Test method for determining possibility of lymphatic invasion or lymph node metastasis

Abstract

[Problem] To develop a novel method for determining a positive case of lymphatic invasion and / or a case of lymph node metastasis in a breast cancer patient. [Solution] Provided is a method comprising analyzing gene expression of a lymphatic endothelial cell extracted from breast adipose tissue of a breast cancer patient.

Description

Testing methods for determining the possibility of lymphatic invasion or lymph node metastasis

[0001] The present invention relates to a testing method and a testing program for determining the possibility of lymphatic vessel invasion or lymph node metastasis of cancer cells in breast cancer patients.

[0002] Lymphatic invasion in breast cancer is a poor prognostic factor (Non-Patent Document 1). After neoadjuvant chemotherapy for breast cancer, cases are occasionally observed in which the main lesion has disappeared but severe lymphatic invasion remains. Progression of lymphatic invasion leads to lymph node metastasis, further worsening the prognosis. Early and accurate identification of cases with positive lymphatic invasion and treatment could potentially contribute to improving the prognosis.

[0003] Lymphatic invasion and lymph node metastasis in breast cancer cases are usually evaluated by histopathological examination. Because samples are cut and prepared at approximately 5 mm intervals, there is a risk that lymphatic invasion within a 5 mm interval may be overlooked. Lymph node samples are also cut and prepared at approximately 2 mm intervals, making it difficult to grasp the overall picture of metastasis.

[0004] Regarding breast cancer, multigene assays (e.g., Oncotype DX (trademark), MammaPrint (trademark), PAM50 (trademark), Curebest 95GC Breast (trademark), etc.) are known to predict the additional effect of chemotherapy and to make post-operative drug therapy more effective, but all of these are tests on the breast cancer tissue itself and are not assays to evaluate or predict lymphatic metastasis.

[0005] Abramowitz MC et al. Am J Clin Oncol. 2009;32(1):30-3.

[0006] An objective of the present invention is to develop a novel method for distinguishing between breast cancer patients with positive lymphatic invasion and / or lymph node metastasis.

[0007] The present inventors discovered that the possibility of lymphatic invasion or lymph node metastasis can be determined by analyzing the gene expression of lymphatic endothelial cells (LECs) extracted from the breast adipose tissue of breast cancer patients, and thus completed the present invention.

[0008] That is, in one embodiment, the present invention relates to a testing method for determining the possibility of lymphatic invasion or lymph node metastasis of cancer cells in a breast cancer patient, the method comprising: (A): a step of analyzing the expression level of at least one gene in lymphatic endothelial cells extracted from the breast adipose tissue of the breast cancer patient; and (B): a step of comparing the expression level of the gene analyzed in step (A) with a predetermined reference value.

[0009] In one embodiment of the present invention, the reference value in step (B) is a reference value set based on the expression level of the gene in lymphatic endothelial cells extracted from the breast adipose tissue of a breast cancer patient (control breast cancer patient) who has neither lymph node metastasis nor lymphatic invasion, and / or a reference value set based on the expression level of the gene in lymphatic endothelial cells extracted from the breast adipose tissue of a human who does not have breast cancer.

[0010] In one embodiment of the present invention, the at least one gene is selected from the group consisting of MMRN1, INS-IGF2, RAMP3, NDRG4, CDKN1C, CARD16, IGF2, FLJ41200, C1orf115, LOC643733, ZDHHC14, DTX4, LRRC70, HES1, LDB2, ARL4A, GIMAP8, BMX, LIMD2, PDK4, ID1, RHOJ, FAM174B, EPHB4, SHC2, P and / or selected from gene group A-1 consisting of DCL, GMFG, FAM213A, TMEM200B, LYVE1, GNG11, CGNL1, CXCL2, GABARAPL1, PCAT19, MYZAP, GRAP, TBX1, SLC40A1, KCTD12, RN7SK, IPO11-LRRC70, PPFIBP1, PIM3, NR2F1, NR2F2, NXN, ATP5E, CDKN1B, and RBM6; The at least one gene is selected from the group consisting of TNFRSF10D, SERPINE1, ANGPTL4, PLOD1, ASPH, STC2, EFEMP2, AXL, FAT1, LDHA, PLXNA1, FLJ23867, GPX8, TRAM2, PLOD2, FBN2, SLC1A5, QS0X1, HDDC2, KRT19, IKBIP, LOXL2, PAPPA, TPM2, OSMR, DCBLD2, FN1, ITGBL1, MT1E, PLAT, STEAP3, VCAN, SLC2A1, FAM129A, PSD3, EGFR, MLPH, M RC2, ADAMTSL1, NRP1, CCBE1, ADAM12, SMOC1, LOX, COL1A2, CCDC80, IGFBP3, TPBG, NPR3, SEMA3C, AOX1, TGFBI, CXCL6, CREB3L1, AIM1, DKK1, GREM1, LOXL4, SLC38A5, CSF3, TMEM158, S1PR3, EMP3, SERPINB2, ASIC1, GFPT2, MYO1F, MYBL2, CCNB2, UHRF1, and LINC01605.

