Biomarker related to breast cancer prognosis, detection kit and biochip

Abstract

The invention relates to a breast cancer prognosis related biomarker, a detection kit and a biochip, the breast cancer prognosis related biomarker is a fusion cell formed by fusing an epithelial tumor cell and a myeloid cell, and the fusion cell comprises an epithelial cell molecular marker and a myeloid cell molecular marker; wherein the epithelial cell molecular marker comprises at least one of EPCAM and PanCK; the myeloid cell molecular marker comprises at least one of CD14 and CD68; the biomarker is used for breast cancer metastasis risk assessment. The liquid biopsy marker has the beneficial effects that the liquid biopsy marker for the metastasis risk of the breast cancer patient is provided and can be used as an independent prognosis index of the breast cancer metastasis risk.

Description

Technical Field

[0001] This invention relates to the technical field of cancer treatment and prognosis evaluation, and in particular to a biomarker and detection kit and biochip related to the prognosis of breast cancer. Background Technology

[0002] Breast cancer is the most common cancer among women worldwide, with distant metastases frequently occurring in the bones, lungs, liver, and brain. Circulating tumor cells (CTCs) are considered precursors to metastasis, shed from primary or metastatic lesions and entering the bloodstream; a very small percentage of these CTCs can lead to distant metastasis and death. As one of the gold standards for liquid biopsies, CTCs play a crucial role in early cancer screening and dynamic monitoring of tumor progression.

[0003] Circulating fusion cells (CFCs) are a type of circulating tumor cell (CTC), specifically tumor cells that simultaneously possess epithelial and immune cell phenotypes in the blood. Preclinical studies have shown that tumor cells can utilize cell fusion processes to promote DNA repair, develop treatment resistance, and evade immune surveillance.

[0004] The FDA-approved Cellsearch platform targets CD45 in blood samples from clinical patients. - EPCAM + Circulating tumor cells (CTCs) identification and counting are used to assess distant metastasis and survival risk in patients. CD45, or Cluster of Differentiation 45, is a protein widely present on all types of leukocytes (lymphocytes, monocytes, granulocytes, etc.). It plays a crucial role in immune cell signaling. Because CD45 primarily marks normal leukocytes in the blood, it is often used as an exclusion marker to exclude leukocytes of non-tumor origin when searching for circulating tumor cells. EPCAM (Epithelial Cell Adhesion Molecule) is another cell surface protein that is typically highly expressed in epithelial tissues. Many types of tumor cells originate from epithelial tissues; therefore, EPCAM is frequently used to label and isolate these circulating tumor cells (CTCs). Therefore, targeting CD45... - EPCAM +Identification of circulating tumor cells (CTCs) allows for more accurate identification of epithelial-originating CTCs from peripheral blood, enabling the assessment of distant metastasis and survival risk in patients. This strategy utilizes EPCAM as a positive selection marker to identify epithelial-derived cells while excluding interference from normal leukocytes expressing CD45.

[0005] However, CD45 - EPCAM + Circulating tumor cells (CTCs) are used to assess patients' distant metastasis and survival risk, but their drawback is that they overlook circulating fusion cells (CFCs), which are potentially associated with metastasis and are double-positive for epithelial-immune markers. Furthermore, there are currently no reliable biomarkers for identifying circulating fusion cells (CFCs) for breast cancer prognosis. Summary of the Invention

[0006] (a) Technical problems to be solved

[0007] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a biomarker and detection kit and biochip related to the prognosis of breast cancer, which solves the problem that there is no reliable biomarker for identifying circulating fusion cell technology in the existing breast cancer prognosis.

[0008] (II) Technical Solution

[0009] To achieve the above objectives, the main technical solutions adopted by the present invention include:

[0010] In a first aspect, embodiments of the present invention provide a biomarker related to the prognosis of breast cancer, wherein the biomarker is a fused cell formed by the fusion of epithelial-originating tumor cells and myeloid cells, and the fused cell contains epithelial cell molecular markers and myeloid cell molecular markers.

[0011] Among them, epithelial cell molecular markers include at least one of EPCAM and PanCK;

[0012] Myeloid cell molecular markers include at least one of CD14 and CD68;

[0013] The biomarkers are used for assessing the risk of breast cancer metastasis.

