs are characterized by the expression of mesenchymal markers, morphological features (spindle shape), and by the absence of non-mesenchymal markers (markers of epithelial, endothelial, immune, and neuronal cells). The latest consensus states that CAFs are collections of cells in the tumors that are negative for epithelial, endothelial, and leukocyte markers; have an elongated morphology; and lack the genetic mutations found in cancer cells [2]. CAFs can be derived from normal fibroblasts, stellate cells, bone marrow-derived mesenchymal stem cells, adipocytes, or endothelial cells [3][4][5][6]. Quiescent PSCs are the main precursors of CAFs in the TME of PDAC. When injury or inflammation occurs, quiescent PSCs will be activated, accompanied by changes in cell morphology and the disappearance of lipid droplets. Subsequently, quiescent PSCs transform into α-smooth muscle actin (α-SMA)-positive myofibroblast-like cells, secreting a large amount of extracellular matrix components and remodeling the matrix of pancreatic tissue [7]. Matthew et al. [8] reported that the fibroblast subpopulations exist in both normal pancreas and pancreatic tumors in KC (Ptf1a-Cre; LSL-Kras G12D) mice, and these fibroblast subpopulations display characteristics of mesenchymal stem cells (MSCs), which can differentiate into chondrocytes, adipocytes, and osteoblasts. This would promote tumor growth by inducing the polarization of macrophages to pro-cancer subtypes. Huang et al. [9] found mesothelial cells could transform into antigen-presenting CAFs (apCAFs) by downregulating mesothelial characteristics and acquiring fibroblast characteristics under the induction of interleukin-1 and transforming growth factor β (TGF-β). The apCAFs can directly ligate and convert the naive CD4+ T cells into regulatory T cells in an antigen-specific way. Miyazaki et al. [10] found that the adipose-derived mesenchymal stem cells (AD-MSCs) could transform into variant CAF subtypes under different co-culture conditions in vitro. When co-cultured with PDAC Capan-1 cells directly, AD-MSCs could transform into myofibroblast CAFs (myCAFs) and inflammatory CAFs (iCAFs), while indirect co-culture induced differentiation into only iCAFs. Waghray et al. [11] identified and characterized cancer-associated MSC (CA-MSC) subpopulations from human PDAC samples and CAFs, highlighting the heterogeneity of fibroblast subpopulations in PDAC. CA-MSCs can significantly enhance the proliferation, invasion, and metastatic potential of PDAC cells by secreting granulocyte-macrophage colony-stimulating factor (GM-CSF). CAFs will then differentiate into different subtypes with similar characteristics to their origin cells, according to the various cell origins and culture conditions. Hence, CAFs in human PDAC show great heterogeneity and exert different functions.

