Use of tumor stem cell vascular mimicry marker TEM8 in the preparation of diagnostic reagents and kits

Abstract

The present invention belongs to the fields of biology and medical testing technology and relates to a tumor stem cell vascular mimicry marker and its application. Specifically, it relates to a protein, TEM8, as a novel tumor stem cell vascular mimicry marker and its use in methods and kits for diagnosing tumor development and prognosis. In particular, it relates to its use in the preparation of diagnostic reagents or kits for triple-negative breast tumors. Experimental studies have confirmed that the tumor stem cell vascular mimicry marker TEM8, combined with the breast tumor stem cell marker ALDH, exhibits significantly greater sensitivity and specificity than the traditional angiogenesis marker CD31, making it suitable for use as a tumor marker for population screening and for the preparation of diagnostic reagents and kits.

Description

Technical Field

[0001] The present invention belongs to the fields of biology and medical testing technology and relates to a tumor stem cell vascular mimicry marker and its application. Specifically, it relates to the tumor stem cell vascular mimicry marker TEM8 and its use in preparing reagents and kits for diagnosing tumor sensitivity to anti-vascular treatment, particularly for preparing reagents and kits for diagnosing malignant breast tumor sensitivity to anti-vascular treatment. Background Art

[0002] Breast cancer is reportedly the most common malignant cancer among women. Surveys show that my country has one of the fastest-growing rates of breast cancer, severely impacting the physical and mental health of women. In 10-20% of breast cancer patients, tumor cells lack expression of the estrogen receptor (ER), progesterone receptor (PR), and HER2 proteins, resulting in triple-negative breast cancer. Studies have shown that this deficiency in hormone receptors and HER2 protein renders triple-negative breast cancer unresponsive to both common hormonal and HER2-targeted therapies, and current treatment strategies for triple-negative breast cancer remain limited.

[0003] There is increasing evidence that there is a group of cells within tumors that have the ability to self-renew and differentiate, which are referred to in the art as cancer stem cells. Cancer stem cells are believed to be an important factor driving the development, metastasis, and treatment resistance of various malignant tumors. Chemotherapy (abbreviated as chemotherapy) is currently one of the most effective means of treating cancer. Because cancer stem cells have stronger drug resistance and DNA damage repair capabilities, traditional chemotherapy can kill ordinary tumor cells but has difficulty killing cancer stem cells, resulting in the enrichment of cancer stem cells, ultimately leading to tumor recurrence and treatment failure.

[0004] Research has shown that the rapid growth of malignant solid tumors requires an efficient blood circulation network to provide them with essential nutrients and oxygen. Therefore, anti-angiogenic therapy is a crucial strategy in the treatment of malignant tumors. Currently, clinically used anti-angiogenic tumor treatment strategies primarily focus on targeting the endothelial growth factor (VEGF) and its receptor (VEGFR) pathway. However, several clinical trials have shown that anti-VEGF angiogenesis therapy has limited efficacy in prolonging overall survival or progression-free survival. Furthermore, because the VEGF / VEGFR pathway also plays a crucial role in normal tissue function, anti-VEGF / VEGFR therapy has significant cytotoxic side effects. Furthermore, the tumor vasculature is a complex system. In addition to endothelial angiogenesis, other important vascular formation processes occur, such as tumor vascular mimicry, which refers to the formation of a network of blood-perfused structures by tumor cells. In the early stages of tumor development, before external endothelial blood vessels have infiltrated the tumor interior, tumor vascular mimicry plays a crucial role in the rapid early growth of the tumor. Recent studies have demonstrated that cancer stem cells play a crucial role in the formation of tumor vascular mimicry. Therefore, clarifying the commonalities of the angiogenesis processes of various tumors and finding strategies that can specifically target tumor-related vascular structures have become urgent tasks in basic research and clinical medicine for the treatment of malignant tumors.

[0005] Based on the current state of the art, the present invention provides a tumor stem cell vascular mimicry marker and its application. Specifically, it relates to the tumor stem cell vascular mimicry marker TEM8 and its use in preparing reagents and kits for diagnosing tumor sensitivity to anti-vascular therapy, particularly for preparing reagents and kits for diagnosing malignant breast tumor sensitivity to anti-vascular therapy. Summary of the Invention

[0006] The present invention aims to address the current state, foundation, and deficiencies of existing technologies by providing a tumor stem cell vascular mimicry marker and its application. Specifically, it relates to the use of the protein TEM8 as a new tumor stem cell vascular mimicry marker and its application in preparing reagents and kits for diagnosing tumor sensitivity to anti-vascular therapy; in particular, its application in preparing reagents and kits for diagnosing malignant breast tumor sensitivity to anti-vascular therapy.