[0014] As a preferred embodiment of the present invention, the biomarker related to breast cancer prognosis, the epithelial cell molecular marker PanCK, includes at least one of KRT5, KRT7, KRT8, KRT9, KRT10, KRT14, KRT15, ​​KRT18 and KRT19.

[0015] In a preferred embodiment of the present invention, the fused cells further comprise lipid-related myeloid cell molecular markers, which are also included in the breast cancer prognosis-related biomarkers.

[0016] As a preferred embodiment of the present invention, the lipid-related myeloid cell molecular markers related to breast cancer prognosis are at least one of TREM2, LIPA, CSTB, CTSL, FABP4, FABP5, LGALS1, LGALS3, CD36, ACP2, ACP5, LGMN, MSR1, APOE, FOLR2, CD63, and APOC1.

[0017] Secondly, embodiments of the present invention provide a breast cancer prognosis-related detection kit, the detection kit comprising reagents for detecting said breast cancer prognosis-related biomarkers.

[0018] In a preferred embodiment of the present invention, the detection kit includes antibodies that specifically target the epithelial cell molecular markers and the myeloid cell molecular markers.

[0019] Thirdly, embodiments of the present invention provide a biochip related to breast cancer prognosis, wherein the biochip is capable of detecting the expression levels of the biomarkers related to breast cancer prognosis.

[0020] As a preferred embodiment of the present invention, the biochip is at least one of tissue microarray, microfluidic chip and circulating tumor cell chip.

[0021] Fourthly, the application of the aforementioned prognostic biomarkers or their specific antibodies in the preparation of diagnostic kits, chips, or devices for breast cancer prognostic assessment.

[0022] Fifthly, the application of the aforementioned prognostic biomarkers or their specific antibodies in constructing a breast cancer metastasis risk assessment system or assessment model.

[0023] (III) Beneficial Effects

[0024] The beneficial effects of this invention are as follows: The biomarkers and detection kits / biochips related to breast cancer prognosis of this invention, wherein the biomarkers are fused cells formed by the fusion of epithelial-originating tumor cells and myeloid cells isolated from the blood, and the fused cells include both epithelial cell molecular markers and myeloid cell molecular markers (hereinafter referred to as epithelial-myeloid marker double-positive fused cells); wherein the epithelial cell molecular markers include at least one of EPCAM and PanCK; and the myeloid cell molecular markers include at least one of CD14 and CD68; the biomarkers are used in the assessment of breast cancer metastasis risk. These breast cancer prognosis-related biomarkers are most abundant in patients with multiple metastases of breast cancer, significantly reduced in patients with a single metastatic site, and almost undetectable in non-metastatic patients. This invention uses epithelial-myeloid marker double-positive fused cells as a breast cancer prognosis-related biomarker, and their quantity in blood and tissues is closely related to whether breast cancer patients have metastasized and whether there are multiple metastases. Compared with the prior art, this invention provides a liquid biopsy biomarker for the risk of metastasis in breast cancer patients, which can serve as an independent prognostic indicator for breast cancer metastasis risk.

[0025] Furthermore, in the blood, circulating fusion cells (CFCs) with double positive epithelial-myeloid markers include circulating fusion cells with lipid-related myeloid cell molecular markers (triple-positive circulating fusion cells). Triple-positive fusion cells constitute the majority of double-positive circulating fusion cells with epithelial-myeloid markers, accounting for 81%. Triple-positive fusion cells are widely distributed in the primary lesion, distant metastases, and blood of breast cancer patients. Their infiltration level is closely related to the progression of breast cancer lesions and can serve as a further prognostic indicator in the prognosis of breast cancer metastasis risk. Attached Figure Description

