Decoding the molecular landscape of keloids: new insights from single-cell transcriptomics

Abstract

Keloids are a fibrotic disease caused by an excessive accumulation of extracellular matrix in the dermis; they have neoplasia-like properties of aggressive growth and high posttreatment recurrence rates. Therefore, it is imperative to gain additional insight into the pathobiology of keloid formation. Single-cell RNA sequencing (scRNA-seq) technology has brought data-driven innovation to understanding the pathogenesis of keloids by breaking the limitations of traditional sequencing technologies to resolve cell composition and to distinguish functional cell subtypes at an unprecedented resolution. The present review aims to cover the application of scRNA-seq technology in keloids and its exploratory findings, including the depiction of the cellular landscape of keloids, fibroblast heterogeneity, the lineage development of Schwann cells and the mesenchymal-activation phenomenon of endothelial cells. Furthermore, scRNA-seq records the transcriptional profiles of fibroblasts and immune cells in a more refined manner, and this gene expression information provides excellent material for inferring intercellular communication networks and lays an important theoretical foundation for future studies.

Keywords

Keloid / scRNA-seq / Fibroblast / Schwann cell / Immune cell / Transcriptomics

References

[1] Ud-Din S, Bayat A. Keloid scarring or disease: unresolved quasi-neoplastic tendencies in the human skin. Wound Repair Regen. 2020; 28: 422-6.

[2] Davies P, Cuttle L, Young A.A scoping review of the methodology used in studies of genetic influences on the development of keloid or hypertrophic scarring in adults and children after acute wounding. Adv Wound Care (New Rochelle). 2021; 10: 557-70.

[3] Carroll W, Patel K. Steroids and fluorouracil for keloids and hypertrophic scars. JAMA Facial Plast Surg. 2015; 17: 77-9.

[4] Ekstein SF, Wyles SP, Moran SL, Meves A. Keloids: a review of therapeutic management. Int J Dermatol. 2021; 60: 661-71.

[5] Carantino I, Florescu IP, Carantino A. Overview about the keloid scars and the elaboration of a non-invasive, unconventional treatment. J Med Life. 2010; 3: 122-7.

[6] Sigaux N, Jacquemart M, Cousin AS, Lorchel F, Breton P. Association of surgical excision and brachytherapy for the management of keloids. J Stomatol Oral Maxillofac Surg. 2017; 118: 161-6.

[7] Wang X, Liang B, Li J, Pi X, Zhang P, Zhou X, et al. Identification and characterization of four immune-related signatures in keloid. Front Immunol. 2022; 13: 942446.

[8] Duan X, Wu Y, Zhang Z, Lu Z. Identification and analysis of dysregulated lncRNA and associated ceRNA in the pathogenesis of keloid. Ann Transl Med. 2020; 8: 222.

[9] Lv W, Ren Y, Hou K, Hu W, Yi Y, Xiong M, et al. Epigenetic modification mechanisms involved in keloid: current status and prospect. Clin Epigenetics. 2020; 12: 183.

[10] Shi J, Yao S, Chen P, Yang Y, Qian M, Han Y, et al. The integrative regulatory network of circRNA and microRNA in keloid scarring. Mol Biol Rep. 2020; 47: 201-9.

[11] Zhong L, Bian L, Lyu J, Jin H, Liu Z, Lyu L, et al. Identification and integrated analysis of microRNA expression profiles in keloid. J Cosmet Dermatol. 2018; 17: 917-24.

[12] Deng Y, Xu Y, Xu S, Zhang Y, Han B, Liu Z, et al. Secondary data mining of GEO database for long non-coding RNA and competing endogenous RNA network in keloid-prone individuals. Aging (Albany NY). 2020; 12: 25076-89.

[13] Liu Y, Wang X, Ni Z, Li Y, Song J, Zhu F, et al. Circular RNA hsa_circ_0043688 serves as a competing endogenous RNA for microRNA-145-5p to promote the progression of keloids via fibroblast growth factor-2. J Clin Lab Anal. 2022; 36: e24528.