    2. Roles of Activated CAFs in the PDAC Microenvironment

    The activated CAFs can express various proteins including α-SMA, fibroblast activation protein (FAP), fibroblast-specific protein 1 (FSP-1), podoplanin (PDPN), and PDGFR [5][12]. Some of these proteins were used as typing markers to classify CAFs into different subtypes, which might exert anti-cancer or pro-cancer roles in PDAC. CAFs with high expression of α-SMA were named myCAFs, whereas CAFs with low α-SMA levels that secrete inflammatory cytokines such as IL-6 and leukemia inhibitory factor (LIF) were defined as iCAF [13]. McAndrews et al. [14] found that depletion of FAP+ CAFs resulted in a survival benefit, while depletion of α-SMA+ CAFs caused increased mortality in mouse models of PDAC. The oncogenic FAP+ CAFs and anti-tumor α-SMA+ CAFs were proved to regulate cancer-related pathways and regulatory T cells via different mechanisms. Elyada et al. [15] identified a subtype of apCAFs with MHCII expression and antigen-presenting capability in KPC PDAC mice, which could present the model antigen to the CD4+ T cells, decrease the CD8+T/Treg ratio, and contribute to immune suppression in the TME. The apCAFs could also transform into myCAFs under appropriate culture conditions, which implied that CAFs were in a dynamic cell state. Lin et al. [16] confirmed the presence of three CAF subtypes (myCAF, iCAF, and apCAF) in human PDAC tissues by single-cell transcriptomic technology. The subtype of CAFs with high Meflin expression was reported to correlate with a favorable outcome in both mouse models or patients with PDAC, and deficiency or low expression of Meflin resulted in straightened stromal collagen fibers, indicating that Meflin was a marker of cancer-restraining CAFs that suppressed the progression of PDAC [17]. Chen et al. [18] identified a subset of complement-secreting CAFs (csCAFs) which were located in the tissue stroma adjacent to tumor cells only in early PDAC and specifically expressed complement system components.
    PSCs were found to produce myeloid-derived suppressor cell (MDSC)-promoting cytokines (interleukins, IL-6; VEGF; macrophage colony-stimulating factor, M-CSF) and chemokines (SDF-1, MCP-1) to promote differentiation of immune cells into the functional MDSC phenotypes to prevent innate or adaptive immune responses against cancer cells, among which IL-6 was the key factor contributing to STAT3 signaling and MDSC differentiation [19]. CAFs were also reported to upregulate the expression of immune checkpoints PD-1, cytotoxic lymphocyte-associated antigen-4 (CTLA-4), T-cell immunoglobulin, mucin-domain containing-3 (TIM-3), and lymphocyte-activation gene-3 (LAG-3) in both CD4+ and CD8+ T cells, thereby inhibiting the proliferation of T cells and diminishing immune function in PDAC [20].
    The specific mechanisms by which CAFs regulate tumor progression have not yet been fully clarified. More and more evidence has shown that the activation mechanisms of different CAF subtypes varied widely, and the dominant subtype of CAFs could dynamically change in response to different biological signals in the TME of PDAC or different in vitro culture conditions [10][21]. Along with the understanding of the differentiation and regulational mechanism of CAFs and their effect on tumor cells in the TME of PDAC, novel therapeutic drugs targeting CAFs modulation may be developed to transform the tumor-promoting microenvironment into a tumor-suppressing microenvironment, thereby increasing the sensitivity of immunotherapy.

    3. Regulatory Mechanism of CAFs Differentiation and Activation in PDAC

    In a healthy pancreas, fibroblasts exist mainly in a quiescent state. During the progress of carcinogenesis, quiescent fibroblasts transform into active CAFs through the activation of distinct signaling pathways with the participation of miRNAs, cytokines, paracrine lactate, and growth factors secreted by tumor cells.

    3.1. Tumor Cells Promote the Differentiation and Activation of CAFs

    Tumor cells can secrete connective tissue growth factor (CTGF) to promote the activation, proliferation, migration, and adhesion of PSCs and fibroproliferation [22]. Biffi et al. [23] found that PDAC cells could secrete IL1α to activate PSCs and promote the formation of iCAFs by activating the IL1/JAK/STAT signaling pathway, and tumor cells could also increase the expression of inflammatory cytokines and chemokines like IL1a, IL6, and LIF in iCAFs, which promoted cancer progression and formed an immunosuppressive microenvironment. Moreover, PDAC cells secreted TGFβ to upregulate the expression of α-SMA in PSCs, and thus transformed the quiescent PSCs to myCAF, promoting the formation of a dense extracellular matrix [23]. The presence of oncogenic KRAS mutation was also reported to be involved in the activation of CAFs and to regulate tumor cell signaling via stromal cells [24]. Awaji [25] found that KRAS mutation could promote the secretion of paracrine factors such as IL4, IL10, and IL13 in tumor cells and simultaneously activate CXCR2 signaling in CAFs, resulting in the formation of a secreted CAF phenotype by activating NF-κB signaling and promoting tumor progression. The paracrine lactate secreted by PDAC cells could increase alpha-ketoglutarate (aKG) production in MSCs, which decreased cytosine methylation and increased hydroxymethylation during the differentiation of MSCs into CAFs by mediating the activation of the demethylase enzyme ten-eleven translocation (TET) [26]. Additionally, the reactive oxygen species (ROS) associated with the hypoxic TME of PDAC could also promote the activation of PSCs by stabilizing HIF-1α and increasing the expression of GLI1 [27].