[0007] The present invention obtains a differentially expressed gene, tumor endothelial marker protein 8 (TEM8), based on mRNA sequencing experimental analysis of 7 pairs of normal adjacent tissues and cancerous tissue samples from breast cancer patients. The mRNA is then verified in 9 normal adjacent tissue samples and 19 cancerous tissue samples from breast cancer patients. The results show that the differentially expressed gene TEM8 has a significantly helpful sensitivity and specificity for diagnosing breast cancer.

[0008] More specifically, the present invention uses a differential genomics quantitative method to set up multiple biological replicates to analyze gene expression in normal adjacent tissues and cancerous tissues of triple-negative breast cancer patients, and obtains an important molecule related to the angiogenesis pathway that is highly expressed in breast tumors, with a P < 0.05 and a difference of more than 2 times;

[0009] The present invention further conducted experimental verification by customizing a rabbit-derived polyclonal antibody specifically targeting the extracellular end of TEM8. Immunohistochemical analysis of TEM8 protein expression in breast cancer tissue sections and adjacent normal tissue sections from breast cancer patients was performed. The results showed that TEM8 protein expression was significantly higher in breast cancer tumor tissue than in adjacent normal tissue. Furthermore, TEM8 protein expression levels gradually increased with increasing malignancy of breast tumor subtypes, reaching the highest expression levels in triple-negative breast cancer cells, the most malignant type. The differentially expressed protein TEM8 can be used as a new diagnostic marker for malignant breast tumors.

[0010] Furthermore, the present invention measured the density of endothelial vessels and tumor vascular mimicry in breast cancer tissues from patients using immunohistochemical staining for the endothelial cell marker CD31 and periodic acid-Schiff staining, and analyzed their relationship with the expression of the TEM8 protein. The results showed no significant correlation between tumor endothelial vessel density and breast cancer subtype or protein expression, while a significant positive correlation was found between tumor vascular mimicry density and the malignancy of breast cancer subtypes and protein expression levels.

[0011] Furthermore, the present invention utilized a patient-derived xenograft (PDX) mouse model of triple-negative breast cancer. Using flow cytometry, fluorescence activated cell sorting (FACS) was used to isolate tumor cells that overexpressed and underexpressed the TEM8 protein. These cells were then injected orally into the fourth mammary fat pad of the mice at a gradient dilution ratio to form tumors. The results showed that cells overexpressing the TEM8 protein not only had a stronger tumor-forming ability, but also formed tumors with a higher density of tumor vascular mimicry.

[0012] Furthermore, the present invention used flow cytometry to stain and analyze the TEM8 protein, along with the classic breast cancer stem cell markers ALDH and CD24 / CD44, using triple-negative breast cancer cell lines and PDX tumors. The results showed that the TEM8 protein was significantly expressed in ALDH+ breast cancer stem cells. After FACS sorting of ALDH+ breast cancer stem cells and ALDH- non-breast cancer stem cells, the TEM8 mRNA and protein were detected, demonstrating that both the TEM8 gene and protein were significantly highly expressed in ALDH+ breast cancer stem cells.

[0013] Furthermore, the present invention used FACS to separate four cell populations: TEM8-high expression and ALDH+, TEM8-high expression and ALDH-, TEM8-low expression and ALDH+, and TEM8-low expression and ALDH-. These populations were then subjected to in vitro angiogenesis and in vivo tumorigenesis experiments. The results demonstrated that breast cancer stem cells with high TEM8 expression and ALDH+ expression had significantly enhanced vascular mimicry and in vivo tumor formation abilities. The TEM8 protein can be used as a novel marker for vascular mimicry in breast cancer stem cells.

[0014] A differentially expressed protein TEM8 of the present invention can be used as a new malignant tumor stem cell vascular mimicry marker for preparing reagents and kits for diagnosing malignant tumors;

[0015] In the present invention, the malignant tumor is a triple-negative breast tumor.

[0016] In the present invention, the malignant tumor diagnostic reagent comprises an antibody or antibody fragment specific to a tumor stem cell vascular mimicry marker protein (anti-TEM8).

