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Single-Cell Transcriptome Analysis Reveals Mesenchymal Stem Cells In bioRxiv preprint doi: https://doi.org/10.1101/2021.09.02.458742; this version posted September 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 Single-cell transcriptome analysis reveals mesenchymal stem cells 2 in cavernous hemangioma 3 Fulong Ji1$, Yong Liu2$, Jinsong Shi3$, Chunxiang Liu1, Siqi Fu1 4 Heng Wang1, Bingbing Ren1, Dong Mi4, Shan Gao2*, Daqing Sun1* 5 1 Department of Paediatric Surgery, Tianjin Medical University General Hospital, Tianjin 300052, P.R. China. 6 China. 7 2 College of Life Sciences, Nankai University, Tianjin, Tianjin 300071, P.R. China; 8 3 National Clinical Research Center of Kidney Disease, Jinling Hospital, Nanjing University School of 9 Medicine, Nanjing, Jiangsu 210016, P.R. China; 10 4 School of Mathematical Sciences, Nankai University, Tianjin, Tianjin 300071, P.R. China; 11 12 13 $ These authors contributed equally to this paper. 14 * Corresponding authors. 15 SG：[email protected] 16 DS：[email protected] 17 bioRxiv preprint doi: https://doi.org/10.1101/2021.09.02.458742; this version posted September 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 18 Abstract 19 A cavernous hemangioma, well-known as vascular malformation, is present at birth, grows 20 proportionately with the child, and does not undergo regression. Although a cavernous hemangioma 21 has well-defined histopathological characteristics, its origin and formation remain unknown. In the present 22 study, we characterized the cellular heterogeneity of cavernous hemangioma using single-cell RNA 23 sequencing (scRNA-seq). The main contribution of this study is the discovery of mesenchymal stem cells 24 (MSCs) that cause tumour formation in cavernous hemangioma and we propose that these MSCs may be 25 abnormally differentiated or incompletely differentiated from epiblast stem cells. 26 Other new findings include the responsive ACKR1 positive endothelial cell (ACKR1+EC) and BTNL9 27 positive endothelial cell (BTNL9+EC) and the BTNL9-caused checkpoint blockade enhanced by the 28 CXCL12-CXCR4 signalling. The activated CD8+T and NK cells may highly express CCL5 for their 29 infiltration in cavernous hemangiomas, independent on the tumor cell-derived CCL5-IFNG-CXCL9 pathway. 30 The highly co-expression of CXCR4 and GZMB suggested that plasmacytoid dendritic cells (pDCs) function 31 for anti-tumour as CD8+T cells in cavernous hemangiomas. The oxidised low-density lipoprotein (oxLDL) 32 in the TME of cavernous hemangiomas may play an important role as a signalling molecular in the immune 33 responses. Notably, we propose that oxLDL induces the oxLDL-OLR1-NLRP3 pathway by over-expression 34 of OLR1 in M1-like macrophages, whereas oxLDL induces the oxLDL-SRs-C1q (SRs are genes encoding 35 scavenger receptors of oxLDL except OLR1) pathway by over-expression of other scavenger receptors in 36 M2-like macrophages. 37 The present study revealed the origin of cavernous hemangiomas and discovered marker genes, cell 38 types and molecular mechanisms associated with the origin, formation, progression, diagnosis or therapy of 39 cavernous hemangiomas. The information from the present study makes important contributions to the 40 understanding of cavernous hemangioma formation and progression and facilitates the development of gold 41 standard for molecular diagnosis and effective drugs for treatment. 42 Keyword: MSC; Vascular malformation; Vascular tumor; Stem cell; scRNA-seq 43 bioRxiv preprint doi: https://doi.org/10.1101/2021.09.02.458742; this version posted September 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 44 Introduction 45 Vascular tumours include hemangioma, hemangioendothelioma, angiosarcoma, and their epithelioid 46 variants [1]. According to the size of the affected vessels, hemangiomas are histologically classified as 47 capillary, cavernous, or mixed-type hemangiomas [2]. A capillary hemangioma (superficial, red, raised), 48 also called strawberry hemangioma, is a tumour of infancy that undergoes a phase of rapid growth and 49 expansion followed by a period of slow but steady regression during childhood. In contrast, a cavernous 50 hemangioma (deep dermal, blue hue), which is now classified as vascular malformation according to the 51 International Society for the Study of Vascular Anomalies (ISSVA) classification [3], is present at birth, 52 grows proportionately with the child, and