Use of gene expression products as markers of partial endothelial-to-mesenchymal transition

Abstract

To provide use of gene expression products as markers of Partial EndoMT being an intermediate stage of Endothelial-to-Mesenchymal transition (EndoMT).SOLUTION: The invention provides the use of at least one gene expression product as a marker of Partial EndoMT being an intermediate stage of Endothelial-to-Mesenchymal transition (EndoMT), where the gene is selected from the group consisting of Rgs2 gene, Sost gene, Sp5 gene, Asap3 gene, Cd40 gene, Nckap5l gene, Hpn gene, Myzap gene, Pdgfb gene, Bambi gene, Mindy4 gene, Dapk2 gene, Limch1 gene, Dock8 gene, Vwf gene, Abhd17a gene, Adora2a gene, Zfp69 gene, Ctnnal1 gene, Mvb12b gene, A530016 L24Rik gene, Mmp28 gene, Rnf144a gene, Scn1b gene, Ypel2 gene, Lhfpl2 gene, Tmem158 gene, Plvap gene, Ctsh gene, Sema6c gene, Arl15 gene, Mafb gene, Eln gene, Il4ra gene, Rgcc gene, Fam181b gene, Septin4 gene, Serinc5 gene, Robo3 gene, Ace gene, Kif5c gene, Adamts17 gene, and Trp53i11 gene.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to the use of the expression product of a gene as a marker for Partial EndoMT (also called Partial EndMT, partial EndoMT, partial endothelial-mesenchymal transition, etc.), which is an intermediate stage of endothelial-mesenchymal transition (EndoMT). The present invention relates to a method for determining whether a cell has undergone Partial EndoMT. The present invention relates to a method for selecting cells that have undergone Partial EndoMT or cells that have not undergone Partial EndoMT. The present invention relates to an inhibitor of induction or enhancement of EndoMT in cells, and particularly to an inhibitor of induction or enhancement of EndoMT in endothelial cells. The present invention relates to a therapeutic agent for cancer, fibrosis, pulmonary arterial hypertension, or atherosclerosis.

Background Art

[0002] The tumor microenvironment (TME) is composed of cancer cells, endothelial cells, cancer-associated fibroblasts (CAFs), immune cells, and other stromal cells [Non-Patent Document 1]. The interaction between these components of the TME is carried out through direct cell-cell contact or the secretion of various cytokines [Non-Patent Documents 1, 2]. Multiple evidences suggest that transforming growth factor-β (TGF-β) plays an important role in the regulation of the TME network [Non-Patent Document 3]. TGF-β binds to serine-threonine kinase type receptors (TGF-β type I and II receptors; TβRI and TβRII) [Non-Patent Document 4] and phosphorylates Smad2 / 3. Phosphorylated Smad2 / 3 forms a complex with Smad4, translocates into the nucleus, and controls the transcription of various target genes including plasminogen activator inhibitor 1 (PAI-1).

[0003] TGF-β contributes to tumor progression and metastasis by inducing epithelial-mesenchymal transition (EMT) in epithelial cancer cells, which causes cancer cell invasion and metastasis [Non-Patent Documents 3, 5-7]. EMT also confers resistance of cancer cells to chemotherapy and radiotherapy. Furthermore, TGF-β induces tumor angiogenesis, immune tolerance, and the formation of cancer-associated fibroblasts (CAFs). Therefore, TGF-β regulates tumor progression and metastasis by influencing various components of the tumor microenvironment (TME).

[0004] TGF-β also induces endothelial-mesenchymal transition (also called EndoMT), which plays a role in tumor progression and metastasis [Non-Patent Documents 8, 9]. During EndoMT, endothelial cells lose characteristics such as strong cell-cell contacts and the expression of endothelial cell-specific markers such as vascular endothelial growth factor receptor 2 (VEGFR2) and tyrosine kinase with immunoglobulin-like and epidermal growth factor-like domains 2 (Tie2), and acquire mesenchymal phenotypes such as high motility and the expression of mesenchymal cell markers such as smooth muscle protein 22α (SM22α) and α-smooth muscle actin (αSMA). The present inventors previously reported that tumor necrosis factor α (TNF-α) enhances TGF-β-induced EndoMT and generates TGF-β2-secreting CAF-like cells that induce EMT in oral squamous cell carcinoma (OSCC) cells. In distant organs, vascular integrity is reduced, promoting intravascular invasion and extravasation of cancer cells. The present inventors previously reported that tumor necrosis factor α (TNF-α) enhances TGF-β-induced EndoMT and generates TGF-β2-secreting CAF-like cells that induce EMT in oral squamous cell carcinoma (OSCC) cells [Non-Patent Document 10]. During the metastasis of cancer to distant organs, vascular integrity is reduced, promoting intravascular invasion and extravasation of cancer cells. The present inventors previously reported that extracellular vesicles released by TGF-β-stimulated OSCC cells decrease the barrier function of endothelial cells with the induction of EndoMT [Non-Patent Document 11]. Also, a recent series of evidence indicates that EndoMT plays a role in tumor angiogenesis [Non-Patent Documents 12, 13]. EndoMT is involved in various stages of cancer progression and metastasis, but the underlying molecular mechanisms of EndoMT have not yet been elucidated.

[0005] In previous studies focusing on EndoMT during its occurrence, the transition from endothelial cells to mesenchymal cells was considered to be a permanent transition between two differentiated cell types. However, recent reports have suggested a high plasticity of EndoMT [Non-Patent Document 9]. It is now widely recognized that cells undergoing EndoMT differentiate through various intermediate stages. This plasticity means that some endothelial cells retain the characteristics of endothelial / mesenchymal hybrids and maintain a Partial EndoMT state, which is an intermediate stage of EndoMT. Notably, this Partial EndoMT state can potentially reverse under specific conditions [Non-Patent Document 9]. Partial EndoMT has also been observed during angiogenesis [Non-Patent Documents 14, 15]. However, the lack of experimental systems and specific markers for identifying cells in Partial EndoMT has hindered a detailed analysis of their characteristics.

[0006] To characterize EndoMT-driven mesenchymal cells, several transgenic mouse models have been developed using endothelial lineage tracing, which genetically labels vascular endothelial cells [Non-Patent Documents 16, 17]. Such endothelial lineage tracing is useful for analyzing EndoMT, but the stepwise transition of EndoMT has not yet been identified.

[0007] Regarding the above, further explanations are provided for supplementation. The microenvironmental network that controls cancer: Tumors consist of not only cancer cells but also various cells that make up the tumor microenvironment, such as endothelial cells that form tumor blood vessels and cancer-associated fibroblasts (CAFs). In the cancer microenvironment, the formation and progression of tumors are spatiotemporally regulated by direct interactions between various cells and indirect interactions via humoral factors such as cytokines. These interactions between cancer cells and the microenvironment have attracted attention as new therapeutic targets. However, many aspects of these interactions remain unclear, and the development of therapeutic methods targeting these interactions is still in its infancy.

[0008] Interaction between cancer cells and cancer-associated fibroblasts (CAFs) derived from tumor blood vessels via endothelial-mesenchymal transition (EndoMT): "CAF", a constituent factor in the cancer microenvironment, has been shown to induce cancer cell proliferation and malignancy by secreting various humoral factors, thus inducing cancer progression. Therefore, it is urgent to elucidate its formation mechanism. CAF was thought to be formed by the activation of resident normal fibroblasts by cytokines such as TGF-β, but it has been shown by genetic methods that about 30% of CAF is derived from tumor vascular endothelial cells. The phenomenon in which these tumor vascular endothelial cells acquire the properties of mesenchymal cells is called "Endothelial-Mesenchymal Transition (EndoMT)", and it has been clarified by the representative researchers that it plays an important role in cancer progression and metastasis. However, there are still many unclear parts about its detailed molecular mechanism.

[0009] EndoMT is also involved in the development of diseases such as tissue fibrosis, pulmonary arterial hypertension, and atherosclerosis: EndoMT may contribute to the development of various diseases including fibrotic diseases and cancer as follows. Cardiac fibrosis: Endothelial cells undergoing EndoMT may differentiate into cardiac fibroblasts that enable cardiac fibrosis. Pulmonary arterial hypertension (PAH): The accumulation of SMA-overexpressing fibrotic cells derived from EndoMT thickens and narrows the arterial wall, promoting the development of PAH. Atherosclerosis (AS): The accumulation of fibroblasts derived from EndoMT leads to plaque growth and promotes the thickening of AS plaques. Organ fibrosis: It has been proven that EndoMT-induced fibroblasts are a source of fibroblast-like cells in liver fibrosis, kidney fibrosis, and intestinal fibrosis.

[0010] Factors that control partial EndoMT become therapeutic targets: EndoMT is the process by which vascular endothelial cells lose their properties and acquire the properties of mesenchymal cells. In recent years, it has been becoming clear that the intermediate stage of EndoMT (Partial EndoMT) plays an important role in cancer metastasis and tumor angiogenesis. However, there has been no experimental system that can detect Partial EndoMT until now.

Prior Art Documents

Non-Patent Documents

[0011] [Non-Patent Document 1] Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011; 144: 646-674. [Non-Patent Document 2] Oikawa Y, Michi Y, Tsushima F, et al. Management of retropharyngeal lymph node metastasis in oral cancer. Oral Oncol. 2019; 99: 104471. [Non-Patent Document 3] Miyazono K, Katsuno Y, Koinuma D, Ehata S, Morikawa M. Intracellular and extracellular TGF-β signaling in cancer: some recent topics. Front Med. 2018; 12: 387-411. [Non-Patent Document 4] Derynck R, Budi EH. Specificity, versatility, and control of TGF-β family signaling. Sci Signal. 2019; 12: eaav5183. [Non-Patent Document 5] Pickup M, Novitskiy S, Moses HL. The roles of TGFβ in the tumour microenvironment. Nat Rev Cancer. 2013; 13: 788-799. [Non-Patent Document 6] Liu S, Ren J, Ten Dijke P. Targeting TGFβ signal transduction for cancer therapy. Signal Transduct Target Ther. 2021; 6: 8. [Non-Patent Document 7] Derynck R, Turley SJ, Akhurst RJ. TGFβ biology in cancer progression and immunotherapy. Nat Rev Clin Oncol. 2021; 18: 9-34. Non-Patent Document 8 Yoshimatsu Y, Watabe T. Emerging roles of inflammation-mediated endothelial-mesenchymal transition in health and disease. Inflamm Regen. 2022; 42: 9. Non-Patent Document 9 Watabe T, Takahashi K, Pietras K, Yoshimatsu Y. Roles of TGF-β signals in tumor microenvironment via regulation of the formation and plasticity of vascular system. Semin Cancer Biol. 2023; 92: 130-138. Non-Patent Document 10 Yoshimatsu Y, Wakabayashi I, Kimuro S, et al. TNF-α enhances TGF-β-induced endothelial-to-mesenchymal transition via TGF-β signal augmentation. Cancer Sci. 2020; 111: 2385-2399. Non-Patent Document 11 Kobayashi M, Fujiwara K, Takahashi K, et al. Transforming growth factor-β-induced secretion of extracellular vesicles from oral cancer cells evokes endothelial barrier instability via endothelial-mesenchymal transition. Inflamm Regen. 2022; 42: 38.

Non-Patent Document 12

Non-Patent Document 13

Non-Patent Document 14

Non-Patent Document 15

Non-Patent Document 16

Non-Patent Document 17

Summary of the Invention

Problems to be Solved by the Invention

[0012] Therefore, in the present invention, in order to solve this problem, an experimental system capable of detecting Partial EndoMT was established to identify its specific markers and develop a treatment method targeting EndoMT regulators.

[0013] That is, an object of the present invention is to provide the use of an expression product of a gene as a Partial EndoMT marker, which is an intermediate stage of EndoMT. An object of the present invention is to provide a method for determining whether a cell has undergone Partial EndoMT. An object of the present invention is to provide a method for sorting cells that have undergone Partial EndoMT or cells that have not undergone Partial EndoMT. An object of the present invention is to provide an inhibitor of induction or enhancement of EndoMT in cells, and particularly to provide an inhibitor of induction or enhancement of EndoMT in endothelial cells. An object of the present invention is to provide a therapeutic agent for cancer, fibrosis, pulmonary arterial hypertension, or atherosclerosis.

Means for Solving the Problems

[0014] In the present invention, EndoMT reporter endothelial cells (EMREC), which is a new EndoMT reporter system that enables visualization of continuous changes during EndoMT, was established. Using EMREC, gene profiles of multiple EndoMT stages were characterized, and successful identification of novel Partial EndoMT-specific markers was achieved. Also, using single-cell RNA sequencing (scRNA-seq) analysis, it was shown that cells representing the Partial EndoMT state can be detected within human tumors.

[0015] This will be specifically described below. Tumor progression and metastasis are controlled by endothelial cells undergoing endothelial-mesenchymal transition (EndoMT), a cell differentiation process in which endothelial cells lose their characteristics and differentiate into mesenchymal cells. Cells undergoing EndoMT differentiate through various intermediate stages. This suggests that some cells remain in a Partial EndoMT state, an intermediate stage of EndoMT, and exhibit an endothelial / mesenchymal phenotype. However, detailed analysis of Partial EndoMT has been hampered by the lack of specific markers. Transforming growth factor-β (TGF-β) plays a central role in the induction of EndoMT. Here, the inventors showed that EndoMT is suppressed by inhibition of TGF-β signaling in a human oral cancer cell xenograft mouse model. By using genetic labeling of the endothelial cell lineage, a new EndoMT reporter cell system, EndoMT reporter endothelial cells (EMREC), was also established to enable visualization of the continuous changes during TGF-β-induced EndoMT. Using EMREC, the gene profiles of multiple EndoMT stages were characterized, and CD40 was identified as a novel Partial EndoMT-specific marker. CD40 expression was upregulated in cells undergoing Partial EndoMT but decreased in fully EndoMT cells. Furthermore, single-cell RNA sequencing analysis of human tumors revealed that CD40 expression was abundant in cell populations expressing both endothelial and mesenchymal markers. Additionally, reduced expression of CD40 in EMREC enhanced TGF-β-induced EndoMT, suggesting that CD40 expressed during Partial EndoMT inhibits the transition of cells to full EndoMT. This discovery provides a better understanding of the mechanisms underlying TGF-β-induced EndoMT and will facilitate the development of new therapeutic strategies targeting EndoMT-induced cancer progression and metastasis.