[0017] In the present invention, the tumor stem cell vascular mimicry marker TEM8 is TEM8 gene, mRNA, cDNA or protein.

[0018] In the present invention, the unique peptides (Unique Peptide) are specific to each protein of the tumor stem cell vascular mimicry marker (TEM8).

[0019] In the present invention, the TEM8 protein is a protein expressed in tissues.

[0020] In the present invention, the malignant tumor diagnostic kit includes an antibody specific to the tumor stem cell vascular mimicry marker protein TEM8, a fluorescent secondary antibody, and magnetic beads that can adsorb this protein and protein combinations; the tumor stem cell vascular mimicry marker protein TEM8 protein unique characteristic peptide segment and its isotope-labeled peptide segment.

[0021] The present invention provides a protein, TEM8, as a novel tumor stem cell vascular mimicry marker and its use in methods and kits for diagnosing tumor development and prognosis. In particular, the protein is used in the preparation of diagnostic reagents or kits for triple-negative breast tumors. Experimental studies have demonstrated that the tumor stem cell vascular mimicry marker, TEM8, combined with the breast tumor stem cell marker ALDH, exhibits significantly greater sensitivity and specificity than the traditional angiogenesis marker CD31, making it suitable for use as a tumor marker for population-based screening and for the preparation of diagnostic reagents and kits.

[0022] The present invention is further described in detail below through the accompanying drawings and specific embodiments. The protection scope of the present invention is not limited by the specific embodiments but is defined by the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It was shown that the relative mRNA level of TEM8 was significantly increased in triple-negative breast cancer cells.

[0024] Figure 2 Results showed that the relative mRNA levels of TEM8 were significantly increased in various types of malignant tumors.

[0025] Figure 3 It was shown that the protein level of TEM8 was significantly increased in triple-negative breast cancer cells.

[0026] Figure 4 It showed that the tumor vascular mimicry density of breast cancer patients was significantly positively correlated with tumor malignancy and TEM8 protein levels.

[0027] Figure 5 It was shown that triple-negative breast tumor PDX cells with high expression of TEM8 had significantly enhanced tumorigenesis and angiogenesis capabilities.

[0028] Figure 6 It was shown that TEM8 was significantly overexpressed on ALDH+ breast cancer stem cells in triple-negative breast tumors.

[0029] Figure 7 It was shown that ALDH+ breast cancer stem cells with high expression of TEM8 had significantly enhanced tumorigenesis and tumor vascular mimicry formation capabilities. DETAILED DESCRIPTION

[0030] Example 1:

[0031] Detection of differential expression of angiogenesis-related genes in cancer cells and adjacent normal cells of triple-negative breast cancer patients

[0032] Seven pairs of triple-negative breast cancer (TNBC) cancer cells and adjacent normal cells were selected. Total RNA was extracted using Trizol reagent, quantified using a 2100 Bioanalyzer, and libraries constructed using the NEB Next Ultra Directional RNA Library Prep Kit for Illumina. RNA sequencing was then performed on the HiSeq3000 platform. Sequencing results were quality-controlled using FastQC. Genes with a cancer cell to normal cell ratio greater than 2 and a p-value less than 0.05 were screened to identify a gene set highly expressed in TNBC cells. This gene set was then intersected with two previously reported angiogenesis-related gene sets, GSE86788 and GSE43742, to identify two angiogenesis-related genes, TEM8 and SPOCK1, that were highly expressed in TNBC cells.

[0033] Example 2:

[0034] Detection of TEM8 protein expression in cancerous tissue and adjacent tissues of breast cancer patients

[0035] The TEM8 extracellular end (AA33-321) peptide sequence was cloned into the pET8a expression vector, expressed and purified to obtain 3 mg of TEM8 extracellular peptide. The peptide was used to immunize New Zealand white rabbits, and the immune serum was collected. After verification of the effect, the antibody was purified to obtain a rabbit-derived polyclonal antibody specific for the TEM8 extracellular end. Using this antibody, TEM8 protein immunohistochemical staining was performed on 81 breast cancer tissue sections (including 45 luminal, 20 HER2+, and 16 triple-negative breast cancers) and 18 breast cancer patients' adjacent normal tissues. Based on the intensity of immunohistochemical staining and the proportion of positive staining, the corresponding immunohistochemical score was calculated according to the following formula to reflect the level of TEM8 protein expression:

[0036] Percentage of weakly stained cells + 2 × percentage of moderately stained cells + 3 × percentage of strongly stained cells

[0037] The results showed that the TEM8 protein level in breast cancer tissue was significantly higher than that in adjacent normal tissue, and the TEM8 expression level in triple-negative breast cancer with high malignancy was significantly higher than that in luminal breast cancer with low malignancy.