[0026] Figure 1 The following are the detection results from Example 1 of this invention: A, Distribution of epithelial cell subsets in the primary lesion of breast cancer patients; B, Distribution of epithelial markers EPCAM and KRT19 and myeloid markers CD14 and CD68 in the epithelial cell subsets of the primary lesion of breast cancer patients; C, Ridgeline map showing SNX10 in primary breast cancer. + D. Genomic instability score of malignant cells and other malignant cells; E. Multiple immunohistochemical staining of DAPI (blue), EPCAM (purple), and CD68 (yellow) in primary breast cancer; G. Distribution map of epithelial cell subsets in brain and liver metastases of breast cancer; G. TREM2 in brain and liver metastases of breast cancer. +Ridge plot of genomic instability scores for malignant cells and other malignant cell subtypes; HI, multiplex immunohistochemical staining for breast cancer brain metastases (H), liver metastases, bone metastases, and lung metastases (I); J, distribution map of two different CTCs (epithelial-like CTCs and myeloid-like CTCs); K, heatmap showing matching WES (whole exome sequencing) sites for C2 (myeloid-like CTCs) CTCs in Pt2; L, pie chart showing the proportion of C2 CTCs in Pt2 that match genomic mutations; M, immunofluorescence staining for CTCs from Pt3 and Pt4; N, comparison of CD68 in breast cancer patients. + EPCAM + / EPCAM + The proportion of cells; O, comparing EPCAM in breast cancer patients with no metastasis, a single detectable metastatic lesion, and multiple detectable metastatic lesions. + CD68 + Number of fused cells;

[0027] Figure 2 The following are the detection results from Example 2 of this invention: A, a Venn diagram based on differentially expressed gene (DEG) analysis (logFC>0.58 and p<0.05), showing the overlap of highly expressed genes in the Epi_TREM2, Epi_SNX10, and CTC_Myeloid_like subsets; B, gene set enrichment analysis (GSEA) showing enrichment of lipid metabolism pathways in groups with higher fusion scores; C, a scatter plot showing the correlation between LAM feature scores and fusion scores in the breast cancer patient cohort of the Cancer Genome Atlas Project (TCGA); DE, multiplex immunohistochemical staining of primary breast cancer (D) and breast cancer brain, liver, bone, and lung metastases (E) samples; F, immunofluorescence staining of CTCs isolated from Pt3 and Pt4; G, CD68 in primary lesions, brain metastases, lung metastases, bone metastases, and liver metastases of non-metastatic breast cancer patients and metastatic breast cancer patients. + TREM2 + EPCAM + / EPCAM + Comparison of cell proportions;

[0028] Figure 3 This is a graph showing the detection results in Example 3 of the present invention, indicating the presence of EPCAM in CTCs in the blood of breast cancer patients. + CD68 - Circulating tumor cells, EPCAM + CD68 + Circulating fused cells (CFC) and EPCAM + CD68 + TREM2 +The proportion of circulating fusion cells (CFC). Detailed Implementation

[0029] To better explain and facilitate understanding of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0030] To better understand the above technical solutions, exemplary embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although exemplary embodiments of the present invention are shown in the drawings, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that the present invention can be understood more clearly and thoroughly, and that the scope of the present invention can be fully conveyed to those skilled in the art.

[0031] Example 1

[0032] First, we integrated single-cell transcriptome data from the primary epithelial lesions of breast cancer patients and performed multi-omics analysis on human clinical samples to identify fusion subpopulations carrying molecular markers of epithelial and myeloid cells (fusion cells formed by the fusion of epithelial-origin tumor cells and myeloid cells). Specifically, we visualized the epithelial cell subpopulations through cluster analysis (such as t-SNE and UMAP) and labeled the subpopulations with different characteristic genes. (Refer to...) Figure 1 Image A shows the distribution of epithelial cell subsets in the primary lesion of a breast cancer patient. Furthermore, Figure 1 Chinese image B shows the epithelial markers (molecular markers of epithelial cells) EPCAM and KRT19, and myeloid markers (molecular markers of myeloid cells) CD14 and CD68 in the epithelial cell subsets of the primary lesion in breast cancer patients. Figure 1 The distribution of A) in the middle, where, corresponding to Figure 1 SNX10 circled in A + In the malignant cell subpopulation, both epithelial cell molecular markers and epithelial cell molecular markers were densely distributed, indicating co-localization of expression. It was preliminarily determined that this subpopulation may be epithelial-myeloid fusion cells.