[0011] In one embodiment of the present invention, the step (A) is characterized in that the expression levels of a plurality of genes contained in the gene groups A-1 and A-2 are analyzed.

[0012] In one embodiment of the present invention, if the expression level of one or more genes selected from gene group A-1 in lymphatic endothelial cells extracted from the breast adipose tissue of the breast cancer patient is significantly higher than the reference value, it is determined that there is a high possibility of lymphatic invasion or lymph node metastasis, and / or if the expression level of one or more genes selected from gene group A-2 in lymphatic endothelial cells extracted from the breast adipose tissue of the breast cancer patient is significantly lower than the reference value, it is determined that there is a high possibility of lymphatic invasion or lymph node metastasis.

[0013] In one embodiment of the present invention, the at least one gene is selected from gene group B-1 consisting of MMRN1, INS-IGF2, RAMP3, NDRG4, CDKN1C, CARD16, IGF2, LOC643733, LDB2, ARL4A, GIMAP8, PDK4, FAM174B, SHC2, GMFG, GNG11, KCTD12, RN7SK, and NXN, and / or The at least one gene is selected from gene group B-2 consisting of STC2, AXL, LDHA, PLOD2, HDDC2, LOXL2, TPM2, FN1, MT1E, PLAT, VCAN, EGFR, ADAMTSL1, ADAM12, COL1A2, IGFBP3, SEMA3C, AOX1, TGFBI, CXCL6, CREB3L1, AIM1, DKK1, GREM1, LOXL4, S1PR3, EMP3, SERPINB2, ASIC1, GFPT2, MYBL2, and UHRF1.

[0014] In one embodiment of the present invention, the step (A) is characterized in that the expression levels of a plurality of genes contained in the gene groups B-1 and B-2 are analyzed.

[0015] In one embodiment of the present invention, if the expression level of one or more genes selected from the gene group B-1 in lymphatic endothelial cells extracted from the breast adipose tissue of the breast cancer patient is significantly higher than the reference value, it is determined that there is a high possibility of lymphatic invasion or lymph node metastasis, and / or if the expression level of one or more genes selected from the gene group B-2 in lymphatic endothelial cells extracted from the breast adipose tissue of the breast cancer patient is significantly lower than the reference value, it is determined that there is a high possibility of lymphatic invasion or lymph node metastasis.

[0016] In one embodiment of the present invention, the breast adipose tissue is adipose tissue surrounding lymph nodes draining the mammary gland.

[0017] In one embodiment of the present invention, the mammary gland-draining lymph nodes are axillary lymph nodes, supraclavicular lymph nodes, parasternal lymph nodes, or interpectoral (Rotter) lymph nodes.

[0018] In one embodiment of the present invention, the axillary lymph node is axillary lymph node level I, axillary lymph node level II, or axillary lymph node level III.

[0019] In one embodiment of the present invention, the breast adipose tissue is adipose tissue surrounding a sentinel lymph node.

[0020] Another embodiment of the present invention relates to a program for causing a computer to execute each step included in any of the above methods.

[0021] Another embodiment of the present invention relates to a computer-readable storage medium storing the above program.

[0022] Another embodiment of the present invention relates to a method for obtaining lymphatic endothelial cells (population), comprising the steps of: (i) dissociating cells contained in the breast adipose tissue of a breast cancer patient into single cells to obtain a single-cell suspension; (ii) removing CD45-positive cells from the single-cell suspension; and (iii) selecting only CD31-positive and podoplanin-positive cells from the single-cell suspension from which the CD45-positive cells have been removed.

[0023] Another embodiment of the present invention relates to an isolated lymphatic endothelial cell (population) obtained by the above method.

[0024] It should be noted that inventions that combine one or more of the features of the present invention described above are also included within the scope of the present invention.

[0025] Lymphatic invasion and lymph node metastasis in breast cancer cases are typically evaluated by histopathological examination. In the case of lymphatic invasion, samples are cut and prepared at approximately 5 mm intervals, which carries the risk of overlooking lymphatic invasion within a 5 mm interval. Lymph node samples are also cut and prepared at approximately 2 mm intervals, making it difficult to grasp the overall picture of metastasis. The present invention overcomes these limitations of pathological diagnosis and enables more precise diagnosis. Because the test of the present invention can predict the progression of lymphatic metastasis, the test results can also be used to predict the prognosis of breast cancer. Furthermore, because lymphatic invasion and lymph node metastasis are related to the malignancy of breast cancer, the test results of the present invention can also be used to determine the need for postoperative treatment and to consider the content of treatment. As a result, the present invention can increase the survival chances of breast cancer patients.