    3.2. Aberrant Expression of miRNAs Was Involved in the Activation of CAFs

    Studies have revealed that the aberrant expression of miRNAs in PDAC played important roles in the activation of CAFs in the TME. Xu et al. [28] reported that miR-200a could attenuate the activation of PSC induced by TGF-β1 and inhibit ECM formation through the PTEN/Akt/mTOR pathway. Inhibition of miR-199a-3p and miR-214-3p was found to be involved in the de-differentiation of CAFs which could reduce the differentiation of PSCs into myofibroblasts [29]. Chu et al. indicated that the overexpression of miR-224 in pancreatic fibroblasts can significantly increase their proliferation, migration, and invasion ability, thus promoting the activation of CAFs [30]. Overexpression of miR-21 contributed to the activation of CAFs by regulating the PDCD4 gene and increased the invasion ability of PDAC cells by stimulating the secretion of MMP-3, MMP-9, CCL-7, and PDGF in CAFs [31]. In a mouse model of alcoholic chronic pancreatitis (CP), Charrier et al. [32] reported that the expression of miR-21 was significantly elevated in activated PSCs, which promoted the expression of connective tissue growth factor (CCN2), and a positive feedback loop between miR-21 and CCN2 was found to increase the expression of collagen and the activation of quiescent PSCs. MiR-301a was also involved in the activation of PSCs by inhibiting Gadd45g expression and promoting STAT3 activation during the pancreatic intraepithelial neoplasia lesion formation [33].

    3.3. Exosomal miRNAs Promote the Activation of CAFs in TME of PDAC

    The specifically enriched miRNAs in the exosomes play vital roles as messengers of cell-to-cell interaction in the TME. Pang et al. [34] reported that PDAC cells delivered miR-155 into fibroblasts via exosomes, which could inhibit the expression of the target gene TP53INP1 and reprogram the quiescent fibroblasts into CAFs. MiR-1246 and miR-1290 were highly enriched in PDAC cell-derived exosomes, which could be transferred into PSCs to induce the activation of PSCs and the occurrence of pro-fibrosis, including proliferation, migration, collagen production, and expression of α-SMA [35].
    In summary, the activation of CAFs is essential to form the immunosuppressive TME in PDAC. In the process of carcinogenesis, the differentiation and activation of CAFs was modulated by complex mechanisms, among which exosomal miRNAs derived from tumor cells play important roles, although the specific mechanism remains to be further studied.