[0033] Figure 1 The ridgeline map of the middle C shows SNX10 in primary breast cancer. + Genomic instability scores of cell subpopulations and other cell subpopulations can determine SNX10. + The cell subsets and other cell subsets are all malignant cells (tumor cells).

[0034] Furthermore, multiple immunohistochemical staining was performed on the primary breast cancer lesion for DAPI (blue), EPCAM (purple), and CD68 (yellow), and the combined images showed EPCAM. + CD68 + Fusion cells, see details Figure 1 D, arrow indicates EPCAM + CD68 + Fusion cells. Magnification 40x, indicating the presence of EPCAM in the primary breast cancer lesion. + CD68 + Fusion cells.

[0035] Then, a subset of malignant cells expressing both epithelial and myeloid markers was identified in distant metastases of breast cancer patients:

[0036] Single-cell transcriptome analysis, cluster analysis, and UMAP / t-SNE visualization analysis were performed on epithelial cells from breast cancer brain and liver metastases. For detailed results, please refer to [link to relevant documentation]. Figure 1 Distribution map of epithelial cell subsets in brain and liver metastases of breast cancer in the central E region. Each point represents a single cell, and the subsets are labeled with different characteristic genes. Based on Figure 1 Distribution of epithelial cell subsets in E, see corresponding [reference needed]. Figure 1 The image shows the expression distribution of EPCAM, KRT18, CD14, and CD68 in epithelial cells of breast cancer brain and liver metastases, indicating that these cells express EPCAM and KRT19, molecular markers of epithelial cells, as well as CD14 and CD68, molecular markers of myeloid cells. Furthermore, all four molecular markers are simultaneously expressed in TREM2. + Fusion cell subsets ( Figure 1 (E is circled). See also Figure 1 Middle G, showing TREM2 in brain and liver metastases of breast cancer. + Both the fusion cell subpopulation and other malignant cell subtypes are tumor cells.

[0037] Further, see Figure 1 Multiple immunohistochemical staining was performed on brain metastases (H), liver metastases, bone metastases, and lung metastases (I) of breast cancer in the H and I images, respectively, staining with DAPI (blue), EPCAM (purple), and CD68 (yellow); the merged images showed the presence of EPCAM, indicated by arrows, in all breast cancer brain metastases, liver metastases, bone metastases, and lung metastases. + CD68 + Fusion cells.

[0038] Furthermore, single-cell transcriptomic analysis of circulating tumor cells (CTCs) in breast cancer was conducted, and subpopulations of CTCs were analyzed. (See [link to relevant documentation]). Figure 1The image shows the distribution of two different CTC subpopulations (epithelial-like CTCs and myeloid-like CTCs). The myeloid subpopulation is circled near a cluster of myeloid-like cell subpopulations (C2-CTCs), indicating the presence of myeloid-like CTCs in the blood of breast cancer patients, suggesting the presence of circulating tumor cells with similar expression profiles to myeloid cells.

[0039] See Figure 1 The heatmap of K shows the matching WES (whole exome sequencing) sites of the myeloid-like cell subset in Pt2 (patient sample 2). The results show that the mutations carried by the myeloid-like CTCs subset are consistent with the primary lesion of Pt2, and the myeloid-like CTCs subset is a malignant tumor cell.

[0040] Combination Figure 1 In the L-C2 CTCs, the proportion of cells carrying mutations reached 80%, supporting the use of C2CTCs in liquid biopsy to reveal tumor heterogeneity.

[0041] See Figure 1 Immunofluorescence staining was performed on breast cancer CTCs from Pt3 (patient sample 3) and Pt4 (patient sample 4) samples, staining for DAPI (blue), EPCAM (green), and CD68 (red), respectively; each staining was displayed individually and then combined. Scale bar, 5 μm. The results indicate the presence of CTCs co-expressing EPCAM and CD68 in the blood of breast cancer patients.

[0042] See Figure 1 N, comparing EPCAM in breast cancer patients + CD68 + EPCAM + The proportions of cells were analyzed in a sample including 8 patients with non-metastatic breast cancer (n=8), 6 patients with primary metastatic breast cancer (n=6), 5 patients with brain metastases (n=5), 5 patients with lung metastases (n=5), 4 patients with bone metastases (n=4), and 6 patients with liver metastases (n=6). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, and 1 / 2 p<0.0001 were considered. One-way ANOVA with Dunnett's post-hoc test was used.