[0026] Figure 1 shows an outline of the method for extracting lymphatic endothelial cells from breast adipose tissue. Figure 2 shows that extracted lymphatic endothelial cells form tubular structures on BME-coated dishes. Figure 3 shows that adding VEGF-C to extracted lymphatic endothelial cells improves their proliferation ability. Figure 4 shows that extracted lymphatic endothelial cells express LEC markers. Figure 5 shows that there is a clear difference in gene expression profile between a case without lymph node metastasis or lymphatic invasion (321F-LEC) and a case with at least one of lymph node metastasis and lymphatic invasion (325F-LEC, 328F-LEC). Figure 6 shows that there is a clear difference in gene expression profiles between cases without lymph node metastasis or lymphatic vessel invasion (343F-LEC, 347F-LEC, 349F-LEC) and cases with at least one of lymph node metastasis and lymphatic vessel invasion (351F-LEC, 353F-LEC).

[0027] The present disclosure relates to a testing method for determining the possibility of lymphatic invasion or lymph node metastasis of cancer cells in a breast cancer patient, the method comprising a step of analyzing the expression level of at least one gene in lymphatic endothelial cells extracted from breast adipose tissue of the breast cancer patient.

[0028] The breast is primarily composed of glandular tissue, adipose tissue (mammary adipose tissue) that fills the gaps between the glandular tissue, and the connective tissue that supports them. The glandular tissue is divided into 15 to 20 "lobules," which branch into numerous "lobules," and milk is produced in the lobules. Thin "milk ducts" emerge from the lobules to carry milk to the nipple, and these gradually merge to form a single milk duct that drains to the nipple. In the present disclosure, breast cancer may be cancer that has developed in the glandular tissue, and may be cancer that has developed in the milk ducts or lobules, for example.

[0029] Multiple lymph nodes are distributed in the breast, and these lymph nodes are called mammary-regional lymph nodes. When breast cancer occurs, the site of metastasis is most likely to occur in the mammary-regional lymph nodes. In the present disclosure, the mammary-regional lymph nodes may be axillary lymph nodes, supraclavicular lymph nodes, parasternal lymph nodes, or interpectoral (Rotter) lymph nodes. Anatomically, axillary lymph nodes are classified into axillary lymph node levels I to III. Axillary lymph node level I may refer to lymph nodes distributed in the area outside the outer edge of the pectoralis minor muscle, axillary lymph node level II may refer to lymph nodes distributed dorsal to the pectoralis minor muscle and between the pectoral muscles, and axillary lymph node level III may refer to lymph nodes distributed in the area inside the inner edge of the pectoralis minor muscle under the clavicle.

[0030] Breast cancer cells first invade lymphatic vessels surrounding cancer tissue (lymphatic invasion) and then metastasize to lymph nodes via these lymphatic vessels (lymph node metastasis). Most typically, lymph node metastasis of breast cancer progresses from axillary lymph node level I to level II and then to level III. From the viewpoint of early detection of lymphatic invasion or lymph node metastasis, the adipose tissue used in the examination method of the present disclosure is preferably adipose tissue surrounding axillary lymph node levels I to III. However, depending on the site of breast cancer, the adipose tissue used in the examination method of the present disclosure may also be adipose tissue surrounding the supraclavicular lymph nodes, parasternal lymph nodes, or interpectoral (Rotter) lymph nodes.

[0031] A "sentinel lymph node" is defined as the lymph node (or nodes) to which breast cancer cells first arrive from within the breast. A series of tests to discover and remove this sentinel lymph node and then pathologically examine whether cancer cells are present (presence or absence of metastasis) is generally called a "sentinel lymph node biopsy." The breast fat cells used in the method of the present disclosure may be adipose tissue collected together with the sentinel lymph node in a sentinel lymph node biopsy (adipose tissue surrounding the sentinel lymph node). In other words, among the tissues collected in a sentinel lymph node biopsy, the lymph node is subjected to pathological examination, and the remaining adipose tissue after removing the lymph node is subjected to the method of the present disclosure, thereby improving the accuracy of tests for lymphatic invasion / lymph node metastasis.

[0032] In the present disclosure, the method for identifying the sentinel lymph node is not limited, and any method commonly used by those skilled in the art may be used. For example, the sentinel lymph node may be identified by applying a dye (e.g., isosulfan blue, patent blue, indocyanine green, indigo carmine) or a radioactive drug (e.g., tin colloid, albumin colloid, phytic acid) to the breast cancer lesion or its surrounding tissue to visualize the lymphatic vessels and lymph nodes.

[0033] In the present disclosure, the method for extracting lymphatic endothelial cells (population) from breast adipose tissue is not limited, and lymphatic endothelial cells may be extracted by a combination of techniques known in the art. As a non-limiting example, lymphatic endothelial cells may be extracted by a method including the steps of: (i) dissociating cells contained in the breast adipose tissue of a breast cancer patient into single cells to obtain a single-cell suspension; (ii) removing CD45-positive cells from the single-cell suspension; and (iii) selecting only CD31-positive and podoplanin-positive cells from the single-cell suspension from which the CD45-positive cells have been removed.