[0016] This will be further specifically described below.

[0017] Established EndoMT reporter mice: To observe EndoMT in real time in vivo and examine what molecular events are occurring, the representative created an EndoMT reporter mouse ahead of the world. In this mouse, vascular endothelial cells are genetically labeled with a red fluorescent protein (VE-cadherin-Cre: R26R-loxP-STOP-loxP-tdTomato), and green fluorescent protein (GFP) is expressed by the SMA promoter, which is a mesenchymal cell marker. Thus, it can be identified that the mesenchymal cells formed by EndoMT are derived from vascular endothelial cells, and it is possible to detect cells that are currently undergoing EndoMT migration in real time.

[0018] Established EndoMT reporter vascular endothelial cells (EMREC): EndoMT reporter endothelial cells (EMREC) express tdTomato as a vascular endothelial lineage and express GFP downstream of the mesenchymal marker SMA upon TGF-β stimulation. Therefore, cells labeled from orange to yellow-green could be observed along with the induction of EndoMT. In addition, the expression of endothelial cell markers in EMREC decreased upon TGF-β stimulation, while the expression of mesenchymal cell markers increased, and the motility also increased, indicating that EndoMT was induced.

[0019] In three-dimensional microvessels established from EMREC, TGF-β induced EndoMT: Since blood vessels form a three-dimensional lumen structure in vivo, to examine the effect of TGF-β on intravascular EndoMT, the inventors applied in vitro techniques to create three-dimensional endothelial microvessels using EMREC. Microvessels derived from EMREC were incubated for 72 hours in the absence or presence of TGF-β and stained for VEGFR2. As a result, the expression of VEGFR2 decreased throughout the microvessels upon TGF-β treatment. In contrast, TGF-β-induced αSMA-GFP expression was not uniform and was restricted to some EMREC within the microvessels.

[0020] EndoMT enhanced the permeability of three-dimensional microvessels: To investigate whether TGF-β-induced EndoMT impairs the barrier function of three-dimensional microvessels, two fluorescently labeled dextran probes were injected into the lumen of microvessels to perform a permeability assay. When the spatial redistribution of the dextran probes was monitored for 60 seconds with a confocal microscope, both dextran probes leaked into the collagen gel at higher levels from TGF-β-treated microvessels compared to control microvessels, suggesting that TGF-β-induced EndoMT enhanced the permeability of EMREC-derived microvessels. These results suggest that EMREC exhibits various phenotypes of TGF-β-induced EndoMT.

[0021] Sorted out the cell population during EndoMT migration from EndoMT reporter cells with activated TGF-β signal: For the purpose of identifying specific markers of partial EndoMT, EndoMT reporter endothelial cells were stimulated with TGF-β, and cell sorter sorting was performed on each of the four fractions of cell populations classified by FACS based on the expression of SMA-GFP and VEGFR2 in tdTomato-positive cells. Transcriptome analysis by RNA sequencing and gene expression analysis using specific primers were then carried out.

[0022] Was able to sort out the multi-step EndoMT reporter cell population that migrates from endothelial cells to mesenchymal cells: A heatmap was created from the RNA sequencing data. Each of the four cell populations showed different gene expression patterns from one another. To examine whether each cell population showed characteristic changes similar to EndoMT, Gene Set Enrichment Analysis (GSEA) was performed between each cell population. Since there is no gene set showing the characteristics of EndoMT in the database, the "HALLMARK EMT" gene set related to epithelial-mesenchymal transition (EMT), which shows mesenchymal gene expression changes similar to EndoMT, was used. As a result, the expression of the gene group of mesenchymal cell traits was enriched step by step in each cell population, suggesting that EndoMT occurred step by step in each sorted cell fraction as expected. Furthermore, from the results of GSEA, it was considered that EndoMT was induced not only in the Partial fraction but also in the Tβ-EC fraction.

[0023] In the cell population during EndoMT migration, the expression of endothelial cell markers and mesenchymal cell markers changed step by step: Next, quantitative RT-PCR analysis was performed on each cell population from which the RNA sequence analysis was originally performed to evaluate EndoMT. The expression of PAI-1 was increased in all of the TGF-β-treated cell populations. The expression of VEGFR2 decreased step by step, while the expression of SM22α increased step by step. Here, regarding the Tβ-EC fraction, since the expression of VEGFR2 decreased while the expression of αSMA hardly increased and the expression of SM22α increased slightly, the Tβ-EC fraction was considered to be in the early stage of Partial EndoMT. Also, in the Partial fraction, since the expression of αSMA and SM22α increased while the expression of VEGFR2 was slightly maintained, the Partial fraction was considered to show the late stage of Partial EndoMT. Therefore, it was proven that the experimental system using EMRECs is a system that can visually identify and isolate cells at each transition stage of EndoMT. Regarding the above, in particular, refer to Fig. 17.

[0024] Identified 43 factors as partial EndoMT markers: Next, using this experimental system, we attempted to identify novel Partial EndoMT markers. Candidate genes for Partial EndoMT markers were considered to be genes whose expression was specifically increased in the Tβ-EC fraction or the Partial fraction indicating Partial EndoMT. Therefore, candidate genes showing such changes were searched using Gene Spring software, and 43 genes were selected as those with a change in expression of 1.75-fold or more. Thus, CD40 and Ace were found as genes whose expression increased at the stage of Partial EndoMT based on the actual responsiveness to TGF-β and various bioinformatics analyses using RNA-seq data. Among them, in this study, we focused on CD40 and proceeded with more detailed analysis.

[0025] (Reference) Regarding CD40: CD40 is a member of the tumor necrosis factor receptor (TNFR) superfamily and is mainly expressed in antigen-presenting cells such as B cells and macrophages, as well as vascular endothelial cells. CD40 is involved in inflammatory and immune responses by binding to the CD40 ligand. As for its association with blood vessels, it has been reported to be related to vascular diseases such as angiogenesis, the formation of intimal hyperplasia, and atherosclerosis. In addition, the anti-human CD40 agonist antibody (ABBV-927: Giloralima) is currently undergoing clinical trials as a single agent or in combination with an anti-PD-L1 antibody (ABBV 181: Budigalimab) for advanced solid cancers.

[0026] The expression of CD40 transiently increased at the partial EndoMT stage: First, we examined the effect of TGF-β stimulation on CD40 expression. The expression of CD40 was most increased in the Tβ-EC fraction, and its expression was lower in the Full fraction than in the EC fraction. Next, to examine the change in the expression level of CD40 at each stage of EndoMT transition, FACS two-dimensional analysis was performed with VEGFR2 and αSMA, and the expression level of CD40 was color-coded to examine the distribution of CD40-high expressing cells in each cell population. CD40-positive cells were also slightly present in the EC fraction, but particularly, many CD40-high expressing cells were distributed in both the Tβ-EC fraction and the Partial fraction after TGF-β treatment. Also, when examining the transition of the CD40-high expressing cell population from the histogram showing the expression level of CD40, the expression of CD40 increased in the Tβ-EC fraction and the Partial fraction and then decreased in the Full fraction, revealing that the expression of CD40 transiently increased at the Partial EndoMT stage. Regarding the above, in particular, refer to Fig. 18.

[0027] (Reference) By analyzing the single-cell RNA sequencing data of tumor tissues from 10 types and 226 cancer patients, a cluster of CAFs derived from EndoMT was identified: To verify the clinical significance of the results obtained in the previous in vitro experimental system, we re-analyzed the publicly available data of single cell RNA sequencing of tumor tissues from 226 patients with 10 types of solid cancers reported in Nature Communication last year, and identified clusters of five types of cell populations based on the expression of various specific markers. Among these, in addition to the endothelial cell cluster and the fibroblast cluster, a Partial EndoMT cluster was found.

[0028] Re-analyzed the publicly available single-cell RNA sequencing data of tumor tissues from 10 types and 226 cancer patients to identify the cluster of partial EndoMT: To verify the clinical significance of the results obtained from previous in vitro experimental systems, we re-analyzed the publicly available single cell RNA sequencing data of tumor tissues from 226 patients with 10 types of solid cancers reported in Nature Communication last year. Based on the expression of various specific markers, we identified clusters of five cell populations. Among these, in addition to the endothelial cell cluster and the fibroblast cluster, we found a cluster of Partial EndoMT.

[0029] CD40 was specifically expressed in the cell fraction of partial EndoMT in human tumor tissues as well: When only the endothelial cluster and the CAF cluster were extracted from the scRNA seq data and the expression of several markers was shown in violin plots, the endothelial marker and the fibroblast marker were specifically expressed in these clusters. However, in the cell population shown near the center, both the endothelial marker and the fibroblast marker were expressed, so we think this might be the cluster of Partial EndoMT. Actually, in the pseudotime analysis following the progression of EndoMT from the gene expression pattern, it became clear that it progresses from the endothelial cluster, through this Partial EndoMT cluster, to the CAF cluster. Therefore, when we examined the expression of CD40, it was not expressed in the endothelial cluster, transiently increased in the Partial EndoMT cluster, and then decreased again in the CAF cluster. From the above, it was shown that CD40 is a marker of Partial EndoMT in human cancers.

[0030] The induction of EndoMT by TGF-β was inhibited by the decreased expression of CD40: CD40 is originally a surface antigen expressed in dendritic cells and B cells involved in immunity. Since immunity is activated by activation with CD40L expressed in T cells and the like, clinical trials are underway for the combination therapy of an agonist antibody of CD40 with an immune checkpoint inhibitor. Therefore, in order to clarify the role of CD40 in the induction of EndoMT, when endothelial cells with reduced CD40 expression by siRNA were treated with TGF-β, the expression of mesenchymal cell markers increased, that is, EndoMT was enhanced, and the expression of Activin having EndoMT-inducing activity increased. Since CD40 suppresses EndoMT, the agonist antibody, for which clinical trials are currently underway for solid cancers, may become a therapeutic agent targeting EndoMT. Regarding the above, in particular, refer to FIG. 19.

[0031] Sorted out the cell population of partial EndoMT from TGF-β-treated EndoMT reporter cells and identified CD40 as a specific marker: With the EndoMT reporter cell EMREC established in the present invention, the transition of EndoMT can be gradually visualized. This suggests that there are two stages, an early stage and a late stage, in Partial EndoMT. Furthermore, a specific marker factor CD40 whose expression increases only in Partial EndoMT was identified. Also, it became clear that CD40 suppresses EndoMT.