    References

    1. Ziani, L.; Chouaib, S.; Thiery, J. Alteration of the Antitumor Immune Response by Cancer-Associated Fibroblasts. Front. Immunol. 2018, 9, 414.
    2. Sahai, E.; Astsaturov, I.; Cukierman, E.; DeNardo, D.G.; Egeblad, M.; Evans, R.M.; Fearon, D.; Greten, F.R.; Hingorani, S.R.; Hunter, T.; et al. A framework for advancing our understanding of cancer-associated fibroblasts. Nat. Rev. Cancer 2020, 20, 174–186.
    3. Kanzaki, R.; Pietras, K. Heterogeneity of cancer-associated fibroblasts: Opportunities for precision medicine. Cancer Sci. 2020, 111, 2708–2717.
    4. Bu, L.; Baba, H.; Yoshida, N.; Miyake, K.; Yasuda, T.; Uchihara, T.; Tan, P.; Ishimoto, T. Biological heterogeneity and versatility of cancer-associated fibroblasts in the tumor microenvironment. Oncogene 2019, 38, 4887–4901.
    5. Kobayashi, H.; Enomoto, A.; Woods, S.L.; Burt, A.D.; Takahashi, M.; Worthley, D.L. Cancer-associated fibroblasts in gastrointestinal cancer. Nat. Rev. Gastroenterol. Hepatol. 2019, 16, 282–295.
    6. Nurmik, M.; Ullmann, P.; Rodriguez, F.; Haan, S.; Letellier, E. In search of definitions: Cancer-associated fibroblasts and their markers. Int. J. Cancer 2020, 146, 895–905.
    7. Bynigeri, R.R.; Jakkampudi, A.; Jangala, R.; Subramanyam, C.; Sasikala, M.; Rao, G.V.; Reddy, D.N.; Talukdar, R. Pancreatic stellate cell: Pandora’s box for pancreatic disease biology. World J. Gastroenterol. 2017, 23, 382–405.
    8. Mathew, E.; Brannon, A.L.; Del Vecchio, A.; Garcia, P.E.; Penny, M.K.; Kane, K.T.; Vinta, A.; Buckanovich, R.J.; di Magliano, M.P. Mesenchymal Stem Cells Promote Pancreatic Tumor Growth by Inducing Alternative Polarization of Macrophages. Neoplasia 2016, 18, 142–151.
    9. Huang, H.; Wang, Z.; Zhang, Y.; Pradhan, R.N.; Ganguly, D.; Chandra, R.; Murimwa, G.; Wright, S.; Gu, X.; Maddipati, R.; et al. Mesothelial cell-derived antigen-presenting cancer-associated fibroblasts induce expansion of regulatory T cells in pancreatic cancer. Cancer Cell 2022, 40, 656–673 e657.
    10. Miyazaki, Y.; Oda, T.; Mori, N.; Kida, Y.S. Adipose-derived mesenchymal stem cells differentiate into pancreatic cancer-associated fibroblasts in vitro. FEBS Open Bio 2020, 10, 2268–2281.
    11. Waghray, M.; Yalamanchili, M.; Dziubinski, M.; Zeinali, M.; Erkkinen, M.; Yang, H.; Schradle, K.A.; Urs, S.; Pasca Di Magliano, M.; Welling, T.H.; et al. GM-CSF Mediates Mesenchymal-Epithelial Cross-talk in Pancreatic Cancer. Cancer Discov. 2016, 6, 886–899.
    12. Park, D.; Sahai, E.; Rullan, A. SnapShot: Cancer-Associated Fibroblasts. Cell 2020, 181, 486–486.e1.
    13. Ohlund, D.; Handly-Santana, A.; Biffi, G.; Elyada, E.; Almeida, A.S.; Ponz-Sarvise, M.; Corbo, V.; Oni, T.E.; Hearn, S.A.; Lee, E.J.; et al. Distinct populations of inflammatory fibroblasts and myofibroblasts in pancreatic cancer. J. Exp. Med. 2017, 214, 579–596.
    14. McAndrews, K.M.; Chen, Y.; Darpolor, J.K.; Zheng, X.; Yang, S.; Carstens, J.L.; Li, B.; Wang, H.; Miyake, T.; Correa de Sampaio, P.; et al. Identification of Functional Heterogeneity of Carcinoma-Associated Fibroblasts with Distinct IL6-Mediated Therapy Resistance in Pancreatic Cancer. Cancer Discov. 2022, 12, 1580–1597.