[0034] The method for obtaining lymphatic endothelial cells may further include, after step (i), a step of lysing red blood cells contained in the single-cell suspension. The step of lysing red blood cells contained in the single-cell suspension may be carried out, for example, by adding a commercially available red blood cell lysis buffer to the single-cell suspension.

[0035] The removal of CD45-positive cells in step (ii) of the above-described method for obtaining lymphatic endothelial cells may be carried out, for example, using a commercially available kit designed to remove CD45-positive cells from a cell population. Specifically, this may be carried out, for example, by adding a labeled molecule containing an anti-CD45 antibody (or an antigen-binding fragment thereof) to the single-cell suspension and removing only cells to which the labeled molecule is bound. The labeled molecule may be, for example, a magnetically labeled molecule, and the removal of cells to which the magnetically labeled molecule is bound may be carried out, for example, by magnetic cell sorting (MACS).

[0036] In step (iii) of the above-described method for obtaining lymphatic endothelial cells, the selection of only CD31-positive and podoplanin-positive cells may be performed, for example, by sorting only cells that bind to both an anti-CD31 antibody and an anti-podoplanin antibody from a cell population. Specifically, for example, the selection may be performed by adding an anti-CD31 antibody, an anti-podoplanin antibody, and fluorescently labeled secondary antibodies against each antibody to a single-cell suspension, and then sorting only cells stained with both the anti-CD31 antibody and the anti-podoplanin antibody. The selection of the stained cells may be performed, for example, by fluorescence-activated cell sorting (FACS).

[0037] That is, the lymphatic endothelial cells (or cell population) that can be used in the testing method of the present disclosure may be isolated lymphatic endothelial cells (or cell population) obtained by a method including the steps of: (i) breaking down cells contained in the breast adipose tissue of a breast cancer patient into single cells to obtain a single-cell suspension; (ii) removing CD45-positive cells from the single-cell suspension; and (iii) selecting only CD31-positive and podoplanin-positive cells from the single-cell suspension from which the CD45-positive cells have been removed.

[0038] The method for confirming that the obtained cells are lymphatic endothelial cells is not particularly limited, and may be performed using methods known in the art, such as the formation of tubular structures on a cell culture dish, improved cell proliferation upon addition of VEGF-C, or expression of known LEC markers (e.g., LYVE1, FLT4, KDR, FLT1, PECAM1, and PDPN).

[0039] Although there have been reports of collecting LECs from skin, which is rich in LECs, and from LEC-derived tumors, there have been no reports of isolating LECs derived from tissues that can directly interact with breast cancer. Skin-derived LEC cell lines (e.g., commercially available human dermal LECs (HDLECs)) and LECs collected from LEC-derived tumors are not LECs derived from tissues that can directly interact with breast cancer, and therefore cannot be used in the tests disclosed herein for predicting lymph node metastasis and lymphatic invasion.

[0040] As demonstrated in the Examples of the present application described later, the isolated lymphatic endothelial cells (or cell populations) obtained by the above method have a gene expression profile that is clearly different from that of lymphatic endothelial cells (or cell populations) extracted from breast adipose tissue of breast cancer patients (control breast cancer patients) who have neither lymph node metastasis nor lymphatic invasion. Therefore, the isolated lymphatic endothelial cells (or cell populations) obtained by the above method can be used in various experiments and studies related to breast cancer (e.g., in vitro and in vivo experiments, transcriptome, epigenomic, and metabolomic profiling, etc.).

[0041] In the method of the present disclosure, the method for analyzing gene expression levels in lymphatic endothelial cells is not limited, and various methods capable of quantitatively analyzing gene expression levels can be used. For example, bulk RNA-seq analysis may be used to obtain an average gene expression profile in a cell population. Furthermore, quantitative PCR (e.g., real-time PCR) analysis may be used to analyze the expression levels of one or more specific genes.

[0042] In the method disclosed herein, the expression levels of one or more genes in lymphatic endothelial cells extracted from the breast adipose tissue of a breast cancer patient are compared with a predetermined reference value. The reference value may be a reference value set based on the expression levels of the genes in lymphatic endothelial cells extracted from the breast adipose tissue of a breast cancer patient (control breast cancer patient) who has neither lymph node metastasis nor lymphatic invasion, or may be a reference value set based on the expression levels of the genes in lymphatic endothelial cells extracted from the breast adipose tissue of a human who does not have breast cancer. Which reference value to use can be determined by a person skilled in the art from the perspective of technical effectiveness and medical ethics, and multiple reference values ​​may be set.