[0032] That is, the present invention provides the following. [Aspect 1] Use of the gene expression product as a Partial EndoMT marker, which is an intermediate stage of EndoMT, wherein the gene is selected from the group consisting of the Rgs2 gene, Sost gene, Sp5 gene, Asap3 gene, Cd40 gene, Nckap5l gene, Hpn gene, Myzap gene, Pdgfb gene, Bambi gene, Mindy4 gene, Dapk2 gene, Limch1 gene, Dock8 gene, Vwf gene, Abhd17a gene, Adora2a gene, Zfp69 gene, Ctnnal1 gene, Mvb12b gene, A530016L24Rik gene, Mmp28 gene, Rnf144a gene, Scn1b gene, Ypel2 gene, Lhfpl2 gene, Tmem158 gene, Plvap gene, Ctsh gene, Sema6c gene, Arl15 gene, Mafb gene, Eln gene, Il4ra gene, Rgcc gene, Fam181b gene, Septin4 gene, Serinc5 gene, Robo3 gene, Ace gene, Kif5c gene, Adamts17 gene, and Trp53i11 gene. [Aspect 2] The use according to Aspect 1, wherein the gene expression product is RNA or protein. [Aspect 3] A method for determining whether a cell has undergone Partial EndoMT, wherein the expression product of a gene in the cell is used as an indicator for the determination, and the gene is selected from the group consisting of the Rgs2 gene, Sost gene, Sp5 gene, Asap3 gene, Cd40 gene, Nckap5l gene, Hpn gene, Myzap gene, Pdgfb gene, Bambi gene, Mindy4 gene, Dapk2 gene, Limch1 gene, Dock8 gene, Vwf gene, Abhd17a gene, Adora2a gene, Zfp69 gene, Ctnnal1 gene, Mvb12b gene, A530016L24Rik gene, Mmp28 gene, Rnf144a gene, Scn1b gene, Ypel2 gene, Lhfpl2 gene, Tmem158 gene, Plvap gene, Ctsh gene, Sema6c gene, Arl15 gene, Mafb gene, Eln gene, Il4ra gene, Rgcc gene, Fam181b gene, Septin4 gene, Serinc5 gene, Robo3 gene, Ace gene, Kif5c gene, Adamts17 gene, and Trp53i11 gene. [Aspect 4] The method according to aspect 3, wherein using the expression product of the gene in the cell as an indicator for the determination is to use the amount of the expression product of the gene in the cell as an indicator for the determination. [Aspect 5] The method according to aspect 4, wherein when using the amount of the expression product of the gene in the cell as an indicator for the determination, the amount of the expression product of the gene in the cell is compared with a predetermined amount. [Aspect 6] The method according to aspect 5, wherein it is determined that the cell has undergone Partial EndoMT using as an indicator that the amount of the expression product of the gene in the cell is greater compared to a predetermined amount. [Aspect 7] The method according to aspect 5, wherein it is determined that the cell has not undergone Partial EndoMT using as an indicator that the amount of the expression product of the gene in the cell is less compared to a predetermined amount. [Aspect 8] The method according to embodiment 3, wherein the gene expression product is RNA or protein. [Embodiment 9] The method according to embodiment 3, wherein the cell is a tumor vascular endothelial cell. [Embodiment 10] A method for determining whether a cell has undergone Partial EndoMT, comprising the step of measuring the gene expression product in the cell, wherein the gene is selected from the group consisting of the Rgs2 gene, Sost gene, Sp5 gene, Asap3 gene, Cd40 gene, Nckap5l gene, Hpn gene, Myzap gene, Pdgfb gene, Bambi gene, Mindy4 gene, Dapk2 gene, Limch1 gene, Dock8 gene, Vwf gene, Abhd17a gene, Adora2a gene, Zfp69 gene, Ctnnal1 gene, Mvb12b gene, A530016L24Rik gene, Mmp28 gene, Rnf144a gene, Scn1b gene, Ypel2 gene, Lhfpl2 gene, Tmem158 gene, Plvap gene, Ctsh gene, Sema6c gene, Arl15 gene, Mafb gene, Eln gene, Il4ra gene, Rgcc gene, Fam181b gene, Septin4 gene, Serinc5 gene, Robo3 gene, Ace gene, Kif5c gene, Adamts17 gene, and Trp53i11 gene. [Embodiment 11] The method according to embodiment 10, wherein the step of measuring the gene expression product in the cell is a step of measuring the amount of the gene expression product in the cell. [Embodiment 12] The method according to embodiment 11, comprising the step of comparing the measured amount of the gene expression product in the cell with a predetermined amount. [Embodiment 13] The method according to embodiment 12, wherein, using as an indicator that the measured amount of the gene expression product in the cell is greater than the predetermined amount, it is determined that the cell has undergone Partial EndoMT. [Embodiment 14] The method according to aspect 12, which is a method for determining that a cell has not undergone Partial EndoMT, using as an indicator that the amount of the gene expression product in the measured cell is less than a predetermined amount. [Aspect 15] The method according to aspect 10, wherein the gene expression product is RNA or protein. [Aspect 16] The method according to aspect 10, wherein the cell is a tumor vascular endothelial cell. [Aspect 17] A method for sorting cells that have undergone Partial EndoMT or cells that have not undergone Partial EndoMT, comprising a step of sorting cells in which the amount of the gene expression product is more than a predetermined amount or cells in which the amount of the gene expression product is less than a predetermined amount, wherein the gene is selected from the group consisting of the Rgs2 gene, Sost gene, Sp5 gene, Asap3 gene, Cd40 gene, Nckap5l gene, Hpn gene, Myzap gene, Pdgfb gene, Bambi gene, Mindy4 gene, Dapk2 gene, Limch1 gene, Dock8 gene, Vwf gene, Abhd17a gene, Adora2a gene, Zfp69 gene, Ctnnal1 gene, Mvb12b gene, A530016L24Rik gene, Mmp28 gene, Rnf144a gene, Scn1b gene, Ypel2 gene, Lhfpl2 gene, Tmem158 gene, Plvap gene, Ctsh gene, Sema6c gene, Arl15 gene, Mafb gene, Eln gene, Il4ra gene, Rgcc gene, Fam181b gene, Septin4 gene, Serinc5 gene, Robo3 gene, Ace gene, Kif5c gene, Adamts17 gene, and Trp53i11 gene. [Aspect 18] The method according to aspect 17, wherein the gene expression product is RNA or protein. [Aspect 19] The method according to aspect 17, wherein the cell is a tumor vascular endothelial cell. [Aspect 20] A method for selecting cells that have undergone partial EndoMT or cells that have not undergone partial EndoMT, the method including a step of measuring the amount of the expression product of a gene in the cells, wherein the gene is selected from the group consisting of the Rgs2 gene, the Sost gene, the Sp5 gene, the Asap3 gene, the Cd40 gene, the Nckap5l gene, the Hpn gene, the Myzap gene, the Pdgfb gene, the Bambi gene, the Mindy4 gene, the Dapk2 gene, the Limch1 gene, the Dock8 gene, the Vwf gene, the Abhd17a gene, the Adora2a gene, the Zfp69 gene, the Ctnnal1 gene, the Mvb12b gene, the A530016L24Rik gene, the Mmp28 gene, the Rnf144a gene, the Scn1b gene, the Ypel2 gene, the Lhfpl2 gene, the Tmem158 gene, the Plvap gene, the Ctsh gene, the Sema6c gene, the Arl15 gene, the Mafb gene, the Eln gene, the Il4ra gene, the Rgcc gene, the Fam181b gene, the Septin4 gene, the Serinc5 gene, the Robo3 gene, the Ace gene, the Kif5c gene, the Adamts17 gene, and the Trp53i11 gene. [Aspect 21] The method according to aspect 20, wherein the expression product of the gene is RNA or protein. [Aspect 22] The method according to aspect 20, wherein the cells are tumor vascular endothelial cells. [Aspect 23] A program for causing an information processing apparatus to execute the method according to any one of aspects 3 to 22. [Aspect 24] An information processing apparatus storing the program according to aspect 23. [Aspect 25] An inhibitor for inducing or enhancing EndoMT of cells, comprising as an active ingredient a substance that acts on the expression products of genes selected from the group consisting of the Rgs2 gene, Sost gene, Sp5 gene, Asap3 gene, Cd40 gene, Nckap5l gene, Hpn gene, Myzap gene, Pdgfb gene, Bambi gene, Mindy4 gene, Dapk2 gene, Limch1 gene, Dock8 gene, Vwf gene, Abhd17a gene, Adora2a gene, Zfp69 gene, Ctnnal1 gene, Mvb12b gene, A530016L24Rik gene, Mmp28 gene, Rnf144a gene, Scn1b gene, Ypel2 gene, Lhfpl2 gene, Tmem158 gene, Plvap gene, Ctsh gene, Sema6c gene, Arl15 gene, Mafb gene, Eln gene, Il4ra gene, Rgcc gene, Fam181b gene, Septin4 gene, Serinc5 gene, Robo3 gene, Ace gene, Kif5c gene, Adamts17 gene, and Trp53i11 gene. [Aspect 26] The inhibitor according to claim 25, wherein the substance that acts on the expression product of the gene is an agonist of the expression product of the gene. [Aspect 27] A therapeutic agent for treating cancer, fibrosis, pulmonary arterial hypertension, or atherosclerosis, which comprises as an active ingredient a substance that acts on the expression products of genes selected from the group consisting of the Rgs2 gene, Sost gene, Sp5 gene, Asap3 gene, Cd40 gene, Nckap5l gene, Hpn gene, Myzap gene, Pdgfb gene, Bambi gene, Mindy4 gene, Dapk2 gene, Limch1 gene, Dock8 gene, Vwf gene, Abhd17a gene, Adora2a gene, Zfp69 gene, Ctnnal1 gene, Mvb12b gene, A530016L24Rik gene, Mmp28 gene, Rnf144a gene, Scn1b gene, Ypel2 gene, Lhfpl2 gene, Tmem158 gene, Plvap gene, Ctsh gene, Sema6c gene, Arl15 gene, Mafb gene, Eln gene, Il4ra gene, Rgcc gene, Fam181b gene, Septin4 gene, Serinc5 gene, Robo3 gene, Ace gene, Kif5c gene, Adamts17 gene, and Trp53i11 gene. [Aspect 28] The therapeutic agent according to claim 27, wherein the substance that acts on the expression product of the gene is an agonist of the expression product of the gene. [Aspect 29] A cell comprising a first exogenous gene expressed based on the promoter of an endothelial marker gene and a second exogenous gene expressed based on the promoter of a mesenchymal marker gene. [Aspect 30] The cell according to aspect 29, wherein the first exogenous gene expressed based on the promoter of the endothelial marker gene is expressed based on the expression of the expression product of the gene expressed based on the promoter of the endothelial marker gene. [Aspect 31] The cell according to aspect 30, wherein the expression product of the gene expressed based on the promoter of the endothelial marker gene is derived from Cre recombinase. [Aspect 32] The cell according to aspect 29, wherein the first exogenous gene is a gene that expresses a fluorescent protein and the second exogenous gene is a gene that expresses a fluorescent protein. [Aspect 33] The cell according to aspect 29, wherein the endothelial marker gene is the Cdh5 gene and the mesenchymal marker gene is the Acta2 (αSMA) gene. [Aspect 34] A blood vessel comprising the cell according to any one of aspects 29 to 33. [Aspect 35] A non-human animal comprising the cell according to any one of aspects 29 to 33. [Aspect 36] Use of the cell, blood vessel, or non-human animal according to any one of aspects 29 to 35 in the manufacture of an inhibitor for enhancing or inducing EndoMT of a cell, or in the manufacture of a therapeutic agent for cancer, fibrosis, pulmonary arterial hypertension, or atherosclerosis. [Advantages of the Invention]

[0033] The expected results and prospects of the present invention are as follows. 1) Since it is expected that the detailed molecular mechanism of EndoMT, which has many unclear parts so far, will be clarified, its academic significance is great. 2) By using a therapeutic agent targeting EndoMT, which plays an important role in the progression and metastasis of cancer, metastasis, which accounts for 90% of cancer deaths, can be suppressed, and many cancer patients can be saved. 3) EndoMT is a factor contributing to the worsening of many diseases such as cancer and pulmonary fibrosis. If a treatment method targeting EndoMT is developed according to the present invention, many patients suffering from EndoMT-related diseases can be saved, and thus a great social impact is expected. [Brief Description of the Drawings]

[0034]

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Mode for Carrying Out the Invention

[0035] Abbreviations: CAF, cancer-associated fibroblasts; CD40L, CD40 ligand; EMREC, EndoMT reporter endothelial cells; EMT, epithelial-mesenchymal transition; EndoMT, endothelial-mesenchymal transition; GO, Gene Ontology; GSEA, gene set enrichment analysis; NES, normalized enrichment score; NG2, nerve / glia antigen 2; OSCC, oral squamous cell carcinoma; PAI-1, plasminogen activator inhibitor-1; PECAM-1, platelet and endothelial cell adhesion molecule-1; qRT-PCR, quantitative RT-PCR; scRNA-seq, single-cell RNA sequencing; αSMA, α-smooth muscle actin; SM22α, smooth muscle protein 22α; Tie2, endomucin endothelial cell kinase 2; TβRI, transforming growth factor β type I receptor; TβRII, transforming growth factor β type II receptor; TGF-β, transforming growth factor-β; TME, tumor microenvironment; TNF-α, tumor necrosis factor-α; VEGFR2, vascular endothelial growth factor receptor 2.

[0036] The first aspect: The present invention provides the use of an expression product of a gene as a Partial EndoMT marker, which is an intermediate stage of endothelial-mesenchymal transition, wherein the gene is selected from the group consisting of the Rgs2 gene, Sost gene, Sp5 gene, Asap3 gene, Cd40 gene, Nckap5l gene, Hpn gene, Myzap gene, Pdgfb gene, Bambi gene, Mindy4 gene, Dapk2 gene, Limch1 gene, Dock8 gene, Vwf gene, Abhd17a gene, Adora2a gene, Zfp69 gene, Ctnnal1 gene, Mvb12b gene, A530016L24Rik gene, Mmp28 gene, Rnf144a gene, Scn1b gene, Ypel2 gene, Lhfpl2 gene, Tmem158 gene, Plvap gene, Ctsh gene, Sema6c gene, Arl15 gene, Mafb gene, Eln gene, Il4ra gene, Rgcc gene, Fam181b gene, Septin4 gene, Serinc5 gene, Robo3 gene, Ace gene, Kif5c gene, Adamts17 gene, and Trp53i11 gene.

[0037] Examples of the use of the gene expression product as a partial EndoMT marker include the use of the gene expression product for determining whether a cell has undergone partial EndoMT.

[0038] Examples of the use of the gene expression product include detection of the gene expression product, measurement of the gene expression product, and the like.

[0039] Examples of the use of the gene expression product include the use of the amount of the gene expression product.

[0040] The gene expression product is, for example, RNA, protein, or the like.

[0041] In the use of the gene expression product, for example, a substance that binds to the gene expression product is used. Examples of the substance that binds to the gene expression product include nucleic acid probes, antibodies, and the like.

[0042] The use of the present invention described above may include the step of selecting a gene from the above-mentioned group, but this is not essential.

[0043] In the use of the present invention described above, the number of genes selected from the above-mentioned group is, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, or 43.

[0044] The third aspect: The present invention provides a method for determining whether a cell has undergone Partial EndoMT, using the expression product of a gene in the cell as an indicator for the determination, wherein the gene is selected from the group consisting of the Rgs2 gene, Sost gene, Sp5 gene, Asap3 gene, Cd40 gene, Nckap5l gene, Hpn gene, Myzap gene, Pdgfb gene, Bambi gene, Mindy4 gene, Dapk2 gene, Limch1 gene, Dock8 gene, Vwf gene, Abhd17a gene, Adora2a gene, Zfp69 gene, Ctnnal1 gene, Mvb12b gene, A530016L24Rik gene, Mmp28 gene, Rnf144a gene, Scn1b gene, Ypel2 gene, Lhfpl2 gene, Tmem158 gene, Plvap gene, Ctsh gene, Sema6c gene, Arl15 gene, Mafb gene, Eln gene, Il4ra gene, Rgcc gene, Fam181b gene, Septin4 gene, Serinc5 gene, Robo3 gene, Ace gene, Kif5c gene, Adamts17 gene, and Trp53i11 gene.

[0045] Regarding using the expression product of a gene in the cell as an indicator for the determination, for example, in addition to using the presence or absence of the expression product of the gene in the cell as an indicator for the determination, using the amount of the expression product of the gene in the cell as an indicator for the determination and the like can be mentioned.

[0046] When using the amount of the gene expression product in the cell as an indicator for the determination, for example, the amount of the gene expression product in the cell is compared with a predetermined amount. The predetermined amount may be a pre-determined amount, but this is not essential. For example, it may be an amount revealed by being measured simultaneously with the measurement of the amount of the gene expression product in the cell. When the amount of the gene expression product in the cell is compared with the predetermined amount, the amount of the gene expression product in the cell may be specified as a numerical value, but this is not essential. For example, although the amount of the gene expression product in the cell is not specified as a numerical value, it may be relatively specified by comparison with a control or the like.

[0047] The method of the present invention described above determines, for example, that the cell has undergone Partial EndoMT, which is an intermediate stage of EndoMT, using as an indicator that the amount of the gene expression product in the cell is greater compared to a predetermined amount. Examples of the amount of the gene expression product in the cell being greater compared to the predetermined amount include, in addition to the amount of the gene expression product in the cell exceeding the predetermined amount, the amount of the gene expression product in the cell being equal to or greater than the predetermined amount.

[0048] The method of the present invention described above determines, for example, that the cell has not undergone Partial EndoMT, using as an indicator that the amount of the gene expression product in the cell is less compared to a predetermined amount. Examples of the amount of the gene expression product in the cell being less compared to the predetermined amount include, in addition to the amount of the gene expression product in the cell being less than the predetermined amount, the amount of the gene expression product in the cell being equal to or less than the predetermined amount.

[0049] The gene expression product is, for example, RNA, protein, or the like.

[0050] When using the gene expression product in the cell as an indicator for the determination, for example, a substance that binds to the gene expression product is used. Examples of the substance that binds to the gene expression product include nucleic acid probes and antibodies.