[14] Jones LR, Greene J, Chen KM, Divine G, Chitale D, Shah V, et al. Biological significance of genome-wide DNA methylation profiles in keloids. Laryngoscope. 2017; 127: 70-8.

[15] Shen X, Zhao Y, Wang Z, Shi Q. Recent advances in high-throughput single-cell transcriptomics and spatial transcriptomics. Lab Chip. 2022; 22: 4774-91.

[16] Ke Y, Jian-Yuan H, Ping Z, Yue W, Na X, Jian Y, et al. The progressive application of single-cell RNA sequencing technology in cardiovascular diseases. Biomed Pharmacother. 2022; 154: 113604.

[17] Vegliante R, Pastushenko I, Blanpain C. Deciphering functional tumor states at single-cell resolution. EMBO J. 2022; 41: e109221.

[18] Andrews TS, Hemberg M. Identifying cell populations with scRNASeq. Mol Asp Med. 2018; 59: 114-22.

[19] Andrews TS, Kiselev VY, McCarthy D, Hemberg M. Tutorial: guidelines for the computational analysis of single-cell RNA sequencing data. Nat Protoc. 2021; 16: 1-9.

[20] Svensson V, Natarajan KN, Ly LH, Miragaia RJ, Labalette C, Macaulay IC, et al. Power analysis of single-cell RNA-sequencing experiments. Nat Methods. 2017; 14: 381-7.

[21] Kurimoto K, Yabuta Y, Ohinata Y, Saitou M. Global single-cell cDNA amplification to provide a template for representative high-density oligonucleotide microarray analysis. Nat Protoc. 2007; 2: 739-52.

[22] Islam S, Zeisel A, Joost S, La Manno G, Zajac P, Kasper M, et al. Quantitative single-cell RNA-seq with unique molecular identifiers. Nat Methods. 2014; 11: 163-6.

[23] Ziegenhain C, Vieth B, Parekh S, Reinius B, Guillaumet-Adkins A, Smets M, et al. Comparative analysis of single-cell RNA sequencing methods. Mol Cell. 2017; 65: 631-643.e4.

[24] Chen G, Ning B, Shi T. Single-cell RNA-Seq technologies and related computational data analysis. Front Genet. 2019; 10: 317.

[25] Zhang Z, Cui F, Wang C, Zhao L, Zou Q. Goals and approaches for each processing step for single-cell RNA sequencing data. Brief Bioinform. 2021; 22: bbaa314.

[26] Herring CA, Chen B, McKinley ET, Lau KS. Single-cell computational strategies for lineage reconstruction in tissue systems. Cell Mol Gastroenterol Hepatol. 2018; 5: 539-48.

[27] Liu J, Fan Z, Zhao W, Zhou X. Machine intelligence in single-cell data analysis: advances and new challenges. Front Genet. 2021; 12: 655536.

[28] Sommerfeld SD, Cherry C, Schwab RM, Chung L, Maestas DR Jr, Laffont P, et al. Interleukin-36γ-producing macrophages drive IL-17-mediated fibrosis. Sci Immunol. 2019; 4: eaax4783.

[29] Martos SN, Campbell MR, Lozoya OA, Wang X, Bennett BD, Thompson IJB, et al. Single-cell analyses identify dysfunctional CD16(+) CD8 T cells in smokers. Cell Rep Med. 2020; 1: 100054.

[30] Limandjaja GC, Niessen FB, Scheper RJ, Gibbs S. The keloid disorder: heterogeneity, histopathology, mechanisms and models. Front Cell Dev Biol. 2020; 8: 360.

[31] Deng CC, Hu YF, Zhu DH, Cheng Q, Gu JJ, Feng QL, et al. Single-cell RNA-seq reveals fibroblast heterogeneity and increased mesenchymal fibroblasts in human fibrotic skin diseases. Nat Commun. 2021; 12: 3709.