[0043] In the method of the present disclosure, the expression levels of genes contained in the gene sets A-1, A-2, B-1, and / or B-2 are analyzed. Gene sets A-1 and B-1 are groups of genes whose expression is likely to be increased in lymphatic endothelial cells interacting with breast cancer (i.e., lymphatic endothelial cells at sites where lymph node metastasis or lymphatic invasion has occurred). Gene sets A-2 and B-2 are groups of genes whose expression is likely to be decreased in lymphatic endothelial cells interacting with breast cancer (i.e., lymphatic endothelial cells at sites where lymph node metastasis or lymphatic invasion has occurred).

[0044] In lymphatic endothelial cells extracted from the breast adipose tissue of a breast cancer patient, if the expression levels of genes included in gene set A-1 or gene set B-1 are significantly higher than a set reference value, or if the expression levels of genes included in gene set A-2 or gene set B-2 are significantly lower than a set reference value, the cells may be determined to have a "high possibility of lymphatic invasion or lymph node metastasis (high risk)." On the other hand, if the expression levels of all genes are not significantly different from the set reference value, or if the number of genes significantly different from the reference value is below a certain number, the cells may be determined to have a "low possibility of lymphatic invasion or lymph node metastasis (low risk)." Alternatively, the risk of lymphatic invasion or lymph node metastasis may be assessed in stages (low risk to high risk) depending on the number (or percentage) of genes significantly different from the reference value.

[0045] Each step included in the inspection method of the present disclosure may be executed by a computer. A program or computer program according to some embodiments of the present disclosure may cause a computer to execute each step included in the inspection method of the present disclosure, or may be read and executed by a computer. A computer-readable storage medium according to some embodiments of the present disclosure may store a program or computer program for causing a computer to execute each step included in the inspection method of the present disclosure.

[0046] The testing method of the present disclosure may be used in combination with known genetic tests for breast cancer. For example, known multigene assays for predicting the likelihood of recurrence, predicting the additional effect of chemotherapy, determining the need for postoperative drug therapy, etc. include Oncotype DX™ (Genomic Health, USA), MammaPrint™ (Agendia, Netherlands), PAM50™ (NanoString Technologies, USA), and Curebest 95GC Breast™ (Sysmex Corporation, Japan). The testing method of the present disclosure is a test from a different perspective (determining the likelihood of lymphatic invasion or lymph node metastasis) than these tests, and therefore, when used in combination with these tests, it enables multifaceted judgments regarding breast cancer treatment strategies and prognosis.

[0047] It should be noted that the terms used in this specification are used to describe particular embodiments and are not intended to limit the invention.

[0048] Furthermore, the term "comprise" used in this specification intends that the described items (components, steps, elements, numbers, etc.) are present, unless the context clearly dictates otherwise, and does not exclude the presence of other items (components, steps, elements, numbers, etc.).

[0049] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by a person skilled in the art to which this disclosure belongs. Terms used herein should be interpreted as having a meaning consistent with the meaning in the present specification and the related technical field, and should not be interpreted in an idealized or overly formal sense, unless otherwise defined.

[0050] Although terms such as "first" and "second" may be used to describe various elements, it is understood that these elements should not be limited by these terms. These terms are used only to distinguish one element from another, and for example, a first element can be referred to as a second element, and similarly, a second element can be referred to as a first element, without departing from the scope of the present disclosure.

[0051] The present disclosure will be described in more detail below with reference to examples. However, the present disclosure can be embodied in various forms and should not be construed as being limited to the examples set forth herein.

[0052] Example 1: Obtaining lymphatic endothelial cells derived from breast adipose tissue of breast cancer patients Adipose tissue draining into the axilla was collected from the breast excised during breast cancer surgery, and LECs were isolated using MACS (removal of CD45-positive cells) and FACS (CD31-positive, podoplanin-positive) (Figure 1). This adipose tissue was obtained from the adipose tissue surrounding the lymph nodes during sentinel lymph node biopsy in breast cancer surgery, and is usually discarded after lymph node removal.

[0053] More specifically, lymphatic endothelial cells derived from breast adipose tissue of a breast cancer patient were obtained by the following method.

[0054] Sentinel lymph nodes are collected from the fatty tissue draining from the breast of a breast cancer patient into the armpit, then wrapped in saline gauze and transported to the laboratory on ice.

[0055] Adipose tissue cells were dissociated into single cells using a MACS tumor dissociation kit and a MACS dissociator (Miltenyl Biotec). After filtration and centrifugation, the cells were collected and purified using the red blood cell lysis solution lysis buffer (cat#130-094-183, Miltenyl Biotec, Germany).

[0056] The cells were washed twice with PBS (cat#14190-144, Thermo Fisher Scientific, USA) and seeded onto a 6-well microplate coated with collagen type I (cat#4810-010, AGC Techno Glass (Iwaki), Japan) or a 100 mm collagen type I-coated dish (cat#4020-010, AGC Techno Glass (Iwaki), Japan), depending on the number of cells separated. The medium used was LECs after sorting were grown in endothelial cell growth medium MV 2 (Ready-to-use) (cat# C-22022, Promo Cell, Germany) supplemented with penicillin-streptomycin antibiotic solution (cat# 168-23191, Fujifilm, Japan).