[0051] Examples of the cell include endothelial cells such as tumor vascular endothelial cells.

[0052] The method of the present invention described above may include the step of selecting a gene from the above group, but this is not essential.

[0053] In the method of the present invention described above, the number of genes selected from the above group is, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, or 43.

[0054] The tenth aspect: The present invention provides a method for determining whether a cell has undergone Partial EndoMT, the method including a step of measuring an expression product of a gene in the cell, wherein the gene is selected from the group consisting of the Rgs2 gene, the Sost gene, the Sp5 gene, the Asap3 gene, the Cd40 gene, the Nckap5l gene, the Hpn gene, the Myzap gene, the Pdgfb gene, the Bambi gene, the Mindy4 gene, the Dapk2 gene, the Limch1 gene, the Dock8 gene, the Vwf gene, the Abhd17a gene, the Adora2a gene, the Zfp69 gene, the Ctnnal1 gene, the Mvb12b gene, the A530016L24Rik gene, the Mmp28 gene, the Rnf144a gene, the Scn1b gene, the Ypel2 gene, the Lhfpl2 gene, the Tmem158 gene, the Plvap gene, the Ctsh gene, the Sema6c gene, the Arl15 gene, the Mafb gene, the Eln gene, the Il4ra gene, the Rgcc gene, the Fam181b gene, the Septin4 gene, the Serinc5 gene, the Robo3 gene, the Ace gene, the Kif5c gene, the Adamts17 gene, and the Trp53i11 gene.

[0055] The step of measuring the expression product of the gene in the cell may be, for example, a step of measuring the presence or absence of the expression product of the gene in the cell, or may be a step of measuring the amount of the expression product of the gene in the cell.

[0056] The method of the present invention described above may include, for example, a step of comparing the measured amount of the expression product of the gene in the cell with a predetermined amount. The predetermined amount may be a predetermined amount, but this is not essential, and may be, for example, an amount clarified by being measured simultaneously with the measurement of the amount of the expression product of the gene in the cell. In the step of comparing the amount of the expression product of the gene in the cell with a predetermined amount, the amount of the expression product of the gene in the cell may be specified as a numerical value, but this is not essential, and for example, although the amount of the expression product of the gene in the cell is not specified as a numerical value, it may be relatively specified by comparison with a control or the like.

[0057] The method of the present invention described above is, for example, a method for determining that a cell has undergone Partial EndoMT using, as an index, that the amount of the gene expression product in the measured cell is greater compared to a predetermined amount. Examples of the amount of the gene expression product in the cell being greater compared to a predetermined amount include, for example, not only that the amount of the gene expression product in the cell exceeds the predetermined amount, but also that the amount of the gene expression product in the cell is equal to or greater than the predetermined amount.

[0058] The method of the present invention described above is, for example, a method for determining that a cell has not undergone Partial EndoMT using, as an index, that the amount of the gene expression product in the measured cell is less compared to a predetermined amount. Examples of the amount of the gene expression product in the cell being less compared to a predetermined amount include, for example, not only that the amount of the gene expression product in the cell is less than the predetermined amount, but also that the amount of the gene expression product in the cell is equal to or less than the predetermined amount.

[0059] The gene expression product is, for example, RNA, protein, etc.

[0060] In the step of measuring the gene expression product in the cell, for example, a substance that binds to the gene expression product is used. Examples of the substance that binds to the gene expression product include, for example, nucleic acid probes, antibodies, etc.

[0061] Examples of the cell include endothelial cells such as tumor vascular endothelial cells.

[0062] The method of the present invention described above may include the step of selecting a gene from the above-mentioned group, but this is not essential.

[0063] In the method of the present invention described above, the number of genes selected from the above group is, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, or 43.

[0064] The seventeenth aspect: The present invention provides a method for selecting cells that have undergone Partial EndoMT or cells that have not undergone Partial EndoMT, the method including a step of selecting cells in which the amount of the gene expression product is greater than a predetermined amount or cells in which the amount of the gene expression product is less than a predetermined amount, wherein the gene is selected from the group consisting of the Rgs2 gene, Sost gene, Sp5 gene, Asap3 gene, Cd40 gene, Nckap5l gene, Hpn gene, Myzap gene, Pdgfb gene, Bambi gene, Mindy4 gene, Dapk2 gene, Limch1 gene, Dock8 gene, Vwf gene, Abhd17a gene, Adora2a gene, Zfp69 gene, Ctnnal1 gene, Mvb12b gene, A530016L24Rik gene, Mmp28 gene, Rnf144a gene, Scn1b gene, Ypel2 gene, Lhfpl2 gene, Tmem158 gene, Plvap gene, Ctsh gene, Sema6c gene, Arl15 gene, Mafb gene, Eln gene, Il4ra gene, Rgcc gene, Fam181b gene, Septin4 gene, Serinc5 gene, Robo3 gene, Ace gene, Kif5c gene, Adamts17 gene, and Trp53i11 gene.

[0065] The predetermined amount may be a predetermined amount in advance, but this is not essential. For example, it may be an amount revealed by being measured simultaneously with the measurement of the amount of the gene expression product in the cell. In the step of selecting a cell in which the amount of the gene expression product is more than the predetermined amount or a cell in which the amount of the gene expression product is less than the predetermined amount, the amount of the gene expression product in the cell may be specified as a numerical value, but this is not essential. For example, although the amount of the gene expression product in the cell is not specified as a numerical value, it may be relatively specified by comparison with a control or the like.

[0066] Examples of cells in which the amount of the gene expression product is more than the predetermined amount include, for example, cells in which the amount of the gene expression product exceeds the predetermined amount, and cells in which the amount of the gene expression product is equal to or more than the predetermined amount.

[0067] Examples of cells in which the amount of the gene expression product is less than the predetermined amount include, for example, cells in which the amount of the gene expression product is less than the predetermined amount, and cells in which the amount of the gene expression product is equal to or less than the predetermined amount.

[0068] The gene expression product is, for example, RNA, protein, or the like.

[0069] In the step of selecting a cell in which the amount of the gene expression product is more than the predetermined amount or a cell in which the amount of the gene expression product is less than the predetermined amount, for example, a substance that binds to the gene expression product is used. Examples of the substance that binds to the gene expression product include nucleic acid probes, antibodies, and the like.

[0070] Examples of the cell include endothelial cells such as tumor vascular endothelial cells.

[0071] The method of the present invention described above may include the step of selecting a gene from the above group, but this is not essential.

[0072] In the method of the present invention described above, the number of genes selected from the group described above is, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, or 43.

[0073] The twentieth aspect: The present invention provides a method for sorting cells that have undergone partial EndoMT or cells that have not undergone partial EndoMT, the method including a step of measuring the amount of the expression product of a gene in the cells, wherein the gene is selected from the group consisting of the Rgs2 gene, the Sost gene, the Sp5 gene, the Asap3 gene, the Cd40 gene, the Nckap5l gene, the Hpn gene, the Myzap gene, the Pdgfb gene, the Bambi gene, the Mindy4 gene, the Dapk2 gene, the Limch1 gene, the Dock8 gene, the Vwf gene, the Abhd17a gene, the Adora2a gene, the Zfp69 gene, the Ctnnal1 gene, the Mvb12b gene, the A530016L24Rik gene, the Mmp28 gene, the Rnf144a gene, the Scn1b gene, the Ypel2 gene, the Lhfpl2 gene, the Tmem158 gene, the Plvap gene, the Ctsh gene, the Sema6c gene, the Arl15 gene, the Mafb gene, the Eln gene, the Il4ra gene, the Rgcc gene, the Fam181b gene, the Septin4 gene, the Serinc5 gene, the Robo3 gene, the Ace gene, the Kif5c gene, the Adamts17 gene, and the Trp53i11 gene.

[0074] The method of the present invention described above may include a step of comparing the measured amount of the gene expression product in a cell with a predetermined amount. The predetermined amount may be a predetermined amount, but this is not essential, and for example, it may be an amount revealed by being measured simultaneously with the measurement of the amount of the gene expression product in a cell. When comparing the measured amount of the gene expression product in a cell with a predetermined amount, the amount of the gene expression product in a cell may be specified as a numerical value, but this is not essential. For example, although the amount of the gene expression product in a cell is not specified as a numerical value, it may be relatively specified by comparison with a control or the like.

[0075] The method of the present invention described above is, for example, a method for selecting a cell as a cell that has undergone Partial EndoMT when the measured amount of the gene expression product in a cell is greater than a predetermined amount when compared with the predetermined amount. That the amount of the gene expression product in a cell is greater than a predetermined amount includes, for example, not only that the amount of the gene expression product in a cell exceeds the predetermined amount, but also that the amount of the gene expression product in a cell is equal to or greater than the predetermined amount.

[0076] The method of the present invention described above is, for example, a method for selecting a cell as a cell that has not undergone Partial EndoMT when the measured amount of the gene expression product in a cell is less than a predetermined amount when compared with the predetermined amount. That the amount of the gene expression product in a cell is less than a predetermined amount includes, for example, not only that the amount of the gene expression product in a cell is less than the predetermined amount, but also that the amount of the gene expression product in a cell is equal to or less than the predetermined amount.

[0077] The gene expression product is, for example, RNA, protein, or the like.

[0078] In the step of measuring the amount of the gene expression product in cells, for example, a substance that binds to the gene expression product is used. Examples of the substance that binds to the gene expression product include nucleic acid probes, antibodies, and the like.

[0079] Examples of the cells include endothelial cells such as tumor vascular endothelial cells.

[0080] The method of the present invention may include the step of selecting a gene from the above-mentioned group, but this is not essential.

[0081] In the method of the present invention, the number of genes selected from the above-mentioned group is, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, or 43.

[0082] The twenty-third to twenty-fourth aspects: The present invention provides a program for causing an information processing apparatus to execute the above-mentioned method. Further, the present invention provides an information processing apparatus storing the above-mentioned program.

[0083] The twenty-fifth aspect: The present invention provides an inhibitor of induction or enhancement of EndoMT in cells, particularly an inhibitor of induction or enhancement of EndoMT in endothelial cells, which contains, as an active ingredient, a substance that acts on the expression products of genes selected from the group consisting of the Rgs2 gene, Sost gene, Sp5 gene, Asap3 gene, Cd40 gene, Nckap5l gene, Hpn gene, Myzap gene, Pdgfb gene, Bambi gene, Mindy4 gene, Dapk2 gene, Limch1 gene, Dock8 gene, Vwf gene, Abhd17a gene, Adora2a gene, Zfp69 gene, Ctnnal1 gene, Mvb12b gene, A530016L24Rik gene, Mmp28 gene, Rnf144a gene, Scn1b gene, Ypel2 gene, Lhfpl2 gene, Tmem158 gene, Plvap gene, Ctsh gene, Sema6c gene, Arl15 gene, Mafb gene, Eln gene, Il4ra gene, Rgcc gene, Fam181b gene, Septin4 gene, Serinc5 gene, Robo3 gene, Ace gene, Kif5c gene, Adamts17 gene, and Trp53i11 gene.

[0084] Examples of substances that act on the expression products of genes include substances that activate the expression products of genes, substances that inactivate the expression products of genes, etc. Examples of substances that activate the expression products of genes include substances that increase the expression of the expression products of genes. Examples of substances that inactivate the expression products of genes include substances that decrease the expression of the expression products of genes. Examples of substances that act on the expression products of genes include agonists of the expression products of genes.

[0085] The inhibitor of the present invention may be composed of a substance that acts on the expression product of the gene, but when it contains a substance other than the substance that acts on the expression product of the gene, the inhibitor of the present invention may contain, for example, a pharmaceutically acceptable carrier and additives. The pharmaceutically acceptable carrier or additive is not particularly limited and can be appropriately selected according to, for example, the dosage form, etc., and can be any carrier, diluent, excipient, suspending agent, lubricant, adjuvant, medium, delivery system, emulsifier, tablet disintegrating substance, absorbent, preservative, surfactant, coloring agent, flavor or sweetener, etc. The dosage form of the inhibitor of the present invention is not particularly limited and can be appropriately selected according to the desired administration method, and examples include injections (solutions, suspensions, solid preparations for in-use dissolution, etc.), solid preparations (tablets, capsules, suppositories, powders, etc.). As an injection, for example, a pH adjuster, buffer, stabilizer, isotonic agent, local anesthetic, etc. can be added to the composition, and injections for subcutaneous, intra-articular, intramuscular, intravenous use, etc. can be produced by a conventional method. Examples of the pH adjuster and the buffer include sodium citrate, sodium acetate, sodium phosphate, etc. Examples of the stabilizer include sodium pyrosulfite, EDTA, thioglycolic acid, thiolactic acid, etc. Examples of the isotonic agent include sodium chloride, glucose, etc. Examples of the local anesthetic include procaine hydrochloride, lidocaine hydrochloride, etc. The solid preparation may be provided with an enteric coating. That is, the inhibitor of the present invention may be prepared as a liquid preparation or a solid preparation, and can be formulated into various dosage forms such as injections, jelly preparations, spray preparations, tablets, granules, etc. When preparing as a liquid preparation, sterilized water, physiological saline, glucose aqueous solution, etc. can be used as a carrier, and further, if desired, a bactericide, isotonic agent, stabilizer, etc. can be blended. When preparing as a solid preparation, additives such as starch, lactose, mannitol, inorganic salts, etc. can be blended, and further, if desired, a binder, disintegrant, lubricant, etc. can be blended.

[0086] The inhibitor of the present invention can be administered, for example, by any administration method suitable for the dosage form, such as oral administration, intravenous administration, subcutaneous administration, transdermal administration, intramuscular administration, intra-articular administration, nasal administration, intraperitoneal administration, direct injection into the target tissue, inhalation administration, enteral administration, enema administration, parenteral nutrition, etc. The dosage of the inhibitor of the present invention may vary depending on the age, weight, health condition, etc. of the subject. The inhibitor of the present invention may be administered once a day or multiple times a day, and the administration frequency can be, for example, 1 to 100 times per month.