[32] Direder M, Weiss T, Copic D, Vorstandlechner V, Laggner M, Pfisterer K, et al. Schwann cells contribute to keloid formation. Matrix Biol. 2022; 108: 55-76.

[33] Griffin MF, desJardins-Park HE, Mascharak S, Borrelli MR, Longaker MT. Understanding the impact of fibroblast heterogeneity on skin fibrosis. Dis Model Mech. 2020; 13: dmm044164.

[34] Andrews JP, Marttala J, Macarak E, Rosenbloom J, Uitto J. Keloids: the paradigm of skin fibrosis-Pathomechanisms and treatment. Matrix Biol. 2016; 51: 37-46.

[35] Wang Q, Zhong Y, Li Z, Zhu D, Lu H, Chen P, et al. Multi-transcriptome analyses of keloid fibroblasts reveal the role of the HIF-1α/HOXC6/ERK axis in keloid development. Burns Dent Traumatol. 2022; 10: tkac013.

[36] Solé-Boldo L, Raddatz G, Schütz S, Mallm JP, Rippe K, Lonsdorf AS, et al. Single-cell transcriptomes of the human skin reveal age-related loss of fibroblast priming. Commun Biol. 2020; 3: 188.

[37] Shim J, Oh SJ, Yeo E, Park JH, Bae JH, Kim SH, et al. Integrated analysis of single-cell and spatial Transcriptomics in keloids: highlights on Fibrovascular interactions in keloid pathogenesis. J Invest Dermatol. 2022; 142: 2128-2139.e11.

[38] Dehne T, Karlsson C, Ringe J, Sittinger M, Lindahl A.Chondrogenic differentiation potential of osteoarthritic chondrocytes and their possible use in matrix-associated autologous chondrocyte transplantation. Arthritis Res Ther. 2009; 11: R133.

[39] Sieker JT, Proffen BL, Waller KA, Chin KE, Karamchedu NP, Akelman MR, et al. Transcriptional profiling of articular cartilage in a porcine model of early post-traumatic osteoarthritis. J Orthop Res. 2018; 36: 318-29.

[40] Newman AAC, Serbulea V, Baylis RA, Shankman LS, Bradley X, Alencar GF, et al. Multiple cell types contribute to the atherosclerotic lesion fibrous cap by PDGFRβ and bioenergetic mechanisms. Nat Metab. 2021; 3: 166-81.

[41] Lagares D, Ghassemi-Kakroodi P, Tremblay C, Santos A, Probst CK, Franklin A, et al. ADAM10-mediated ephrin-B2 shedding promotes myofibroblast activation and organ fibrosis. Nat Med. 2017; 23: 1405-15.

[42] Wynn TA. Cellular and molecular mechanisms of fibrosis. J Pathol. 2008; 214: 199-210.

[43] Guillamat-Prats R. The role of MSC in wound healing, scarring and regeneration. Cell. 2021; 10: 1729.

[44] Jo H, Brito S, Kwak BM, Park S, Lee MG, Bin BH. Applications of mesenchymal stem cells in skin regeneration and rejuvenation. Int J Mol Sci. 2021; 22: 2410.

[45] Smith AN, Willis E, Chan VT, Muffley LA, Isik FF, Gibran NS, et al. Mesenchymal stem cells induce dermal fibroblast responses to injury. Exp Cell Res. 2010; 316: 48-54.

[46] Tutuianu R, Rosca AM, Iacomi DM, Simionescu M, Titorencu I. Human mesenchymal stromal cell-derived exosomes promote in vitro wound healing by modulating the biological properties of skin keratinocytes and fibroblasts and stimulating angiogenesis. Int J Mol Sci. 2021; 22: 6239.

[47] Tanaka K, Ogino R, Yamakawa S, Suda S, Hayashida K. Role and function of mesenchymal stem cells on fibroblast in cutaneous wound healing. Biomedicine. 2022; 10: 1391.

[48] Takeo M, Lee W, Ito M. Wound healing and skin regeneration. Cold Spring Harb Perspect Med. 2015; 5: a023267.