[0057] When the cells reached 100% confluence, they were harvested using 0.25 w / v% Trypsin-1 mM EDTA 4Na Solution with Phenol Red (FUJIFILM, Tokyo, Japan).

[0058] EasySep ＴＭCD45-positive cells were removed using human CD45 depletion kit II (cat# ST-17898, Stemcell Technologies, Canada), followed by co-staining with anti-podoplanin antibody (catalog #236529, Abcam, UK) and anti-CD31 antibody (catalog #14-0319-82, Invitrogen, USA) for 30 minutes on ice, followed by washing three times with PBS. Podoplanin was stained with Alexa Fluor 488-conjugated anti-rabbit IgG (H+L) secondary antibody (catalog #4412, Cell signaling technology, USA), and CD31 was stained with Alexa Fluor 405 anti-mouse IgG (H+L) cross-absorbed antibody (catalog #A31553, Invitrogen, USA) for 30 minutes on ice in the dark. The labeled cells are resuspended in FACS buffer (5% FBS in PBS) and sorted using a Cell Sorter MA900 (Sony, Tokyo, Japan).

[0059] The selected cells were plated in a 6-well microplate coated with collagen type I (cat# 4810-010, AGC Techno Glass (Iwaki), Japan) or a 100 mm collagen type I-coated dish in endothelial cell growth medium MV 2 (Ready-to-use) (cat# C-22022, Promo Cell, Germany) supplemented with penicillin-streptomycin antibiotic, depending on the cell number. The solution is used for seeding.

[0060] <Confirmation that the collected cells are LECs> LECs collected by the above method were shown to form tubular structures on BME-coated dishes (Figure 2). Furthermore, it was confirmed that the cell proliferation ability of the collected LECs was improved by adding VEGF-C (Figure 3). Furthermore, expression of known LEC markers was confirmed in the collected LECs (Figure 4). These results confirmed that the collected cells were LECs.

[0061] <Discussion> Although there have been reports of collecting LECs from skin, which is rich in LECs, and from LEC-derived tumors, there have been no reports of isolating LECs derived from tissues that can directly interact with breast cancer. Skin-derived LEC cell lines (e.g., commercially available human dermal LECs: HDLECs) and LECs collected from LEC-derived tumors are not LECs derived from tissues that can directly interact with breast cancer, and therefore cannot be used in the tests for predicting lymph node metastasis and lymphatic invasion of the present invention described below.

[0062] Furthermore, in single-cell RNA-seq analysis of nine cases (all derived from breast cancer tissue, total cell count: 60,158) conducted in our laboratory, LECs were detected in only one case, with only 20 LECs (data not shown). In other words, even if we attempted to isolate LECs from breast cancer tissue itself, it was not possible to secure the required number of LECs for testing.

[0063] The present inventors have found that isolating LECs from breast adipose tissue makes it possible to secure tissue-derived LECs capable of directly interacting with breast cancer in sufficient numbers for testing.

[0064] Example 2: Analysis of gene expression in lymphatic endothelial cells Cells collected by the above method were cultured on a 6-well microplate coated with collagen type I (cat#4810-010, AGC Techno Glass (Iwaki), Japan). When the cells reached 90% confluence, they were collected and analyzed using GenNext. ＴＭ , RamDA-Seq ＴＭcDNA was synthesized using Single Cell Kit (cat# RMQ-101, Toyobo, Japan) and bulk RNAseq was performed.

[0065] When cases without lymph node metastasis or lymphatic vessel invasion (321F-LEC) were compared with cases with at least one of lymph node metastasis and lymphatic vessel invasion (325F-LEC, 328F-LEC) (n = 3 each), differences in gene expression were observed (Figure 5).

[0066] Furthermore, when the number of cases was increased and three cases without lymph node metastasis or lymphatic vessel invasion (343F-LEC, 347F-LEC, 349F-LEC; n = 2 for each) were compared with two cases with at least one of lymph node metastasis and lymphatic vessel invasion (351F-LEC, 353F-LEC; n = 2 for each), differences in gene expression were observed (Figure 6).

[0067] Genes with significant differences in expression between the 321F-LEC group and the 325F-LEC + 328F-LEC group were extracted (log2 FC>1, adjusted p value<0.1). Genes with significant differences in expression between the 343F-LEC + 347F-LEC + 349F-LEC group and the 351F-LEC + 353F-LEC group were also extracted (log2 FC>1, adjusted p value<0.1). 121 genes that fell into both sets of extracted gene groups (gene set A) were extracted.

[0068] Of the gene set A, the following 50 genes showed significantly increased expression in samples from cases with at least one of lymph node metastasis and lymphatic vessel invasion (gene set A-1).