[0087] The present invention provides a method for inhibiting the induction or enhancement of EndoMT of cells in a subject, particularly a method for inhibiting the induction or enhancement of EndoMT of endothelial cells in a subject, which comprises administering to the subject a substance that acts on the expression products of genes selected from the group consisting of the Rgs2 gene, Sost gene, Sp5 gene, Asap3 gene, Cd40 gene, Nckap5l gene, Hpn gene, Myzap gene, Pdgfb gene, Bambi gene, Mindy4 gene, Dapk2 gene, Limch1 gene, Dock8 gene, Vwf gene, Abhd17a gene, Adora2a gene, Zfp69 gene, Ctnnal1 gene, Mvb12b gene, A530016L24Rik gene, Mmp28 gene, Rnf144a gene, Scn1b gene, Ypel2 gene, Lhfpl2 gene, Tmem158 gene, Plvap gene, Ctsh gene, Sema6c gene, Arl15 gene, Mafb gene, Eln gene, Il4ra gene, Rgcc gene, Fam181b gene, Septin4 gene, Serinc5 gene, Robo3 gene, Ace gene, Kif5c gene, Adamts17 gene, and Trp53i11 gene.

[0088] Examples of the subject include vertebrates. Examples of vertebrates include mammals such as mice, rats, rabbits, pigs, cows, monkeys, and humans. The mammal is preferably a human. The subject can be of any age, including infants, juveniles, youths, adults, and the elderly.

[0089] The present invention provides the use of a substance that acts on the expression products of genes selected from the group consisting of the Rgs2 gene, Sost gene, Sp5 gene, Asap3 gene, Cd40 gene, Nckap5l gene, Hpn gene, Myzap gene, Pdgfb gene, Bambi gene, Mindy4 gene, Dapk2 gene, Limch1 gene, Dock8 gene, Vwf gene, Abhd17a gene, Adora2a gene, Zfp69 gene, Ctnnal1 gene, Mvb12b gene, A530016L24Rik gene, Mmp28 gene, Rnf144a gene, Scn1b gene, Ypel2 gene, Lhfpl2 gene, Tmem158 gene, Plvap gene, Ctsh gene, Sema6c gene, Arl15 gene, Mafb gene, Eln gene, Il4ra gene, Rgcc gene, Fam181b gene, Septin4 gene, Serinc5 gene, Robo3 gene, Ace gene, Kif5c gene, Adamts17 gene, and Trp53i11 gene, in the manufacture of a pharmaceutical composition for inhibiting the induction or enhancement of EndoMT in cells, particularly in the manufacture of a pharmaceutical composition for inhibiting the induction or enhancement of EndoMT in endothelial cells.

[0090] The twenty-seventh aspect: The present invention provides a therapeutic agent for treating cancer, fibrosis, pulmonary arterial hypertension, or atherosclerosis, which contains, as an active ingredient, a substance that acts on the expression products of genes selected from the group consisting of the Rgs2 gene, Sost gene, Sp5 gene, Asap3 gene, Cd40 gene, Nckap5l gene, Hpn gene, Myzap gene, Pdgfb gene, Bambi gene, Mindy4 gene, Dapk2 gene, Limch1 gene, Dock8 gene, Vwf gene, Abhd17a gene, Adora2a gene, Zfp69 gene, Ctnnal1 gene, Mvb12b gene, A530016L24Rik gene, Mmp28 gene, Rnf144a gene, Scn1b gene, Ypel2 gene, Lhfpl2 gene, Tmem158 gene, Plvap gene, Ctsh gene, Sema6c gene, Arl15 gene, Mafb gene, Eln gene, Il4ra gene, Rgcc gene, Fam181b gene, Septin4 gene, Serinc5 gene, Robo3 gene, Ace gene, Kif5c gene, Adamts17 gene, and Trp53i11 gene.

[0091] Examples of fibrosis include cardiac fibrosis and organ fibrosis. Examples of organ fibrosis include liver fibrosis, kidney fibrosis, and intestinal fibrosis.

[0092] Examples of the therapeutic agent include, in addition to therapeutic agents, prophylactic agents, etc. Examples of treatment include, in addition to complete cure, alleviation of symptoms, etc.

[0093] Examples of the substance that acts on the expression product of a gene include a substance that activates the expression product of a gene, a substance that inactivates the expression product of a gene, etc. Examples of the substance that activates the expression product of a gene include a substance that increases the expression of the expression product of a gene, etc. Examples of the substance that inactivates the expression product of a gene include a substance that decreases the expression of the expression product of a gene, etc. Examples of the substance that acts on the expression product of a gene include an agonist of the expression product of a gene, etc.

[0094] The present invention provides a method for treating cancer, fibrosis, pulmonary arterial hypertension, or atherosclerosis in a subject, the method comprising administering to the subject a substance that acts on the expression product of a gene selected from the group consisting of the Rgs2 gene, Sost gene, Sp5 gene, Asap3 gene, Cd40 gene, Nckap5l gene, Hpn gene, Myzap gene, Pdgfb gene, Bambi gene, Mindy4 gene, Dapk2 gene, Limch1 gene, Dock8 gene, Vwf gene, Abhd17a gene, Adora2a gene, Zfp69 gene, Ctnnal1 gene, Mvb12b gene, A530016L24Rik gene, Mmp28 gene, Rnf144a gene, Scn1b gene, Ypel2 gene, Lhfpl2 gene, Tmem158 gene, Plvap gene, Ctsh gene, Sema6c gene, Arl15 gene, Mafb gene, Eln gene, Il4ra gene, Rgcc gene, Fam181b gene, Septin4 gene, Serinc5 gene, Robo3 gene, Ace gene, Kif5c gene, Adamts17 gene, and Trp53i11 gene.

[0095] The present invention provides the use of a substance that acts on the expression products of genes selected from the group consisting of the Rgs2 gene, Sost gene, Sp5 gene, Asap3 gene, Cd40 gene, Nckap5l gene, Hpn gene, Myzap gene, Pdgfb gene, Bambi gene, Mindy4 gene, Dapk2 gene, Limch1 gene, Dock8 gene, Vwf gene, Abhd17a gene, Adora2a gene, Zfp69 gene, Ctnnal1 gene, Mvb12b gene, A530016L24Rik gene, Mmp28 gene, Rnf144a gene, Scn1b gene, Ypel2 gene, Lhfpl2 gene, Tmem158 gene, Plvap gene, Ctsh gene, Sema6c gene, Arl15 gene, Mafb gene, Eln gene, Il4ra gene, Rgcc gene, Fam181b gene, Septin4 gene, Serinc5 gene, Robo3 gene, Ace gene, Kif5c gene, Adamts17 gene, and Trp53i11 gene in the manufacture of a pharmaceutical composition for treating cancer, fibrosis, pulmonary arterial hypertension, or atherosclerosis.

[0096] Regarding the 27th aspect described above, the explanation for the 25th aspect is equally applicable.

[0097] The twenty-ninth aspect: The present invention provides a cell comprising a first exogenous gene expressed based on the promoter of an endothelial marker gene and a second exogenous gene expressed based on the promoter of a mesenchymal marker gene.

[0098] The exogenous gene is, for example, a gene introduced into the cell, and more specifically, a gene artificially introduced into the cell. The first exogenous gene may be the same as the second exogenous gene, but preferably is different from the second exogenous gene.

[0099] The first exogenous gene expressed based on the promoter of the endothelial marker gene is preferably expressed based on the expression of the expression product of the gene expressed based on the promoter of the endothelial marker gene. The expression product of the gene expressed based on the promoter of the endothelial marker gene is derived from, for example, Cre recombinase. Examples of those derived from Cre recombinase include Cre recombinase itself and modified Cre recombinase.

[0100] The first exogenous gene is preferably a gene that expresses a fluorescent protein, and the second exogenous gene is preferably a gene that expresses a fluorescent protein.

[0101] Examples of the endothelial marker gene include the Cdh5 gene and the Flk1 (VEGFR2) gene. Examples of the mesenchymal marker gene include the Acta2 (αSMA) gene and the Tagln (SM22α) gene.

[0102] The present invention provides a blood vessel containing the above-mentioned cells.

[0103] The present invention provides a non-human animal containing the above-mentioned cells.

[0104] The present invention provides the use of the above-mentioned cells, blood vessels, or non-human animals in the manufacture of an inhibitor for enhancing or inducing EndoMT of cells, particularly in the manufacture of an inhibitor for enhancing or inducing EndoMT of endothelial cells, or in the manufacture of a therapeutic agent for cancer, fibrosis, pulmonary arterial hypertension, or atherosclerosis.

Example

[0105] Materials and methods: Cell culture and reagents: Mouse EMREC was established as described later and maintained in RPMI 1640 medium (Nacalai Tesque, Kyoto, Japan) containing 10% FBS (Thermo Fisher Scientific, Waltham, Massachusetts, USA), 100 units / ml penicillin and 100 μg / ml streptomycin (Nacalai Tesque). The MS-1, a mouse endothelial cell line, was purchased from ATCC (Manassas, Virginia, USA) and maintained in α-minimum essential medium (αMEM, Fujifilm Wako Pure Chemical, Osaka, Japan) supplemented with 10% FBS, 100 units / ml penicillin and 100 μg / ml streptomycin. The SAS, a human OSCC cell line, was obtained from the RIKEN BioResource Center Cell Bank (Tsukuba, Japan) and maintained in DMEM (Nacalai Tesque) supplemented with 10% FBS, 100 units / ml penicillin and 100 μg / ml streptomycin. TGF-β2 was purchased from Peprotech (Rocky Hill, New Jersey, USA).

[0106] RNA isolation and quantitative RT-PCR (qRT-PCR): Total RNA from MS-1 cells or EMREC was isolated using Sepasol I-RNA I SuperG (Nacalai Tesque), RNeasy Plus Mini Kit (QIAGEN, Hilden, Germany), or RNeasy FPE Kit (QIAGEN), and reverse-transcribed using PrimeScript II 1st strand cDNA Synthesis Kit (TaKaRaBio, Otsu, Japan), PrimeScript IV 1st strand cDNA Synthesis Kit (TaKaRa), or ReverTraAce qPCR RT Mix (TOYOBO, Osaka, Japan). qRT-PCR was performed using gene-specific primers and Fast Start Universal SYBR Green Master (Roche, Basel, Switzerland) or PowerUp SYBR Green Master Mix (Applied Biosystems) on a Step One Plus real-time PCR system (Applied Biosystems, Waltham, Massachusetts, USA) or a QuantStudio 3 (Applied Biosystems, Switzerland). All expression data were normalized to the expression of β-actin. The primers used for qRT-PCR are listed in Table 1 (List of primer sequences used for qPCR: shown in SEQ ID NO: 1 to SEQ ID NO: 14 in order from the top).

Table 1

[0107] Fabrication of three-dimensional (3D) microvascular on-chip 25×25×5mm 3 The 25×25×5 mm polydimethylsiloxane (PDMS)-based chip was prepared as before. To prepare 3D microvessels, the PDMS chip was O 2Filled with a collagen solution containing 3 mg / ml type I collagen (Cellmatrix TypeI-A; pH 3.0; Nitta Gelatin, Osaka, Japan), 10× Hank's buffer (Sigma-Aldrich, St. Louis, Missouri, USA), and reconstitution buffer (Nitta Gelatin) at 8:1:1 (v / v / v), which was treated with plasma, sterilized with UV light, and neutralized with ice cooling. A 1% BSA-coated needle Φ200 needle (No.02, 0.20×30 mm 2 , J type, Seirin, Shizuoka Prefecture) was inserted to form channels in the collagen gel, and the device was incubated at 37 °C for 90 minutes. The needle was gently removed, and the luminal surface of the channels in the collagen gel was coated with 1 mg / ml fibronectin, followed by injection of the EMREC suspension (1.0×10 7 cells / ml). EMREC was allowed to adhere to the luminal surface at 37 °C for 90 minutes in a humidified incubator. Next, the device was filled with medium, and the cells were cultured overnight to form microvessels. The microvessels were treated with or without TGF-β2 for 72 hours, and immunocytochemical analysis and permeability assays were performed as described later.

[0108] Flow cytometry analysis and cell sorting EMREC cultured in a RepCell culture dish (CellSeed Inc., Tokyo, Japan) was treated for 68 hours without or with TGF-β2 and harvested using accutase (Innovative Cell Technologies, San Diego, CA, USA) in ice-cold cell dissociation buffer containing 3 mM EDTA (Thermo Fisher Scientific). Next, EMREC was fixed with 2% paraformaldehyde (PFA) and incubated with allophycocyanin (APC)-conjugated anti-CD309 (VEGFR2) antibody, brilliant violet 421 (BV421)-conjugated anti-CD40 antibody, or each fluorescent-conjugated isotype IgG (Table 2: List of antibodies used in the present invention). VEGFR2, CD40, and GFP were analyzed by flow cytometry using an ID7000 Spectral Cell Analyzer (SONY, Tokyo, Japan) and FlowJo software (BD Biosciences, Franklin Lakes, NJ, USA). [Table 2]

[0109] Cell sorting was performed using a MoFlo XDP flow cytometer (Beckman Coulter, Brea, CA, USA). Cells were collected in a medium containing 2% FBS and RNase inhibitor. Next, the sorted cells were subjected to qRT-PCR and bulk RNA-seq analysis as described later.

[0110] Statistical analysis The significant differences between the means were determined using unpaired two-sided Student's t-test, one-way analysis of variance with Tukey's multiple comparison test, or Bonferroni's multiple comparison test using Prism 10 software version 10.1.0 (GraphPad Software, Boston, Massachusetts, USA). The numerical values are presented as mean ± standard deviation (SD). A difference between the means was considered statistically significant at p < 0.05. The significance of gene sets from gene set enrichment analysis (GSEA) was based on the normalized enrichment score (NES) and nominal p-value.

[0111] Preparation of recombinant Fc protein The plasmid construction procedure has been approved by the Recombinant DNA Safety Committee of Tokyo Medical and Dental University (Registration number: G2019-026C6). Recombinant Control-Fc and TβRI-TβRII-Fc proteins were purified from the conditioned medium of Epi293F cells expressing the recombinant proteins [Takahashi K, Akatsu Y, Podyma-Inoue KA, et al. Targeting all transforming growth factor-β isoforms with an Fc chimeric receptor impairs tumor growth and angiogenesis of oral squamous cell cancer. J Biol Chem. 2020; 295: 12559-12572.]. Recombinant Fc proteins were purified using Protein G Sepharose 4 Fast Flow (Cytiva, Marlborough, Massachusetts, USA) equilibrated with wash buffer (20 mM Tris-HCl containing 1 M NaCl, pH 7.5). The bound Fc proteins were eluted with 0.1 M glycine buffer, pH 2.7 and immediately neutralized with 1 M Tris-HCl buffer, pH 10 (1 / 10 volume). Proteins were concentrated using Amicon Ultra Centrifugal filters (30 or 50 kDa NMWL, Merck Millipore, Darmstadt, Germany) and sterilized through a 0.22 μm pore size Millex-GV filter (Merck Millipore).