does not undergo regression [4]. Major features used to 53 discriminate cavernous hemangiomas from capillary hemangiomas include the observation of “normal” 54 vascular endothelial cells in cavernous hemangiomas and the over-expression of vascular endothelial growth 55 factor A (VEGFA) and fibroblast growth factor receptor 1 (FGFR1) in capillary hemangiomas during the 56 proliferative stage [6]. Cavernous hemangiomas have been reported to arise at various sites, including the 57 skin and subcutaneous layers of the head and neck, face, extremities, liver, gastrointestinal tract, and even 58 the thymus [5]. The tumours are composed of dilated vascular spaces, with thinned smooth muscle walls 59 separated by a variable amount of fibroconnective tissue. 60 Three classes of cavernous hemangiomas hepatic cavernous hemangioma (HCH), retinal cavernous 61 hemangioma (RCH), and cerebral cavernous hemangioma (CCH), also known as cerebral cavernous 62 malformation (CCM), are comparatively well studied. HCH, the most common benign tumour of the liver, is 63 present in up to 7% of individuals that participate in autopsy studies. Histological examination of the lesions 64 revealed a network of vascular spaces lined by endothelial cells and separated by a thin fibrous stroma. 65 Large HCHs may be associated with thrombosis, scarring, and calcification [7]. RCH is composed of 66 clusters of saccular aneurysms filled with dark blood. Microscopic examination of the lesions revealed 67 multiple thin-walled interconnected vascular spaces lined by flat endothelial cells, with red cell necrosis and 68 partially organised intravascular thrombosis. Vascular spaces are bordered by thin, fibrous septa, with 69 occasional nerve fibers and glial cells. Although mutations in KRIT1 and CCM1 genes have been found in 70 patients with both RCH and CCH [8], the cause of RCH remains unknown. CCHs that occur in the central 71 nervous system, most often in the brain, can cause intracranial hemorrhage, seizures, neurological deficits, 72 and even death. Ultrastructural studies revealed abnormal or absent blood–brain barrier components, poorly 73 formed tight junctions with gaps between endothelial cells, lack of astrocytic foot processes, and few bioRxiv preprint doi: https://doi.org/10.1101/2021.09.02.458742; this version posted September 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 74 pericytes in the lesions. CCH has sporadic and familial forms; familial CCHs often display multiple lesions 75 and autosomal dominant inheritance. According to the current theory, capillary hemangiomas originate from 76 neogenesis or revival of dormant embryonic angioblasts and arise through hormonally driven vessel growth 77 [4], while familial CCHs may be caused by loss-of-function mutations in three genes, KRIT1, CCM2, and 78 PDCD10 [9]. However, the origins of capillary and cavernous hemangiomas remain controversial and 79 unknown, respectively. 80 Although capillary hemangiomas are mainly treated via surgery, several drugs (e.g., propranolol and 81 glucocorticoids) have been developed to avoid the risks of intraoperative profuse bleeding, postoperative 82 recurrence, long-term scarring, and other complications. The treatment of cavernous hemangiomas is still 83 dependent on excision and venous embolization therapy [3]; however, inadequate excision causes recurrence 84 of cavernous hemangiomas [10]. To develop drugs or other new therapies, more basic research must be 85 conducted to understand cavernous hemangiomas at the molecular level. In the present study, we 86 characterised the cellular heterogeneity of cavernous hemangioma using single-cell RNA sequencing 87 (scRNA-seq) [11]. Through further analysis of the scRNA-seq data, we aimed to: (1) reveal the 88 comprehensive cellular composition and gene expression profile of a cavernous hemangioma at the 89 single-cell level, and (2) discover marker genes, cell types, and molecular mechanisms associated with the 90 origin, formation, progression, diagnosis, and therapy of cavernous hemangiomas. 91 92 Results 93 Single-cell RNA sequencing and basic analyses 94 The lesion was obtained as tumour tissue from a 6-year-old patient diagnosed with cavernous 95 hemangioma (Supplementary File 1). Using tissue in the center of the lesion, scRNA-seq libraries (10x 96 Genomics, USA) were constructed and sequenced to produce ~163 Gbp of raw data (Materials and 97
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