[0069] [Gene set A-1] MMRN1, INS-IGF2, RAMP3, NDRG4, CDKN1C, CARD16, IGF2, FLJ41200, C1orf115, LOC643733, ZDHHC14, DTX4, LRRC70, HES1, LDB2, ARL4A, GIMAP8, BMX, LIMD2, PDK4, ID1, RHOJ, FAM174B, EPHB4, SHC2, PD CL, GMFG, FAM213A, TMEM200B, LYVE1, GNG11, CGNL1, CXCL2, GABARAPL1, PCAT19, MYZAP, GRAP, TBX1 , SLC40A1, KCTD12, RN7SK, IPO11-LRRC70, PPFIBP1, PIM3, NR2F1, NR2F2, NXN, ATP5E, CDKN1B, RBM6

[0070] Of the gene set A, the following 71 genes showed significantly decreased expression in samples from cases with at least one of lymph node metastasis and lymphatic vessel invasion (gene set A-2).

[0071] [Gene set A-2] TNFRSF10D, SERPINE1, ANGPTL4, PLOD1, ASPH, STC2, EFEMP2, AXL, FAT1, LDHA, PLXNA1, FLJ23867, GPX8, TRAM2, PLOD2, FBN2, SLC1A5, QSOX1, HDDC2, KRT19, IKBIP, LOXL2, PAPPA, TPM2, OSMR, DCBLD2, FN1, ITGBL1, MT1E, PLAT, STEAP3, VCAN, SLC2A1, FAM129A, PSD3, EGFR , MLPH, MRC2, ADAMTSL1, NRP1, CCBE1, ADAM12, SMOC1, LOX, COL1A2, CCDC80, IGFBP3, TPBG, NPR3, SEMA3C, AOX1, TGFBI, CXCL6, CREB3L 1, AIM1, DKK1, GREM1, LOXL4, SLC38A5, CSF3, TMEM158, S1PR3, EMP3, SERPINB2, ASIC1, GFPT2, MYO1F, MYBL2, CCNB2, UHRF1, LINC01605

[0072] Using a similar method, the significance level was raised and further strict narrowing was performed. That is, genes with significant differences in expression between the 321F-LEC group and the 325F-LEC + 328F-LEC group were extracted (log2 FC>2, adjusted p value<0.05). Furthermore, genes with significant differences in expression between the 343F-LEC + 347F-LEC + 349F-LEC group and the 351F-LEC + 353F-LEC group were extracted (log2 FC>2, adjusted p value<0.05). 51 genes that corresponded to both of the extracted gene sets (gene set B) were extracted.

[0073] Of the gene set B, the following 19 genes showed significantly increased expression in samples from cases with at least one of lymph node metastasis and lymphatic vessel invasion (gene set B-1).

[0074] [Gene set B-1] MMRN1, INS-IGF2, RAMP3, NDRG4, CDKN1C, CARD16, IGF2, LOC643733, LDB2, ARL4A, GIMAP8, PDK4, FAM174B, SHC2, GMFG, GNG11, KCTD12, RN7SK, NXN

[0075] Of the gene set B, the following 32 genes showed significantly decreased expression in samples from cases with at least one of lymph node metastasis and lymphatic vessel invasion (gene set B-2).

[0076] [Gene set B-2] STC2, AXL, LDHA, PLOD2, HDDC2, LOXL2, TPM2, FN1, MT1E, PLAT, VCAN, EGFR, ADAMTSL1, ADAM12, COL1A2, IGFBP3, SEMA3C, AOX1, TGFBI, CXCL6, CREB3L1, AIM1, DKK1, GREM1, LOXL4, S1PR3, EMP3, SERPINB2, ASIC1, GFPT2, MYBL2, UHRF1

[0077] <Discussion> From the above results, if the gene expression of gene set A-1 is significantly high or the gene expression of gene set A-2 is significantly low in lymphatic endothelial cells extracted from the breast adipose tissue of a breast cancer patient, it can be predicted that there is a high possibility of lymph node metastasis or lymphatic invasion. Similarly, if the gene expression of gene set B-1 is significantly high or the gene expression of gene set B-2 is significantly low in lymphatic endothelial cells extracted from the breast adipose tissue of a breast cancer patient, it can be predicted that there is a high possibility of lymph node metastasis or lymphatic invasion.

Claims

1. A testing method for determining the possibility of lymphatic invasion or lymph node metastasis of cancer cells in a breast cancer patient, comprising: (A): a step of analyzing the expression level of at least one gene in lymphatic endothelial cells extracted from the breast adipose tissue of the breast cancer patient; and (B): a step of comparing the expression level of the gene analyzed in step (A) with a predetermined reference value.

2. The method according to claim 1, wherein the reference value in step (B) is a reference value set based on the expression level of the gene in lymphatic endothelial cells extracted from the breast adipose tissue of a breast cancer patient (control breast cancer patient) who has neither lymph node metastasis nor lymphatic invasion, and / or a reference value set based on the expression level of the gene in lymphatic endothelial cells extracted from the breast adipose tissue of a human who does not have breast cancer.