[0043] The above indicates that EPCAM is present in non-metastatic breast cancer patients. + CD68 + Fusion cells were almost undetectable, while in cases of metastatic breast cancer, this invention demonstrates that only 0.20% of EPCAM were present in the primary lesions of non-metastatic patients. + CD68 + Fusion cells. And CD68 in both primary and metastatic lesions. + EPCAM + EPCAM+ The proportion of cells increased significantly: this proportion rose significantly to 3.71% in the primary lesion of metastatic breast cancer patients, and even higher to 5.70% in different metastatic lesions.

[0044] See Figure 1 In a study comparing the EPCAM of breast cancer patients with no distant metastases in the blood (6 patients, n=6), a single detectable metastatic lesion (9 patients, Mono_M, n=9), and multiple detectable metastatic lesions (Multi_M, n=10), the results were analyzed. + CD68 + Number of circulating fusion cells (CFCs). It can be seen that EPCAM... + CD68 + The number of circulating fusion cells (CFCs) is most abundant in patients with multiple metastases, significantly reduced in patients with a single metastatic site, and almost undetectable in non-metastatic patients. Therefore, circulating fusion cells (CFCs) have been established as an independent prognostic indicator, and their quantity is closely related to whether a breast cancer patient has metastases and whether there are multiple metastases.

[0045] Example 2

[0046] EPCAM + CD68 + TREM2 + Fusion cells (tumor-lipid-associated myeloid fusion cells) are widely distributed in the primary lesions, metastases, and blood of breast cancer patients. This study integrates the characteristic gene sets of fusion cell subsets carrying both epithelial and myeloid classical markers from the primary lesions, metastases, and blood of breast cancer patients. Pathway enrichment and expression profile similarity analysis confirmed that the fusion cell subset carrying both epithelial and myeloid classical markers is similar to lipid-associated myeloid cells. Multiplex immunofluorescence staining of patient clinical slides further validated the tumor-lipid-associated myeloid fusion cells (EPCAM). + CD68 + TREM2 + Distribution of fusion cells in primary lesions, metastatic lesions, and blood. EPCAM was discovered. + CD68 + TREM2 + The level of fusion cell infiltration in tissues is closely associated with breast cancer progression. The specific results are explained below:

[0047] See Figure 2Figure A is a Venn plot based on differentially expressed gene (DEG) analysis (logFC > 0.58 and p < 0.05), showing the overlap of highly expressed genes in the Epi_TREM2, Epi_SNX10, and CTC_Myeloid_like subsets. Overlapping genes are defined as the fusion feature gene set. The p-values ​​in Figure A were calculated using a two-sided paired Wilcoxon signed-rank test.

[0048] Furthermore, based on the breast cancer patient cohort from the Cancer Genome Atlas (TCGA) project, deconvolution analysis was performed on patient tumor tissues. Differential gene enrichment analysis was then conducted by grouping patients according to the levels of fusion-characteristic gene sets in their tumor tissues. (See [link to relevant documentation]). Figure 2 In the B group, gene set enrichment analysis (GSEA) showed that lipid metabolism pathways were enriched in the group with higher fusion feature gene set scores.

[0049] Further, see Figure 2 The scatter plot in C shows the correlation between LAM feature scores and fusion scores in the breast cancer patient cohort of the Cancer Genome Atlas Project (TCGA). Fusion scores were calculated using the MCP_counter and fusion features in A. LAM feature scores were calculated using the MCP_counter and LAM features. P-values ​​were calculated using the Spearman correlation test. This indicates that the fusion feature gene set is highly similar to the lipid-related myeloid feature gene set.

[0050] Figure 2 Integrated single-cell omics information from A, B, and C identified EPCAMs associated with breast cancer progression. + CD68 + The molecular characteristics of fusion cells are high expression of lipid-related myeloid cell molecular markers such as TREM2, APOE, and LIPA.