[0112] Orthotopic mouse model of oral cancer cells Animal procedures were approved by the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University (Registration number A2021-133C5). SAS cells (1×10 3 cells / 100 μl Matrigel; Corning, Corning, NY, USA) were inoculated subcutaneously into the left inguinal region of 6-week-old male immunodeficient BALB / c nude mice (Sankyo Labo, Shizuoka Prefecture). On day 33 after inoculation, palpable tumors (approximate tumor volume: 50 mm 3) After the occurrence of , the mice were divided into two groups (n = 5 per group), and then recombinant Fc protein, control-Fc, or TβRI-TβRII-Fc (50 μg / 50 μl PBS) was administered intravenously twice a week for 6 weeks. On day 76 after inoculation, the tumors were collected and subjected to immunohistochemical analysis as follows.

[0113] Immunohistochemical analysis Immunohistochemistry was performed as before. The collected tumors were embedded in OCT compound (Sakuratech Fine, Kanagawa, Japan) and instantaneously frozen in a dry ice / acetone-hexane bath. Tumor xenograft samples were sectioned into 10 slices with a thickness of 5 μm using a cryostat CMV 1950 (Leica Microsystems GmbH, Wetzlar, Germany) (3 slices per tumor tissue), fixed with 4% PFA for 15 minutes, blocked with 2% FBS containing 0.01% Tween20 for 40 minutes, and then incubated with specific antibodies (Table 2: List of antibodies used in the present invention). The nuclei were stained with Hoechst33342 (Cell Signaling Technology, Danvers, Massachusetts, USA). Images (3 fields of view for one specimen; a total of 9 fields of view for each tumor tissue) were taken with a Dmi8 THUNDER Imager (Leica Microsystems GmbH, Wetzlar, Germany). Quantification was performed using the co-localized regions detected for each Z-stack by FIJI (ImageJ) software.

[0114] Establishment of EndoMT reporter mice Animal experiments were approved by the Institutional Animal Care and Use Committee of Tokyo University of Pharmacy and Life Sciences (approval number: L16-13, LS27-007) and the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University (registration number: A2021-133C5). The experiments were conducted in accordance with the guidelines of the animal management standards of both institutions. αSMA-promoter-EGFP (hereinafter referred to as "αSMA-GFP") mice [Nishimura G, Manabe I, Tsushima K, et al. δEF1 mediates TGF-β signaling in vascular smooth muscle cell differentiation. Dev Cell. 2006; 11: 93-104.] were obtained from the University of Tokyo (donated by Dr. Katsuhito Fujio). Cdh5-BAC-Cre ERT2 (Cdh5-Cre ERT2 )[Okabe K, Kobayashi S, Yamada T, et al. Neurons limit angiogenesis by titrating VEGF in retina. Cell. 2014; 159: 584-596.] mice were crossed with R26Rosa-loxP-Stop-loxP-tdTomato mice (LSL-tdTomato; The Jackson Laboratory, Bar Harbor, ME, USA) to obtain Cdh5-Cre ERT2 -LSL-tdTomato double transgenic mice. These mice were further crossed with αSMA-GFP mice to obtain αSMA-GFP;Cdh5-Cre ERT2 -LSL-tdTomato triple transgenic mice. Both Cdh5-Cre ERT2 -LSL-tdTomato mice and αSMA-GFP;Cdh5-Cre ERT2 -LSL-tdTomato mice showed normal phenotypes and lifespans.

[0115] Establishment of EndoMT reporter endothelial cells αSMA-GFP;Cdh5-CRE ERT2;The liver of LSL-tdTomato mice was perfused from the portal vein with perfusion fluid and collagenase, homogenized, and then plated on collagen-coated dishes (IWAKI, Tokyo, Japan) of the EBM-2MV Bullet Kit (Lonza, Basel, Switzerland). Next, cells were infected with a retrovirus expressing the PymT oncogene that specifically immortalizes endothelial cells. Cells were grown until the morphology of endothelial cells became clear. Next, the medium was replaced with RPMI 1640 (Nacalai, Kyoto, Japan) supplemented with 10% FBS (Thermo Fisher Scientific, Waltham, MA, USA), 100 units / ml penicillin, and 100 μg / ml streptomycin (Nacalai). Molecular biology experiments have received approval from the President of Tokyo University of Pharmacy and Life Sciences (approval number: LSR3-015).

[0116] Immunocytochemistry EMREC cultured on collagen-coated cover glasses (IWAKI) was fixed with 4% PFA for 15 minutes and permeabilized with 0.2% TritonX-100 for 10 minutes. To detect CD40, cells were fixed with ice-cold methanol for 5 minutes. Next, samples were blocked with 2% FBS for 40 minutes and incubated with specific antibodies (Table 2: List of antibodies used in the present invention). Nuclei were visualized using Hoechst 33342 (Cell Signaling Technology). Stained EMREC was observed with a TCS SP8 confocal laser scanning microscope (Leica Microsystems GmbH) or an FV3000 confocal laser scanning microscope (Olympus). An all-in-one fluorescence microscope BZ-X710 (KEYENCE, Osaka, Japan) was used to observe Ki67. Ki67-positive cells were quantified with BZ-X Analyzer software version 1.3.0.3 (KEYENCE).

[0117] Transwell migration assay EMREC was seeded into the upper chamber of a 24-well culture insert coated with collagen using an 8-μm pore filter (BD Bioscience). The migration of EMREC was evaluated after 24 hours. Next, the filter was stained with DiffQuik (Sysmex, Kobe, Japan), and non-migrating cells were removed using a cotton swab. The migrating cells were photographed with an all-in-one fluorescence microscope BZ-X710 (KEYENCE). The migrating cells were quantified using ImageJ software.

[0118] Immunocytochemistry of 3D microvessels The 3D microvessels of the PDMS-based chip were fixed with 4% PFA for 30 minutes and permeabilized with 0.5% TritonX-100 for 10 minutes. Next, the PDMS chip was blocked overnight at 4 °C using 1% BSA, incubated with specific primary antibodies (Table 2: List of antibodies used in the present invention), and then visualized with secondary antibodies. The nuclei were stained with Hoechst 33342. The stained EMREC microvessels within the PDMS chip were observed with an FV3000 confocal laser scanning microscope (Olympus).

[0119] Permeability assay of 3D microvessels The microvessel chip was placed on stereolithography printed by 3D printing to fix a polycarbonate CNC engraving attachment. The attachment was filled with 1% PBS, and uniform pressure was applied inside and outside the microvessels. To evaluate the permeability of the 3D microvessels, a 30-μl 1% PBS solution of 10 mg / ml 4 kDa fluorescein isothiocyanate (FITC)-dextran (Sigma-Aldrich, St. Louis, Missouri, USA) and 70 kDa tetramethylrhodamine isothiocyanate (TRITC)-dextran (Sigma-Aldrich) was used. Diffusion time-lapse videos were taken with lasers at 488 μm and 555 μm (20× magnification at 0.5 zoom, observation area: 640.17 × 640.17 μm 2, with a resolution of 512×512 pixels, was simultaneously used to record for 60 seconds at 3.87 seconds per frame by an LSM 700 confocal microscope (Carl Zeiss, Oberkochen, Germany).

[0120] The movie was analyzed using ImageJ. The average pixel intensity inside and outside each microvessel was obtained for each frame and dextran molecule. The intensity coefficient (I out / I in ) of each frame was calculated using the following equation after subtracting the initial background intensity.

Equation

[0121] Measurement of vessel diameter of 3D microvessels The diameter of the microvessel was measured by analyzing the first frame of the diffusion movie recorded by the confocal microscope. Since the first frame corresponds to the injection of the dextran solution into the microvessel chip, a high signal intensity is detected inside the microvessel, while the outside remains dark. The diameter was measured by using the ImageJ plugin FeatureJ Derivatives to calculate the second derivative of the dextran signal in the radial direction of the image.

[0122] Bulk RNA sequencing and data analysis Total RNA from the selected EMREC was prepared using the RNeasy FPE kit (QIAGEN). Bulk RNA-seq was performed by AZENTA LIFE SCIENCES (Chelmsford, Massachusetts, USA). Libraries were prepared from 1 μg of RNA and sequenced on Illumina HiSeq / Illumina Novaseq / MGI2000. Data analysis was performed using GeneSpring version 14.9.1 (Agilent Technologies, Santa Clara, California, USA). For data analysis and construction of heatmaps, the fragments per kilobase of exon per million mapped reads (FPKM) values were log2-transformed after adding 1, and Z-scores were calculated. Gene set enrichment analysis was performed using GSEA (version 4.3.2) and a previously curated gene set [Mootha VK, Lindgren CM, Eriksson KF, et al. PGC-1α-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nat Genet. 2003; 34: 267-273.; Subramanian A, Tamayo P, Mootha VK, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci. USA. 2005; 102: 15545-15550.]. Gene ontology (GO) analysis was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID). The raw RNA sequence data described herein are available in the Sequence Read Archive (SRA) of the DNA Data Bank of Japan (DDBJ) under accession number: DRA016758.

[0123] RNA interference Mouse CD40 siRNAs (Silencer Select siRNA ID#1: s75281; #2: s75283) and negative control (catalog number 4390846) were purchased from Thermo Fisher Scientific. All siRNAs were introduced into EMRECs using Lipofectamine RNAi Max reagent (Thermo Fisher Scientific) according to the manufacturer's instructions. Briefly, cells were mixed with the siRNA-Lipofectamine complex and seeded onto 6-well culture plates coated with collagen. After 14 hours, the medium was replaced, and the cells were treated for 72 hours in the presence or absence of TGF-β2.

[0124] Processing of pan-cancer single-cell RNA sequencing (scRNA-seq) data Single-cell transcriptome samples (855,271 cells) of 226 were downloaded from the Gene Expression Omnibus (accession number GSE210347) and used for analysis [Luo H, Xia X, Huang LB, et al. Pan-cancer single-cell analysis reveals the heterogeneity and plasticity of cancer-associated fibroblasts in the tumor microenvironment. Nat Commun. 2022; 13: 6619.]. Bioinformatics analysis code was downloaded from github (https: / / github.com / Xiaxy-XuLab / PanCAF). Data integration was performed using Seurat (version 4.1.0), Seurat Wrappers (version 0.3.0), and Signac (version 4.1.0). The top 2000 variable expression genes were used for downstream analysis by the FindVariableFeature function. The identified 50 clusters were visualized by UMAP, and gene expression within each cluster was identified using violin plots. Single-cell trajectory and pseudotime analysis were performed by creating an elastic principal tree on UMAP using Monocle3.

[0125] Results Example 1 Inhibition of TGF-β signaling suppresses EndoMT in the TME That TGF-β plays an important role in tumor progression and metastasis through the induction of EndoMT has been shown by multiple lines of evidence [Non-patent Document 9]. To verify the in vivo induction of EndoMT by TGF-β signaling, a mouse subcutaneous transplantation model of SAS cells, human OSCC, was used. Immunostaining of tumor tissues derived from human OSCC cells identified cells co-expressing platelet and endothelial cell adhesion molecule-1 (PECAM-1) and αSMA, markers of endothelial cells and mesenchymal cells, respectively (Figure 1), suggesting that some endothelial cells within the tumor had undergone EndoMT.

[0126] The present inventors previously developed a chimeric Fc receptor for TGF-β ligands [extracellular domain of the TGF-β receptor fused with the Fc domain of immunoglobulin (IgG): TβRI-TβRII-Fc]. This captures all TGF-β isoforms and inhibits TGF-β signaling [Takahashi K, Akatsu Y, Podyma-Inoue KA, et al. Targeting all transforming growth factor-β isoforms with an Fc chimeric receptor impairs tumor growth and angiogenesis of oral squamous cell cancer. J Biol Chem. 2020; 295: 12559-12572.; Kodama S, Podyma-Inoue KA, Uchihashi T, et al. Progression of melanoma is suppressed by targeting all transforming growth factor-β isoforms with an Fc chimeric receptor. Oncol Rep. 2021; 46: 197.]. When recombinant TβRI-TβRII-Fc protein was administered to mice with tumors derived from oral cancer cells to inhibit TGF-β signaling in the TME, the proportion of αSMA in PECAM-1 positive cells in tumors treated with TβRI-TβRII-Fc was significantly lower than that in tumors treated with Control-Fc (human IgG-Fc) (Figure 1).

[0127] It is widely accepted that normal blood vessels are covered with pericytes. Although some tumor blood vessels also show pericyte coverage, they may be sparse and irregular. Pericytes express nerve / glia antigen 2 (NG2), which is widely considered a pericyte marker, but pericytes may also express mesenchymal cell markers such as αSMA. Therefore, staining was performed with anti-NG2 antibody. Since it was found that cells expressing αSMA were either NG2-positive or NG2-negative (Figure 10A), αSMA-positive and NG2-negative cells detected among PECAM1-positive cells were defined as cells undergoing EndoMT. The inventors discovered that inhibition of the TGF-β signal during TβRI-TβRII-Fc treatment decreased the proportion of such EndoMT cells (Figures 10A - C). These results suggest that TGF-β signaling induces EndoMT in tumors derived from oral cancer cells.