3. The method of claim 1, wherein the at least one gene is selected from the group consisting of MMRN1, INS-IGF2, RAMP3, NDRG4, CDKN1C, CARD16, IGF2, FLJ41200, C1orf115, LOC643733, ZDHHC14, DTX4, LRRC70, HES1, LDB2, ARL4A, GIMAP8, BMX, LIMD2, PDK4, ID1, RHOJ, FAM174B, EPHB4, SHC2, PDCL, and GMFG. , FAM213A, TMEM200B, LYVE1, GNG11, CGNL1, CXCL2, GABARAPL1, PCAT19, MYZAP, GRAP, TBX1, SLC40A1, KCTD12, RN7SK, IPO11-LRRC70, PPFIBP1, PIM3, NR2F1, NR2F2, NXN, ATP5E, CDKN1B, and RBM6; and / or The at least one gene is selected from the group consisting of TNFRSF10D, SERPINE1, ANGPTL4, PLOD1, ASPH, STC2, EFEMP2, AXL, FAT1, LDHA, PLXNA1, FLJ23867, GPX8, TRAM2, PLOD2, FBN2, SLC1A5, QS0X1, HDDC2, KRT19, IKBIP, LOXL2, PAPPA, TPM2, OSMR, DCBLD2, FN1, ITGBL1, MT1E, PLAT, STEAP3, VCAN, SLC2A1, FAM129A, PSD3, EGFR, and ML PH, MRC2, ADAMTSL1, NRP1, CCBE1, ADAM12, SMOC1, LOX, COL1A2, CCDC80, IGFBP3, TPBG, NPR3, SEMA3C, AOX1, TGFBI, CXCL6, CREB3L1, AIM1, DKK1, GREM1, LOXL4, SLC38A5, CSF3, TMEM158, S1PR3, EMP3, SERPINB2, ASIC1, GFPT2, MYO1F, MYBL2, CCNB2, UHRF1, and LINC01605.

4. The method according to claim 3, wherein in step (A), the expression levels of a plurality of genes contained in said gene group A-1 and said gene group A-2 are analyzed.

5. The method according to claim 3, wherein, when the expression level of one or more genes selected from gene group A-1 in lymphatic endothelial cells extracted from the breast adipose tissue of the breast cancer patient is significantly higher than the reference value, it is determined that there is a high possibility of lymphatic invasion or lymph node metastasis, and / or, when the expression level of one or more genes selected from gene group A-2 in lymphatic endothelial cells extracted from the breast adipose tissue of the breast cancer patient is significantly lower than the reference value, it is determined that there is a high possibility of lymphatic invasion or lymph node metastasis.

6. The method of claim 1, wherein the at least one gene is selected from gene group B-1 consisting of MMRN1, INS-IGF2, RAMP3, NDRG4, CDKN1C, CARD16, IGF2, LOC643733, LDB2, ARL4A, GIMAP8, PDK4, FAM174B, SHC2, GMFG, GNG11, KCTD12, RN7SK, and NXN; and / or the at least one gene is selected from gene group B-2 consisting of STC2, AXL, LDHA, PLOD2, HDDC2, LOXL2, TPM2, FN1, MT1E, PLAT, VCAN, EGFR, ADAMTSL1, ADAM12, COL1A2, IGFBP3, SEMA3C, AOX1, TGFBI, CXCL6, CREB3L1, AIM1, DKK1, GREM1, LOXL4, S1PR3, EMP3, SERPINB2, ASIC1, GFPT2, MYBL2, and UHRF1.

7. The method according to claim 6, wherein in step (A), the expression levels of a plurality of genes contained in said gene group B-1 and said gene group B-2 are analyzed.

8. The method according to claim 6, wherein, when the expression level of one or more genes selected from gene group B-1 in lymphatic endothelial cells extracted from the breast adipose tissue of the breast cancer patient is significantly higher than the reference value, it is determined that there is a high possibility of lymphatic invasion or lymph node metastasis, and / or, when the expression level of one or more genes selected from gene group B-2 in lymphatic endothelial cells extracted from the breast adipose tissue of the breast cancer patient is significantly lower than the reference value, it is determined that there is a high possibility of lymphatic invasion or lymph node metastasis.

9. The method of claim 1, wherein the breast adipose tissue is adipose tissue surrounding lymph nodes draining the mammary gland.

10. The method of claim 9, wherein the breast-draining lymph nodes are axillary lymph nodes, supraclavicular lymph nodes, parasternal lymph nodes, or interpectoral (Rotter) lymph nodes.

11. The method of claim 10, wherein the axillary lymph node is axillary lymph node level I, axillary lymph node level II, or axillary lymph node level III.

12. The method of claim 9, wherein the breast adipose tissue is the adipose tissue surrounding a sentinel lymph node.

13. A program for causing a computer to execute each step included in the method according to any one of claims 1 to 12.

14. A computer-readable storage medium storing the program according to claim 13.

Images