[0051] See Figure 2 Multiple immunohistochemical staining was performed on primary breast cancer (D) and breast cancer brain, liver, bone, and lung metastases (E) samples, in sections D and E. Staining markers included DAPI (blue), EPCAM (purple), CD68 (yellow), and TREM2 (red). The staining results are presented separately and then combined. Arrows represent EPCAM. + CD68 + TREM2 + Triple-positive fused cells. Magnification: 40x. See also: Figure 2 In Figure F, CTCs isolated from Pt3 and Pt4 were subjected to immunofluorescence staining. The staining markers included DAPI (blue), EPCAM (green), CD68 (red), and TREM2 (purple). Scale bar, 5 μm. Note: EPCAM... + CD68+ TREM2 + Triple-positive fusion cells are widely distributed in the primary lesion, distant metastases, and blood of breast cancer patients, and their infiltration level is closely related to the progression of breast cancer.

[0052] See Figure 2 G in the middle shows EPCAM + CD68 + TREM2 + / EPCAM + Comparison of cell proportions. The sample included patients with non-metastatic breast cancer (n=8), primary lesions of metastatic breast cancer (n=6), brain metastases (n=5), lung metastases (n=5), bone metastases (n=4), and liver metastases (n=6). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA with Dunnett's post-hoc test was used. Trends in results compared with EPCAM. + CD68 + EPCAM + The cell proportions are similar, so I won't go into detail here.

[0053] Example 3

[0054] The experimental results of Example 1 show that EPCAM + CD68 + It can be used to identify circulating fusion cells (CFCs) in the blood of breast cancer patients. See further details. Figure 3 This shows the presence of EPCAM in CTCs (total cell count 683) in the blood of a breast cancer patient. + CD68 - Circulating tumor cells, EPCAM + CD68 + Circulating fused cells (CFC) and EPCAM + CD68 + TREM2 + The proportion of circulating fused cells (CFCs). EPCAM + CD68 + Circulating fusion cells (CFCs) accounted for 18.59% of all circulating tumor cells. EPCAM levels in the blood were detected by correlating CFCs with clinical metastasis information in breast cancer patients. + CD68 + The number of circulating fusion cells (CFCs) is most abundant in patients with multiple metastases, significantly reduced in patients with a single metastatic site, and almost undetectable in non-metastatic patients. EPCAMs are among the most abundant. + CD68 + TREM2 +Circulating fused cells (CFCs) are EPCAMs + CD68 + Circulating fusion cells (CFCs) constituted the majority of the cell type (81%), accounting for 15.08% of all circulating tumor cells (CTCs). TREM2 was enriched in CTCs from the blood of breast cancer patients. + Fusion cells.

[0055] In addition, EPCAM in the blood of breast cancer patients + CD68 + Circulating fused cells (CFCs) and account for all EPCAMs + The proportion of circulating tumor cells (CTCs) was 18.59%, indicating that CD45 is currently the dominant cell type. - EPCAM + When assessing the risk of distant metastasis and survival in cancer patients using circulating tumor cells (CTCs), information from approximately one-fifth of CTCs is overlooked. Furthermore, based on the data presented in this invention, EPCAM... + CD68 + Circulating fused cells (CFC) and EPCAM + CD68 + TREM2 + Circulating fusion cells (CFCs) serve as a liquid biopsy biomarker for assessing the risk of metastasis in breast cancer patients, providing a reliable biomarker for breast cancer prognosis.

[0056] EPCAM + CD68 + Circulating fused cells (CFC) and EPCAM + CD68 + TREM2 + Compared to circulating fusion cells (CFCs), the latter are more closely associated with the assessment of breast cancer metastasis risk. Further screening with the addition of the molecular marker TREM2 can more accurately identify circulating fusion cells (CFCs) associated with breast cancer metastasis.

[0057] Furthermore, it should be specifically noted that the tumor-lipid-associated myeloid circulating fusion cells (CFCs) in this invention are characterized as circulating tumor cells (CTCs) expressing molecular markers of epithelial cells (EPCAM, PanCK (keratin family)) and lipid-associated myeloid cells (CD14, CD68, TREM2). The examples only demonstrate EPCAM. + CD68 + Circulating fused cells (CFC) and EPCAM + CD68 + TREM2 +Circulating fusion cells (CFCs) serve as a liquid biopsy biopsy marker for the risk of metastasis in breast cancer patients.