[49] Jumper N, Paus R, Bayat A. Functional histopathology of keloid disease. Histol Histopathol. 2015; 30: 1033-57.

[50] El Agha E, Kramann R, Schneider RK, Li X, Seeger W, Humphreys BD, et al. Mesenchymal stem cells in fibrotic disease. Cell Stem Cell. 2017; 21: 166-77.

[51] Hochman B, Nahas FX, Sobral CS, Arias V, Locali RF, Juliano Y, et al. Nerve fibres: a possible role in keloid pathogenesis. Br J Dermatol. 2008; 158: 651-2.

[52] Saffari TM, Bijlard E, Van Bodegraven EAM, Mureau MAM, Hovius SER, Huygen F. Sensory perception and nerve fibre innervation in patients with keloid scars: an investigative study. Eur J Dermatol. 2018; 28: 828-9.

[53] Reinisch CM, Tschachler E. The dimensions and characteristics of the subepidermal nerve plexus in human skin-terminal Schwann cells constitute a substantial cell population within the superficial dermis. J Dermatol Sci. 2012; 65: 162-9.

[54] Campana WM. Schwann cells: activated peripheral glia and their role in neuropathic pain. Brain Behav Immun. 2007; 21: 522-7.

[55] Min Q, Parkinson DB, Dun XP. Migrating Schwann cells direct axon regeneration within the peripheral nerve bridge. Glia. 2021; 69: 235-54.

[56] Nave KA, Werner HB. Myelination of the nervous system: mechanisms and functions. Annu Rev Cell Dev Biol. 2014; 30: 503-33.

[57] Weiss T, Taschner-Mandl S, Janker L, Bileck A, Rifatbegovic F, Kromp F, et al. Schwann cell plasticity regulates neuroblastic tumor cell differentiation via epidermal growth factor-like protein 8. Nat Commun. 2021; 12: 1624.

[58] Clements MP, Byrne E, Camarillo Guerrero LF, Cattin AL, Zakka L, Ashraf A, et al. The wound microenvironment reprograms Schwann cells to invasive mesenchymal-like cells to drive peripheral nerve regeneration. Neuron. 2017; 96: 98-114.e7.

[59] Shea GK, Tsui AY, Chan YS, Shum DK.Bone marrow-derived Schwann cells achieve fate commitment-a prerequisite for remyelination therapy. Exp Neurol. 2010; 224: 448-58.

[60] Gong T, Wang Y, Dong S, Ma X, Du D, Zou C, et al. Single-cell RNA-seq reveals the communications between extracellular matrix-related components and Schwann cells contributing to the earlobe keloid formation. Front Med (Lausanne). 2022; 9: 1000324.

[61] Wang ZC, Zhao WY, Cao Y, Liu YQ, Sun Q, Shi P, et al. The roles of inflammation in keloid and hypertrophic scars. Front Immunol. 2020; 11: 603187.

[62] Wu J, Del Duca E, Espino M, Gontzes A, Cueto I, Zhang N, et al. RNA sequencing keloid transcriptome associates keloids with Th2, Th1, Th17/Th22, and JAK3-skewing. Front Immunol. 2020; 11: 597741.

[63] Ogawa R. Keloid and hypertrophic scars are the result of chronic inflammation in the reticular dermis. Int J Mol Sci. 2017; 18: 606.

[64] Wang HT, Han JT, Hu DH. Research advances on the role and mechanism of inflammatory response in the formation of hypertrophic scars and keloids. Zhonghua Shao Shang Za Zhi. 2021; 37: 490-4.

[65] Zhang D, Li B, Zhao M. Therapeutic strategies by regulating interleukin family to suppress inflammation in hypertrophic scar and keloid. Front Pharmacol. 2021; 12: 667763.

[66] Zhang T, Wang XF, Wang ZC, Lou D, Fang QQ, Hu YY, et al. Current potential therapeutic strategies targeting the TGF-β/Smad signaling pathway to attenuate keloid and hypertrophic scar formation. Biomed Pharmacother. 2020; 129: 110287.