    15. Elyada, E.; Bolisetty, M.; Laise, P.; Flynn, W.F.; Courtois, E.T.; Burkhart, R.A.; Teinor, J.A.; Belleau, P.; Biffi, G.; Lucito, M.S.; et al. Cross-Species Single-Cell Analysis of Pancreatic Ductal Adenocarcinoma Reveals Antigen-Presenting Cancer-Associated Fibroblasts. Cancer Discov. 2019, 9, 1102–1123.
    16. Lin, W.; Noel, P.; Borazanci, E.H.; Lee, J.; Amini, A.; Han, I.W.; Heo, J.S.; Jameson, G.S.; Fraser, C.; Steinbach, M.; et al. Single-cell transcriptome analysis of tumor and stromal compartments of pancreatic ductal adenocarcinoma primary tumors and metastatic lesions. Genome Med. 2020, 12, 80.
    17. Mizutani, Y.; Kobayashi, H.; Iida, T.; Asai, N.; Masamune, A.; Hara, A.; Esaki, N.; Ushida, K.; Mii, S.; Shiraki, Y.; et al. Meflin-Positive Cancer-Associated Fibroblasts Inhibit Pancreatic Carcinogenesis. Cancer Res. 2019, 79, 5367–5381.
    18. Chen, K.; Wang, Q.; Li, M.; Guo, H.; Liu, W.; Wang, F.; Tian, X.; Yang, Y. Single-cell RNA-seq reveals dynamic change in tumor microenvironment during pancreatic ductal adenocarcinoma malignant progression. EBioMedicine 2021, 66, 103315.
    19. Mace, T.A.; Ameen, Z.; Collins, A.; Wojcik, S.; Mair, M.; Young, G.S.; Fuchs, J.R.; Eubank, T.D.; Frankel, W.L.; Bekaii-Saab, T.; et al. Pancreatic cancer-associated stellate cells promote differentiation of myeloid-derived suppressor cells in a STAT3-dependent manner. Cancer Res. 2013, 73, 3007–3018.
    20. Gorchs, L.; Fernandez Moro, C.; Bankhead, P.; Kern, K.P.; Sadeak, I.; Meng, Q.; Rangelova, E.; Kaipe, H. Human Pancreatic Carcinoma-Associated Fibroblasts Promote Expression of Co-inhibitory Markers on CD4(+) and CD8(+) T-Cells. Front. Immunol. 2019, 10, 847.
    21. Sunami, Y.; Haussler, J.; Kleeff, J. Cellular Heterogeneity of Pancreatic Stellate Cells, Mesenchymal Stem Cells, and Cancer-Associated Fibroblasts in Pancreatic Cancer. Cancers 2020, 12, 3770.
    22. Charrier, A.; Brigstock, D.R. Regulation of pancreatic function by connective tissue growth factor (CTGF, CCN2). Cytokine Growth Factor Rev. 2013, 24, 59–68.
    23. Biffi, G.; Oni, T.E.; Spielman, B.; Hao, Y.; Elyada, E.; Park, Y.; Preall, J.; Tuveson, D.A. IL1-Induced JAK/STAT Signaling Is Antagonized by TGFbeta to Shape CAF Heterogeneity in Pancreatic Ductal Adenocarcinoma. Cancer Discov. 2019, 9, 282–301.
    24. Tape, C.J.; Ling, S.; Dimitriadi, M.; McMahon, K.M.; Worboys, J.D.; Leong, H.S.; Norrie, I.C.; Miller, C.J.; Poulogiannis, G.; Lauffenburger, D.A.; et al. Oncogenic KRAS Regulates Tumor Cell Signaling via Stromal Reciprocation. Cell 2016, 165, 910–920.
    25. Awaji, M.; Saxena, S.; Wu, L.; Prajapati, D.R.; Purohit, A.; Varney, M.L.; Kumar, S.; Rachagani, S.; Ly, Q.P.; Jain, M.; et al. CXCR2 signaling promotes secretory cancer-associated fibroblasts in pancreatic ductal adenocarcinoma. FASEB J. 2020, 34, 9405–9418.
    26. Bhagat, T.D.; Von Ahrens, D.; Dawlaty, M.; Zou, Y.; Baddour, J.; Achreja, A.; Zhao, H.; Yang, L.; Patel, B.; Kwak, C.; et al. Lactate-mediated epigenetic reprogramming regulates formation of human pancreatic cancer-associated fibroblasts. Elife 2019, 8, e50663.
    27. Lei, J.; Huo, X.; Duan, W.; Xu, Q.; Li, R.; Ma, J.; Li, X.; Han, L.; Li, W.; Sun, H.; et al. alpha-Mangostin inhibits hypoxia-driven ROS-induced PSC activation and pancreatic cancer cell invasion. Cancer Lett. 2014, 347, 129–138.