[0058] The primary method for sample detection was the use of a clinical blood sample introduction platform (Celutriator LX1 size-based sorting platform (Shenzhen GeneFlow Technology Co., Ltd., Shenzhen, 518000, China)) to separate CTCs from leukocytes, erythrocytes, and other blood components. Cells were fixed with 0.4% formaldehyde, permeabilized with 0.5% Triton X-100, blocked with 1% BSA-PBS, and incubated overnight at 4°C with primary antibody. Subsequently, coverslips were incubated with HRP-labeled secondary antibody at room temperature for 30 minutes, with tyramine signal amplification to stain cells. Antibodies were removed using elution buffer for subsequent antibody incubation. Confocal culture dishes were sealed with mounting medium containing DAPI (abcam, USA) to prevent fluorescence quenching, and fluorescence images were captured using an OLYMPUS FV1000 (OLYMPUS, Japan) confocal scanning microscope.

[0059] Example 4

[0060] A breast cancer prognosis-related diagnostic kit includes reagents for detecting epithelial cell molecular markers and myeloid molecular markers as described in Example 1. The reagents include one or more of antibodies, fluorescent markers, and enzyme-labeled secondary antibodies.

[0061] It should be noted that, for the three molecular markers in Example 2, the detection reagent includes one or more of the following: antibodies, fluorescent markers, and enzyme-labeled secondary antibodies targeting the three molecular markers. The preparation of the detection reagent can refer to existing techniques.

[0062] Example 5

[0063] A breast cancer prognosis-related detection chip is disclosed, capable of detecting epithelial cell molecular markers and myeloid molecular markers as described in Example 1. The detection chip is one of a tissue microarray, a microfluidic chip, and a circulating tumor cell chip.

[0064] It should be noted that a detection chip capable of detecting the three molecular markers in Example 2 can be designed. The fabrication of the detection chip can refer to existing technologies.

[0065] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A biomarker related to breast cancer prognosis, characterized in that, The biomarker is a fused cell formed by the fusion of epithelial-origin tumor cells and myeloid cells, and the fused cell contains both epithelial cell molecular markers and myeloid cell molecular markers. Among them, epithelial cell molecular markers include at least one of EPCAM and PanCK; Myeloid cell molecular markers include at least one of CD14 and CD68; The biomarkers are used for assessing the risk of breast cancer metastasis.

2. The breast cancer prognosis-related biomarker as described in claim 1, characterized in that, The epithelial cell molecular marker PanCK includes at least one of KRT5, KRT7, KRT8, KRT9, KRT10, KRT14, KRT15, ​​KRT18 and KRT19.

3. The breast cancer prognosis-related biomarker as described in claim 1, characterized in that, The fused cells also contain lipid-related myeloid cell molecular markers.

4. The breast cancer prognosis-related biomarker as described in claim 3, characterized in that, The lipid-related myeloid cell molecular markers are at least one of TREM2, LIPA, CSTB, CTSL, FABP4, FABP5, LGALS1, LGALS3, CD36, ACP2, ACP5, LGMN, MSR1, APOE, FOLR2, CD63, and APOC1.

5. A diagnostic kit for breast cancer prognosis, characterized in that, The test kit includes reagents for detecting prognostic biomarkers for breast cancer as described in any one of claims 1-4.

6. The detection kit as described in claim 5, characterized in that, The reagent includes antibodies that specifically target the molecular markers of the epithelial cells and the molecular markers of the myeloid cells.

7. A biochip related to breast cancer prognosis, characterized in that, The biochip can detect the expression levels of the prognostic biomarkers for breast cancer as described in any one of claims 1-4.

8. The biochip as described in claim 7, characterized in that, The biochip is at least one of tissue microarray, microfluidic chip, and circulating tumor cell chip.

9. The use of the breast cancer prognosis-related biomarkers or their specific antibodies as described in any one of claims 1-4 in the preparation of a detection kit, detection chip or detection device for breast cancer prognosis assessment.

10. The use of the breast cancer prognosis-related biomarkers or specific antibodies thereof as described in any one of claims 1-4 in constructing a breast cancer metastasis risk assessment system or assessment model.

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