[0128] Example 2 Establishment of EndoMT reporter endothelial cells (EMREC) The inventors previously reported that TGF-β induces EndoMT in multiple types of human and mouse endothelial cells [Non-Patent Document 10]. However, specific identification of cells in the Partial EndoMT state, which is an intermediate stage of EndoMT, was not achieved. To visualize the stepwise changes during EndoMT, a tamoxifen-inducible endothelial-specific Cre line, Cdh5-BAC-Cre ERT2 (Cdh5-Cre ERT2 ) mice [Okabe K, Kobayashi S, Yamada T, et al. Neurons limit angiogenesis by titrating VEGF in retina. Cell. 2014; 159: 584-596.] were mated with R26Rosa-loxP-Stop-loxP-tdTomato (LSL-tdTomato) mice and αSMA-GFP mice, which are mesenchymal-specific GFP reporter lines that express GFP under the control of the αSMA promoter, to utilize gene labeling of the endothelial cell lineage in combination with live imaging of mesenchymal cells (Figure 2A). Cdh5-Cre ERT2The mouse expresses Cre recombinase under the control of the endothelial cell-specific marker VE-cadherin (Cdh5) promoter, so tdTomato is expressed in cells derived from the endothelial cell lineage. Furthermore, the αSMA-GFP transgene marks mesenchyme-derived αSMA-positive cells and EndoMT-derived cells from the endothelium. Therefore, endothelial cells that have undergone complete EndoMT co-express tdTomato and GFP but do not express endothelial cell markers such as VEGFR2 (Figure 2A). In particular, cells with Partial EndoMT are expected to express tdTomato, GFP, and VEGFR2.

[0129] To characterize the various processes during EndoMT, endothelial cells were isolated from the livers of transgenic mice and immortalized to establish EMRECs that express tdTomato (Figure 2B). When EMRECs were cultured for 72 hours in the presence of TGF-β2 (hereinafter referred to as "TGF-β"), some EMRECs showed GFP expression, suggesting activation of the αSMA promoter via TGF-β (Figure 2B).

[0130] To examine the effect of TGF-β on the expression of various EndoMT markers in EMRECs, qRT-PCR and immunocytochemical analysis were performed. TGF-β upregulated the expression of PAI-1, a target gene of TGF-β signaling (Figure 2C), suggesting that EMRECs respond to TGF-β. TGF-β also upregulated the expression of the mesenchymal cell markers αSMA and SM22α (Figure 2D, E). In contrast, the expression of the endothelial cell markers VEGFR2 and Tie2 decreased upon TGF-β treatment (Figure 2F and Figure 11A). The effect of TGF-β on VEGFR2 expression was also confirmed at the protein level (Figure 2G). In addition, the effect of TGF-β on the RNA expression of various EndoMT markers in the mouse pancreatic endothelial cell line MS-1 was also verified (Figure 11B-F).

[0131] Previous reports have shown that endothelial cells acquire enhanced motility during EndoMT [Non-Patent Document 8]. In the chamber migration assay, TGF-β was found to enhance the motility of EMRECs (Figure 2H), indicating that EMRECs undergo functional EndoMT in response to TGF-β signaling.

[0132] Example 3 TGF-β induces EndoMT in 3D microvessels derived from EMREC Since blood vessels form a 3D lumen structure in vivo, it is necessary to mimic the blood vessel structure in vitro to understand the mechanisms underlying EndoMT in blood vessels. Therefore, to investigate the effect of TGF-β-induced EndoMT in blood vessels, an in vitro model technique for creating 3D endothelial microvessels using EMRECs was applied. After incubating EMREC-derived microvessels for 72 hours in the absence or presence of TGF-β and then staining for VEGFR2, it was revealed that TGF-β treatment decreased VEGFR2 expression throughout the microvessels. In contrast, αSMA-GFP expression was not uniform but sparse and limited to some EMRECs within the microvessels (Figure 3A). Furthermore, the diameter of the microvessels decreased with TGF-β treatment (Figure 12A). This phenomenon was caused by the inhibitory effect of TGF-β on endothelial cell proliferation, as TGF-β decreased the proportion of Ki67-positive cells (Figures 12B, C).

[0133] Previous reports have shown that EndoMT impairs the barrier function of lymphatic vessels [Yoshimatsu Y, Kimuro S, Pauty J, et al. TGF-beta and TNF-alpha cooperatively induce mesenchymal transition of lymphatic endothelial cells via activation of activin signals. PLoS One. 2020; 15: e0232356.]. Permeability assays were performed by simultaneously injecting fluorescently labeled dextran probes (4 kDa and 75 kDa) into the lumen of microvessels. The spatial redistribution of the dextran probes was monitored by confocal microscopy for 60 seconds (Figures 3B, C). Both dextrans leaked at higher levels from TGF-β-treated microvessels to the collagen gel compared to control microvessels (Figures 3B, C), suggesting that TGF-β-induced EndoMT impairs the barrier function of EMREC-derived microvessels. These results suggest that EMREC exhibits various phenotypes of TGF-β-induced EndoMT in 3D microvessels.

[0134] Example 4 Stepwise changes during EndoMT are observed in EMREC Since EMREC enabled visualization of various changes in endothelial cells during TGF-β-induced EndoMT, we next attempted to isolate multiple partial populations of endothelial cells undergoing EndoMT using FACS. After culturing EMREC in the absence or presence of TGF-β, FACS analysis of all tdTomato-positive cells (showing various levels of tdTomato expression from tdTomato low to tdTomato high and tdTomato high cells) was performed to determine the expression of VEGFR2 and αSMA-GFP (Figures 4A and 13). EMREC hardly expressed αSMA-GFP, but TGF-β was present in all dtTomato-positive cells and tdTomato highThe emergence of αSMA-GFP-positive EMRECs was induced in cells (Figures 4A and 13). Furthermore, it was found that αSMA-GFP-positive EMRECs contain partial populations that are VEGFR2-positive and VEGFR2-negative. To isolate multiple subpopulations of endothelial cells undergoing EndoMT, all tdTomato-positive cells were targeted and a large number of cells were collected for further analysis. Therefore, the inventors sorted VEGFR2-positive / αSMA-GFP-negative cells from TGF-β untreated EMRECs and TGF-β treated EMRECs [fractions (1) and (2) respectively; Figure 4A]. Also, VEGFR2-positive / αSMA-GFP-positive cells and VEGFR2-negative / αSMA-GFP-positive cells were sorted from TGF-β treated EMRECs [fractions (3) and (4) respectively; Figure 4A]. To evaluate the mesenchymal state of the sorted fractions, bulk RNA-seq analysis was performed. When comparing the mesenchymal states of the four sorted fractions by GSEA using the EMT hallmark gene set, fraction (2) (TGF-β treated endothelial cells; hereinafter referred to as "Tβ-EC") was found to exhibit a stronger mesenchymal phenotype than fraction (1) (TGF-β untreated endothelial cells; hereinafter referred to as "EC") (Figure 4B). Furthermore, fraction (3), which expressed both VEGFR2 and αSMA-GFP, showed a stronger and weaker mesenchymal phenotype than fraction (2) and fraction (4) respectively (Figures 4C, D), suggesting that fraction (3) and fraction (4) represent cells undergoing Partial EndoMT (hereinafter referred to as "Partial") and complete EndoMT (hereinafter referred to as "Full") respectively (Figure 4A).

[0135] qRT-PCR was used to examine the expression of various EndoMT-related markers in the sorted fractions. PAI-1 was upregulated in all TGF-β-stimulated fractions (Figure 4E), suggesting that the activation of TGF-β signaling is maintained during EndoMT. The expression of the mesenchymal markers αSMA and SM22α was upregulated during the progression of EndoMT (Figure 4F, G). Consistent with the results of the mesenchymal markers, the expression of the endothelial markers, VEGFR2, Tie2, and VE-cadherin, gradually decreased from ECs to the Full fraction (Figure 4H and Figures 14A, B).

[0136] Further analysis of the bulk RNA-seq data revealed that the gene expression profiles of Tβ-ECs and the Partial fraction were similar (Figures 14C, D). The expression profile of Tβ-ECs was comparable to that of ECs (Figures 14C, D). Gene ontology (GO) enrichment analysis of differentially expressed genes (DEGs) showed that GO terms related to angiogenesis and migration were enriched in Tβ-ECs, the Partial, and the Full fractions compared to ECs (Figures 14E–G). GSEA revealed that EndoMT occurs gradually from ECs to the Full fraction via Tβ-ECs and the Partial fraction (Figures 14H, I). Notably, the expression of characteristic angiogenesis- and endothelial migration-related genes was upregulated in Tβ-ECs and the Partial fraction (Figures 14J–M). These results suggest that both Tβ-ECs and the Partial fraction represent a population of endothelial cells undergoing Partial EndoMT.

[0137] Example 5 Identification of CD40 as a novel marker of Partial EndoMT To clarify the molecular characteristics of Partial EndoMT, we attempted to identify genes upregulated in Partial EndoMT. By RNA-seq analysis, 43 genes that were upregulated in cells representing the Partial EndoMT state were identified (Tβ-EC and Partial-induced genes; Figures 5A and B; Table 3: List of 43 genes upregulated in the Tβ-EC and Partial fractions). GO enrichment analysis of the 43 genes revealed that they were involved in migration and cell motility (Figure 5C).

Table 3

[0138] According to a previous report [Non-Patent Document 10] that inflammation is involved in the induction of EndoMT, GSEA revealed the enrichment of genes related to the inflammatory response in the Tβ-EC and Partial fractions, suggesting that the inflammatory response signaling is activated in cells undergoing Partial EndoMT (Figure 5D). Therefore, we focused on genes related to the inflammatory response. Among the top 5 enriched genes of the GSEA gene set related to the inflammatory response, CD40 was identified as a Partial EndoMT-induced gene that controls various immune and inflammatory responses (Figure 5E).

[0139] Example 6 CD40 expression is abundant in the Partial EndoMT state and regulates the progression of EndoMT Our data suggest that CD40 is a potential novel Partial EndoMT-specific marker. CD40 is a member of the TNF receptor superfamily [Elgueta R, Benson MJ, de Vries VC, Wasiuk A, Guo Y, Noelle RJ. Molecular mechanism and function of CD40 / CD40L engagement in the immune system. Immunol Rev. 2009; 229: 152-172.], and its expression in endothelial cells is upregulated in response to TNF-α and other cytokines [Greene JA, Portillo JA, Lopez Corcino Y, Subauste CS. CD40-TRAF signaling upregulates CX3CL1 and TNF-α in human aortic endothelial cells but not in retinal endothelial cells. PLoS One. 2015; 10: e0144133.]. Furthermore, CD40 also contributes to inflammation-induced angiogenesis [Lee A, Papangeli I, Park Y, et al. A PPARγ-dependent miR-424 / 503-CD40 axis regulates inflammation mediated angiogenesis. Sci Rep. 2017; 7: 2528.]. We observed that CD40 was upregulated in TGF-β-treated EMREC and MS-1 cells (Figure 6A and Figure 15A). Furthermore, the expression levels of CD40 mRNA and protein were upregulated in the Tβ-EC and Partial fractions compared to the EC and Full fractions (Figure 6B-D and Figure 15B-D). Immunocytochemical analysis using anti-CD40 antibody revealed low expression of αSMA-GFP or CD40 in EMREC cultured without TGF-β (Figure 6E). Notably, we identified two subsets of TGF-β-treated EMREC with various expressions of αSMA-GFP and CD40, αSMA-GFP low / CD40 high and αSMA-GFP high / CD40 low were observed (Figure 6E). Since CD40 and αSMA-GFP are considered markers for partial and complete EndoMT, respectively, it was concluded that the first and second subsets of EMREC represent cell populations undergoing partial and complete EndoMT, respectively.

[0140] To investigate the role of CD40 in TGF-β-induced EndoMT, siRNA specific to CD40 was used to suppress the expression of CD40 in EMREC treated with TGF-β. CD40-specific siRNA enhanced TGF-β-induced αSMA and SM22α expression (Figure 7B, C) and effectively inhibited CD40 expression (Figure 7A) compared to TGF-β-treated EC.

[0141] Example 7 Single-cell RNA sequencing of human tumors reveals CD40 expression in endothelial cells undergoing Partial EndoMT Next, scRNA-seq data of human tumors were used to examine the clinical significance of the in vitro findings. The inventors previously performed a pan-cancer analysis on 226 samples across 10 types of solid cancers and examined the TME profile at single-cell resolution. To identify subpopulations of cells undergoing Partial EndoMT in the TME, the same scRNA-seq data were used, cells were clustered by principal component analysis, and subpopulations of cells with gene expression profiles of endothelial cells (clusters 1-3) and CAFs (clusters 3-6) were characterized (Figures 8A and 16A-D). Violin plot analysis showed that the expression of endothelial cell markers PECAM-1 and von Willebrand factor (vWF) was associated with the EC cluster, while the expression of mesenchymal cell markers Collagen1A1 and αSMA was associated with the CAF cluster (Figures 8B-E and 16A-D). It was also found that both endothelial cell markers and mesenchymal cell markers were expressed in cluster 3, suggesting that cluster 3 represents cells undergoing Partial EndoMT (Figures 8A-E and 16A-D). Notably, CD40 was expressed in cluster 3 (Figures 8F and 16E).

[0142] Subsequently, to characterize the functional heterogeneity of pan-cancer cells, the pseudotime of each cell was defined along the elastic principal tree calculated by the Monocle function. Pseudotime analysis revealed that the trajectory of EndoMT begins in the EC cluster, the cell fraction progresses to the Partial EndoMT cluster, and finally to the CAF cluster (Figures 8G, H). These results indicate that human tumor tissues contain a population of endothelial cells undergoing Partial EndoMT and cells undergoing Partial and complete EndoMT, confirming that CD40 can be considered a Partial EndoMT marker in human tumors.

[0143] Discussion In the present invention, EMREC was established to visualize the continuous changes during EndoMT. Using EMREC, the inventors discovered that TGF-β induces the continuous transition of endothelial cells from the initial stage (Tβ-EC) of Partial EndoMT, which is an intermediate stage of EndoMT, to the late stage (Partial) of Partial EndoMT and then to the full EndoMT stage (Full) (Figures 4 and 9). Since Partial EndoMT is a stage where the expression of endothelial cell markers and mesenchymal cell markers changes dynamically, these findings may be a valuable source of information for elucidating the molecular mechanisms governing the induction of EndoMT.