    28. Xu, M.; Wang, G.; Zhou, H.; Cai, J.; Li, P.; Zhou, M.; Lu, Y.; Jiang, X.; Huang, H.; Zhang, Y.; et al. TGF-beta1-miR-200a-PTEN induces epithelial-mesenchymal transition and fibrosis of pancreatic stellate cells. Mol. Cell Biochem. 2017, 431, 161–168.
    29. Kuninty, P.R.; Bojmar, L.; Tjomsland, V.; Larsson, M.; Storm, G.; Ostman, A.; Sandstrom, P.; Prakash, J. MicroRNA-199a and -214 as potential therapeutic targets in pancreatic stellate cells in pancreatic tumor. Oncotarget 2016, 7, 16396–16408.
    30. Chu, N.J.; Anders, R.A.; Fertig, E.J.; Cao, M.; Hopkins, A.C.; Keenan, B.P.; Popovic, A.; Armstrong, T.D.; Jaffee, E.M.; Zimmerman, J.W. Inhibition of miR-21 Regulates Mutant KRAS Effector Pathways and Intercepts Pancreatic Ductal Adenocarcinoma Development. Cancer Prev. Res. 2020, 13, 569–582.
    31. Zhang, L.; Yao, J.; Li, W.; Zhang, C. Micro-RNA-21 Regulates Cancer-Associated Fibroblast-Mediated Drug Resistance in Pancreatic Cancer. Oncol. Res. 2018, 26, 827–835.
    32. Charrier, A.; Chen, R.; Chen, L.; Kemper, S.; Hattori, T.; Takigawa, M.; Brigstock, D.R. Connective tissue growth factor (CCN2) and microRNA-21 are components of a positive feedback loop in pancreatic stellate cells (PSC) during and are exported in PSC-derived exosomes. J. Cell Commun. Signal. 2014, 8, 147–156.
    33. Li, F.; Wang, M.; Li, X.; Long, Y.; Chen, K.; Wang, X.; Zhong, M.; Cheng, W.; Tian, X.; Wang, P.; et al. Inflammatory-miR-301a circuitry drives mTOR and Stat3-dcotarget PSC activation in chronic pancreatitis and PanIN. Mol. Ther.-Nucleic Acids 2022, 27, 970–982.
    34. Pang, W.; Su, J.; Wang, Y.; Feng, H.; Dai, X.; Yuan, Y.; Chen, X.; Yao, W. Pancreatic cancer-secreted miR-155 implicates in the conversion from normal fibroblasts to cancer-associated fibroblasts. Cancer Sci. 2015, 106, 1362–1369.
    35. Masamune, A.; Yoshida, N.; Hamada, S.; Takikawa, T.; Nabeshima, T.; Shimosegawa, T. Exosomes derived from pancreatic cancer cells induce activation and profibrogenic activities in pancreatic stellate cells. Biochem. Biophys. Res. Commun. 2018, 495, 71–77.
    More
    Information
    Subjects: Surgery
    Contributors MDPI registered users' name will be linked to their SciProfiles pages. To register with us, please refer to https://encyclopedia.pub/register : , ,
    View Times: 71
    Revisions: 2 times (View History)
    Update Time: 02 Sep 2022
    Table of Contents
      1000/1000

      Confirm

      Are you sure to Delete?

      Video Upload Options

      Do you have a full video?
      Cite
      If you have any further questions, please contact Encyclopedia Editorial Office.
      Chu, X.; Yang, Y.; Tian, X. Roles of CAFs in the TME of PDAC. Encyclopedia. Available online: https://encyclopedia.pub/entry/26772 (accessed on 29 November 2022).
      Chu X, Yang Y, Tian X. Roles of CAFs in the TME of PDAC. Encyclopedia. Available at: https://encyclopedia.pub/entry/26772. Accessed November 29, 2022.
      Chu, Xiangyu, Yinmo Yang, Xiaodong Tian. "Roles of CAFs in the TME of PDAC," Encyclopedia, https://encyclopedia.pub/entry/26772 (accessed November 29, 2022).
      Chu, X., Yang, Y., & Tian, X. (2022, September 01). Roles of CAFs in the TME of PDAC. In Encyclopedia. https://encyclopedia.pub/entry/26772
      Chu, Xiangyu, et al. ''Roles of CAFs in the TME of PDAC.'' Encyclopedia. Web. 01 September, 2022.
      Top
      Feedback