[67] Feng C, Shan M, Xia Y, Zheng Z, He K, Wei Y, et al. Single-cell RNA sequencing reveals distinct immunology profiles in human keloid. Front Immunol. 2022; 13: 940645.

[68] Li Z, Srivastava P. Heat-shock proteins. Curr Protoc Immunol. 2004; Appendix 1. 10.1002/0471142735.ima01ts58.

[69] Schulz R, Moll UM. Targeting the heat shock protein 90: a rational way to inhibit macrophage migration inhibitory factor function in cancer. Curr Opin Oncol. 2014; 26: 108-13.

[70] Constantinides MG, Link VM, Tamoutounour S, Wong AC, Perez-Chaparro PJ, Han SJ, et al. MAIT cells are imprinted by the microbiota in early life and promote tissue repair. Science. 2019; 366: eaax6624.

[71] Legoux F, Salou M, Lantz O. MAIT cell development and functions: the microbial connection. Immunity. 2020; 53: 710-23.

[72] Farhood B, Najafi M, Mortezaee K. CD8(+) cytotoxic T lymphocytes in cancer immunotherapy: a review. J Cell Physiol. 2019; 234: 8509-21.

[73] Halle S, Halle O, Förster R. Mechanisms and dynamics of T cell-mediated cytotoxicity in vivo. Trends Immunol. 2017; 38: 432-43.

[74] Dees S, Ganesan R, Singh S, Grewal IS. Regulatory T cell targeting in cancer: emerging strategies in immunotherapy. Eur J Immunol. 2021; 51: 280-91.

[75] Jiang W, He Y, He W, Wu G, Zhou X, Sheng Q, et al. Exhausted CD8+T cells in the tumor immune microenvironment: new pathways to therapy. Front Immunol. 2020; 11: 622509.

[76] Xu H, Zhu Z, Hu J, Sun J, Wo Y, Wang X, et al. Downregulated cytotoxic CD8(+) T-cell identifies with the NKG2A-soluble HLA-E axis as a predictive biomarker and potential therapeutic target in keloids. Cell Mol Immunol. 2022; 19: 527-39.

[77] Salomé B, Sfakianos JP, Ranti D, Daza J, Bieber C, Charap A, et al. NKG2A and HLA-E define an alternative immune checkpoint axis in bladder cancer. Cancer Cell. 2022; 40: 1027-1043.e1029.

[78] Sivori S, Vacca P, Del Zotto G, Munari E, Mingari MC, Moretta L. Human NK cells: surface receptors, inhibitory checkpoints, and translational applications. Cell Mol Immunol. 2019; 16: 430-41.

[79] van Montfoort N, Borst L, Korrer MJ, Sluijter M, Marijt KA, Santegoets SJ, et al. NKG2A blockade potentiates CD8 T cell immunity induced by cancer vaccines. Cell. 2018; 175: 1744-1755.e1715.

[80] Das A, Sinha M, Datta S, Abas M, Chaffee S, Sen CK, et al. Monocyte and macrophage plasticity in tissue repair and regeneration. Am J Pathol. 2015; 185: 2596-606.

[81] Kratofil RM, Kubes P, Deniset JF. Monocyte conversion during inflammation and injury. Arterioscler Thromb Vasc Biol. 2017; 37: 35-42.

[82] Anderson DA 3rd, Murphy KM, Briseño CG.Development, diversity, and function of dendritic cells in mouse and human. Cold Spring Harb Perspect Biol. 2018; 10: a028613.

[83] Del Prete A, Sozio F, Barbazza I, Salvi V, Tiberio L, Laffranchi M, et al. Functional role of dendritic cell subsets in cancer progression and clinical implications. Int J Mol Sci. 2020; 21: 3930.

[84] Saito Y, Komori S, Kotani T, Murata Y, Matozaki T. The role of Type-2 conventional dendritic cells in the regulation of tumor immunity. Cancers (Basel). 2022; 14: 1976.