[0144] The TGF-β signal is involved in EndoMT induction, which has been confirmed in vitro and in vivo (Figure 1). It has also been shown that TGF-β-induced Partial EndoMT activates inflammatory signaling in EMREC (Figure 5). The inventors previously reported that inflammatory signals enhance TGF-β-induced EndoMT [Non-Patent Document 10]. Inflammatory signals are involved in the decrease in endothelial barrier function after EndoMT induction. In the present invention, it was discovered that TGF-β-induced EndoMT increases the permeability of 3D microvessels (Figure 3B). This effect is thought to be due to a decrease in barrier function and a change in microvessel maturity, as revealed by a decrease in the expression of VE-cadherin (Figure 14B). The inventors' data suggested that CD40, a molecule involved in the inflammatory response, could be considered a Partial EndoMT marker (Figures 5 and 6). CD40 is a member of the TNF receptor superfamily and is expressed in antigen-presenting cells such as B cells, dendritic cells, macrophages, and vascular endothelial cells. Inflammatory cytokines such as TNF-α increase CD40 expression and contribute to inflammation-induced angiogenesis [Lee A, Papangeli I, Park Y, et al. A PPARγ-dependent miR-424 / 503-CD40 axis regulates inflammation mediated angiogenesis. Sci Rep. 2017; 7: 2528.]. Activation of CD40 signaling depends on the presentation of CD40 ligand (CD40L) and is involved in various processes such as leukocyte recruitment and extravasation [Henn V, Slupsky JR, Grafe M, et al. CD40 ligand on activated platelets triggers an inflammatory reaction of endothelial cells. Nature. 1998; 391: 591-594.].Furthermore, although CD40-CD40L may be involved in the induction of EndoMT in embryonic endothelial cells during chicken embryo development [Arciniegas E, Becerra A, De Sanctis JB, Graterol A, Ramirez R. CD40 and CD40L expression in the chicken embryo aorta: possible role in the endothelial-mesenchymal transdifferentiation process. Anat Rec A Discov Mol Cell Evol Biol. 2003; 274: 942-951.], no direct association has been mentioned. Although the involvement of CD40 in EndoMT has been suggested, there are no studies showing a direct correlation between CD40 and EndoMT or Partial EndoMT under pathological conditions. In the present invention, for the first time, it has been shown that CD40 can be considered a Partial EndoMT-specific marker (Figure 6). Furthermore, the inventors have shown that TGF-β-induced EndoMT is enhanced by CD40 silencing in EMREC (Figure 7), suggesting that TGF-β-induced CD40 expression suppresses the transition from Partial EndoMT to complete EndoMT. The inventors' data also revealed that CD40 expression is upregulated by TGF-β and is associated with the population of TGF-β-treated endothelial cells and cells undergoing Partial EndoMT, but is downregulated in endothelial cells and cells undergoing complete EndoMT.

[0145] The present inventors previously demonstrated the existence of heterogeneous CAFs, including EndoMT-derived CAFs in human tumors. From the evolutionary trajectory, it was suggested that such EndoMT-derived CAFs represent a transitional state between tumor endothelial cells and myofibroblastic CAFs. In the present invention, using the same human tumor samples, a cluster of cells undergoing Partial EndoMT (Figure 8) was identified. This likely represents EndoMT-derived CAFs. However, further analysis is required to clarify the correlation between the previously identified EndoMT-derived CAFs and the Partial EndoMT cluster. Furthermore, scRNA-seq analysis revealed that CD40 expression in human tumor tissues is enriched in cell populations undergoing Partial EndoMT (Figure 8). EndoMT is closely associated with tumor progression via angiogenesis and CAF formation. Furthermore, although it is possible to reverse Partial EndoMT to form endothelial cells, multiple lines of evidence suggest that the complete EndoMT process is irreversible, and thus cells that have undergone complete EndMT cannot return to endothelial cells [Non-Patent Document 9]. Therefore, in order to prevent the formation of CAFs from cells that have undergone complete EndoMT, it is important to understand the mechanism that controls the transition from Partial EndoMT to complete EndoMT. Since CD40 was shown to be expressed in cells in the Partial EndoMT state, together with data suggesting that CD40 may suppress the transition of cells to the complete EndoMT state, CD40 is considered a useful Partial EndoMT marker that can be used for the diagnosis and treatment of cancer progression and metastasis (Figure 9).

[0146] Multiple pieces of evidence suggest that EMT transcription factors (EMT-TFs), such as Snail and ZEB1, play important roles not only in EMT but also in the induction of EndoMT. The exact role of EMT-TFs in the series of steps of EndoMT needs to be elucidated in the future. EndoMT is involved not only in tumor progression but also in various diseases such as atherosclerosis, pulmonary arterial hypertension, cardiac fibrosis, and organ fibrosis [Non-Patent Documents 8, 9]. The EMREC (Figure 2) and 3D microvascular model (Figure 3) established in the present invention are used to evaluate the dynamics and distribution of EndoMT in pathological conditions and the efficacy of therapeutic agents by live imaging in a system mimicking the in vivo environment. Furthermore, the imaging technology using EMREC may be used for screening novel therapeutic agents targeting each stage of EndoMT.

Claims

1. Use of the expression product of a gene as a Partial EndoMT marker, which is an intermediate stage of EndoMT, wherein the gene is selected from the group consisting of the Rgs2 gene, Sost gene, Sp5 gene, Asap3 gene, Cd40 gene, Nckap5l gene, Hpn gene, Myzap gene, Pdgfb gene, Bambi gene, Mindy4 gene, Dapk2 gene, Limch1 gene, Dock8 gene, Vwf gene, Abhd17a gene, Adora2a gene, Zfp69 gene, Ctnnal1 gene, Mvb12b gene, A530016L24Rik gene, Mmp28 gene, Rnf144a gene, Scn1b gene, Ypel2 gene, Lhfpl2 gene, Tmem158 gene, Plvap gene, Ctsh gene, Sema6c gene, Arl15 gene, Mafb gene, Eln gene, Il4ra gene, Rgcc gene, Fam181b gene, Septin4 gene, Serinc5 gene, Robo3 gene, Ace gene, Kif5c gene, Adamts17 gene, and Trp53i11 gene.

2. The use according to claim 1, wherein the expression product of the gene is RNA or protein.

3. A method for determining whether a cell has undergone Partial EndoMT, wherein the expression product of a gene in the cell is used as an indicator for the determination, and the gene is selected from the group consisting of the Rgs2 gene, Sost gene, Sp5 gene, Asap3 gene, Cd40 gene, Nckap5l gene, Hpn gene, Myzap gene, Pdgfb gene, Bambi gene, Mindy4 gene, Dapk2 gene, Limch1 gene, Dock8 gene, Vwf gene, Abhd17a gene, Adora2a gene, Zfp69 gene, Ctnnal1 gene, Mvb12b gene, A530016L24Rik gene, Mmp28 gene, Rnf144a gene, Scn1b gene, Ypel2 gene, Lhfpl2 gene, Tmem158 gene, Plvap gene, Ctsh gene, Sema6c gene, Arl15 gene, Mafb gene, Eln gene, Il4ra gene, Rgcc gene, Fam181b gene, Septin4 gene, Serinc5 gene, Robo3 gene, Ace gene, Kif5c gene, Adamts17 gene, and Trp53i11 gene.

4. The method according to claim 3, wherein using the expression product of the gene in the cell as an indicator for the determination is to use the amount of the expression product of the gene in the cell as an indicator for the determination.

5. The method according to claim 4, wherein when using the amount of the expression product of the gene in the cell as an indicator for the determination, the amount of the expression product of the gene in the cell is compared with a predetermined amount.

6. The method according to claim 5, wherein when the amount of the expression product of the gene in the cell is greater than a predetermined amount, it is used as an indicator to determine that the cell has undergone Partial EndoMT.

7. The method according to claim 5, wherein when the amount of the expression product of the gene in the cell is less than a predetermined amount, it is used as an indicator to determine that the cell has not undergone Partial EndoMT.

8. The method according to claim 3, wherein the gene expression product is RNA or protein.

9. The method according to claim 3, wherein the cell is a tumor vascular endothelial cell.

10. A method for determining whether a cell has undergone Partial EndoMT, comprising the step of measuring the gene expression product in the cell, wherein the gene is selected from the group consisting of the Rgs2 gene, Sost gene, Sp5 gene, Asap3 gene, Cd40 gene, Nckap5l gene, Hpn gene, Myzap gene, Pdgfb gene, Bambi gene, Mindy4 gene, Dapk2 gene, Limch1 gene, Dock8 gene, Vwf gene, Abhd17a gene, Adora2a gene, Zfp69 gene, Ctnnal1 gene, Mvb12b gene, A530016L24Rik gene, Mmp28 gene, Rnf144a gene, Scn1b gene, Ypel2 gene, Lhfpl2 gene, Tmem158 gene, Plvap gene, Ctsh gene, Sema6c gene, Arl15 gene, Mafb gene, Eln gene, Il4ra gene, Rgcc gene, Fam181b gene, Septin4 gene, Serinc5 gene, Robo3 gene, Ace gene, Kif5c gene, Adamts17 gene, and Trp53i11 gene.

11. The method according to claim 10, wherein the step of measuring the gene expression product in the cell is a step of measuring the amount of the gene expression product in the cell.

12. The method according to claim 11, comprising the step of comparing the measured amount of the gene expression product in the cell with a predetermined amount.

13. The method according to claim 12, wherein the measured amount of the gene expression product in the cell is used as an indicator that the amount is greater than the predetermined amount to determine that the cell has undergone Partial EndoMT.

14. The method according to claim 12, which is a method for determining that a cell has not undergone Partial EndoMT, using as an indicator that the amount of the gene expression product in the measured cell is less than a predetermined amount.

15. The method according to claim 10, wherein the gene expression product is RNA or protein.

16. The method according to claim 10, wherein the cell is a tumor vascular endothelial cell.

17. A method for sorting cells that have undergone Partial EndoMT or cells that have not undergone Partial EndoMT, comprising a step of sorting cells in which the amount of the gene expression product is more than a predetermined amount or cells in which the amount of the gene expression product is less than a predetermined amount, wherein the gene is selected from the group consisting of the Rgs2 gene, Sost gene, Sp5 gene, Asap3 gene, Cd40 gene, Nckap5l gene, Hpn gene, Myzap gene, Pdgfb gene, Bambi gene, Mindy4 gene, Dapk2 gene, Limch1 gene, Dock8 gene, Vwf gene, Abhd17a gene, Adora2a gene, Zfp69 gene, Ctnnal1 gene, Mvb12b gene, A530016L24Rik gene, Mmp28 gene, Rnf144a gene, Scn1b gene, Ypel2 gene, Lhfpl2 gene, Tmem158 gene, Plvap gene, Ctsh gene, Sema6c gene, Arl15 gene, Mafb gene, Eln gene, Il4ra gene, Rgcc gene, Fam181b gene, Septin4 gene, Serinc5 gene, Robo3 gene, Ace gene, Kif5c gene, Adamts17 gene, and Trp53i11 gene.

18. The method according to claim 17, wherein the gene expression product is RNA or protein.

19. The method according to claim 17, wherein the cell is a tumor vascular endothelial cell.

20. A method for selecting cells that have undergone partial EndoMT or cells that have not undergone partial EndoMT, the method including a step of measuring the amount of the expression product of a gene in the cells, wherein the gene is selected from the group consisting of the Rgs2 gene, the Sost gene, the Sp5 gene, the Asap3 gene, the Cd40 gene, the Nckap5l gene, the Hpn gene, the Myzap gene, the Pdgfb gene, the Bambi gene, the Mindy4 gene, the Dapk2 gene, the Limch1 gene, the Dock8 gene, the Vwf gene, the Abhd17a gene, the Adora2a gene, the Zfp69 gene, the Ctnnal1 gene, the Mvb12b gene, the A530016L24Rik gene, the Mmp28 gene, the Rnf144a gene, the Scn1b gene, the Ypel2 gene, the Lhfpl2 gene, the Tmem158 gene, the Plvap gene, the Ctsh gene, the Sema6c gene, the Arl15 gene, the Mafb gene, the Eln gene, the Il4ra gene, the Rgcc gene, the Fam181b gene, the Septin4 gene, the Serinc5 gene, the Robo3 gene, the Ace gene, the Kif5c gene, the Adamts17 gene, and the Trp53i11 gene.

21. The method according to claim 20, wherein the expression product of the gene is RNA or protein.

22. The method according to claim 20, wherein the cells are tumor vascular endothelial cells.

23. A program for causing an information processing apparatus to execute the method according to any one of claims 3 to 22.

24. An information processing apparatus storing the program according to claim 23.

25. An inhibitor for inducing or enhancing EndoMT of cells, which comprises as an active ingredient a substance that acts on the expression products of genes selected from the group consisting of the Rgs2 gene, Sost gene, Sp5 gene, Asap3 gene, Cd40 gene, Nckap5l gene, Hpn gene, Myzap gene, Pdgfb gene, Bambi gene, Mindy4 gene, Dapk2 gene, Limch1 gene, Dock8 gene, Vwf gene, Abhd17a gene, Adora2a gene, Zfp69 gene, Ctnnal1 gene, Mvb12b gene, A530016L24Rik gene, Mmp28 gene, Rnf144a gene, Scn1b gene, Ypel2 gene, Lhfpl2 gene, Tmem158 gene, Plvap gene, Ctsh gene, Sema6c gene, Arl15 gene, Mafb gene, Eln gene, Il4ra gene, Rgcc gene, Fam181b gene, Septin4 gene, Serinc5 gene, Robo3 gene, Ace gene, Kif5c gene, Adamts17 gene, and Trp53i11 gene. Claim 26 The inhibitor according to claim 25, wherein the substance acting on the expression product of the gene is an agonist of the expression product of the gene. Claim 27 A therapeutic agent for treating cancer, fibrosis, pulmonary arterial hypertension, or atherosclerosis, which comprises, as an active ingredient, a substance that acts on the expression products of genes selected from the group consisting of the Rgs2 gene, Sost gene, Sp5 gene, Asap3 gene, Cd40 gene, Nckap5l gene, Hpn gene, Myzap gene, Pdgfb gene, Bambi gene, Mindy4 gene, Dapk2 gene, Limch1 gene, Dock8 gene, Vwf gene, Abhd17a gene, Adora2a gene, Zfp69 gene, Ctnnal1 gene, Mvb12b gene, A530016L24Rik gene, Mmp28 gene, Rnf144a gene, Scn1b gene, Ypel2 gene, Lhfpl2 gene, Tmem158 gene, Plvap gene, Ctsh gene, Sema6c gene, Arl15 gene, Mafb gene, Eln gene, Il4ra gene, Rgcc gene, Fam181b gene, Septin4 gene, Serinc5 gene, Robo3 gene, Ace gene, Kif5c gene, Adamts17 gene, and Trp53i11 gene.

28. The therapeutic agent according to claim 27, wherein the substance that acts on the expression product of the gene is an agonist of the expression product of the gene.

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