[85] Wang N, Liang H, Zen K. Molecular mechanisms that influence the macrophage m1-m2 polarization balance. Front Immunol. 2014; 5: 614.

[86] Li YL, Wu LW, Zeng LH, Zhang ZY, Wang W, Zhang C, et al. ApoC1 promotes the metastasis of clear cell renal cell carcinoma via activation of STAT3. Oncogene. 2020; 39: 6203-17.

[87] Li X, Liang Y, Lian C, Peng F, Xiao Y, He Y, et al. CST6 protein and peptides inhibit breast cancer bone metastasis by suppressing CTSB activity and osteoclastogenesis. Theranostics. 2021; 11: 9821-32.

[88] Komohara Y, Fujiwara Y, Ohnishi K, Takeya M. Tumor-associated macrophages: potential therapeutic targets for anti-cancer therapy. Adv Drug Deliv Rev. 2016; 99: 180-5.

[89] Mantovani A, Sozzani S, Locati M, Allavena P, Sica A. Macrophage polarization: tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes. Trends Immunol. 2002; 23: 549-55.

[90] Li Y, Lui KO, Zhou B. Reassessing endothelial-to-mesenchymal transition in cardiovascular diseases. Nat Rev Cardiol. 2018; 15: 445-56.

[91] Pober JS, Sessa WC. Evolving functions of endothelial cells in inflammation. Nat Rev Immunol. 2007; 7: 803-15.

[92] Arashiro K, Ohtsuka H, Miki Y. Three-dimensional architecture of human cutaneous vascular lesions: a scanning electron microscopic study of corrosion casts. Acta Derm Venereol. 1995; 75: 257-63.

[93] Li Q, Zhu Z, Wang L, Lin Y, Fang H, Lei J, et al. Single-cell transcriptome profiling reveals vascular endothelial cell heterogeneity in human skin. Theranostics. 2021; 11: 6461-76.

[94] Schupp JC, Adams TS, Cosme C Jr, Raredon MSB, Yuan Y, Omote N, et al. Integrated single-cell atlas of endothelial cells of the human lung. Circulation. 2021; 144: 286-302.

[95] Liu X, Chen W, Zeng Q, Ma B, Li Z, Meng T, et al. Single-cell RNA-sequencing reveals lineage-specific regulatory changes of fibroblasts and vascular endothelial cells in keloids. J Invest Dermatol. 2022; 142: 124-135.e111.

[96] Cowman SJ, Koh MY. Revisiting the HIF switch in the tumor and its immune microenvironment. Trends Cancer. 2022; 8: 28-42.

[97] Masoud GN, Li W. HIF-1α pathway: role, regulation and intervention for cancer therapy. Acta Pharm Sin B. 2015; 5: 378-89.

[98] Jin S, Guerrero-Juarez CF, Zhang L, Chang I, Ramos R, Kuan CH, et al. Inference and analysis of cell-cell communication using CellChat. Nat Commun. 2021; 12: 1088.

[99] Longo SK, Guo MG, Ji AL, Khavari PA. Integrating single-cell and spatial transcriptomics to elucidate intercellular tissue dynamics. Nat Rev Genet. 2021; 22: 627-44.

[100] Meng XM, Nikolic-Paterson DJ, Lan HY. TGF-β: the master regulator of fibrosis. Nat Rev Nephrol. 2016; 12: 325-38.

[101] Tzavlaki K, Moustakas A. TGF-β Signaling. Biomol Ther. 2020; 10: 487.

[102] Murata H, Zhou L, Ochoa S, Hasan A, Badiavas E, Falanga V. TGF-beta3 stimulates and regulates collagen synthesis through TGF-beta1-dependent and independent mechanisms. J Invest Dermatol. 1997; 108: 258-62.

[103] Eickelberg O, Köhler E, Reichenberger F, Bertschin S, Woodtli T, Erne P, et al. Extracellular matrix deposition by primary human lung fibroblasts in response to TGF-beta1 and TGF-beta3. Am J Phys. 1999; 276: L814-24.