[0038] Example 3:

[0039] Correlation analysis between TEM8 protein level and tumor blood vessel density in breast cancer tissue

[0040] Immunohistochemical staining for the endothelial cell marker CD31 combined with periodic acid-Schiff (PAS) staining allows for the differentiation of endothelial vessels (CD31+) from non-endothelial vascular mimicry (CD31-PAS+). Vascular staining was performed on tissue sections from 50 breast cancer patients (including 21 luminal, 15 HER2+, and 14 triple-negative breast cancers). Vascular density was calculated and analyzed in conjunction with immunohistochemical staining for TEM8. The results showed that triple-negative breast cancers with high malignancy had significantly increased vascular mimicry density, and TEM8 protein expression levels were significantly positively correlated with vascular mimicry density.

[0041] Example 4:

[0042] Triple-negative breast cancer PDX mouse model validates that high expression of TEM8 promotes vascular mimicry

[0043] The PDX11 mouse model used was derived from a patient with triple-negative breast cancer. Patient tissue was orthotopically transplanted into the fourth mammary fat pad of severe combined immunodeficient mice to establish tumors. The PDX tumors were stably passaged for at least two generations and immunohistochemically confirmed to lack expression of estrogen receptor, progesterone receptor, and HER2 proteins. The PDX tumors were removed from the mice and minced to obtain single-cell suspensions. Mouse cells were then removed using an H2kd antibody and labeled with an antibody specific for the extracellular domain of TEM8. Cell populations expressing high and low TEM8 expression were isolated by FACS. These cells were then serially diluted and injected into the fourth mammary fat pad of severe combined immunodeficient mice, and their tumor formation was monitored. The experiment was terminated when the tumors reached 10-15 mm in diameter. Tumors were then removed, embedded in paraffin, and sectioned for analysis of tumor vascular density. Results showed that tumor cells with high TEM8 expression had significantly enhanced tumor initiation and vascular mimicry.

[0044] Example 5:

[0045] Triple-negative breast cancer PDX mouse model validates TEM8 protein marker for cancer stem cells with vascular mimicry-forming ability

[0046] Using the PDX15 mouse model, a PDX derived from another triple-negative breast cancer patient, the researchers removed the PDX tumors from the mice and minced and digested them to obtain single-cell suspensions. Mouse cells were then removed using an H2kd antibody. Furthermore, the cells were labeled with an antibody specific for the extracellular end of TEM8 and an Aldefluor assay for ALDH enzyme activity. Four populations of cells were isolated by FACS: TEM8-overexpressing and ALDH+, TEM8-overexpressing and ALDH-, TEM8-underexpressing and ALDH+, and TEM8-underexpressing and ALDH-. These cells were then injected in a serial dilution ratio into the fourth mammary fat pad of severe combined immunodeficient mice and observed for tumor formation. The experiment was terminated when the tumors grew to 10-15 mm in diameter. Tumors were removed, embedded in paraffin, and sectioned for analysis of tumor vascular density. Results showed that TEM8-overexpressing ALDH+ breast cancer stem cells had significantly enhanced tumor initiation and vascular mimicry formation.

[0047] The experimental results described in the above examples show that TEM8 protein has significant sensitivity and specificity for breast cancer patients, especially breast cancer patients and normal people, and TEM8 protein marks a group of breast cancer stem cells with significant vascular mimicry formation ability, and can be used as a breast cancer stem cell vascular mimicry marker for further preparation of diagnostic reagents and diagnostic kits.

Claims

1. Use of a reagent for detecting the tumor stem cell vascular mimicry marker TEM8 in the preparation of diagnostic reagents and kits, characterized in that: The tumor stem cell vascular mimicry marker TEM8 is a TEM8 gene, mRNA, cDNA or protein; The diagnostic reagent and kit are diagnostic reagents and kits for anti-angiogenic therapy sensitivity of malignant tumors; the malignant tumor is triple-negative breast cancer.

2. The use according to claim 1, characterized in that The malignant tumor anti-angiogenic therapy sensitivity diagnostic reagent comprises an antibody or antibody fragment specific to the tumor stem cell vascular mimicry marker protein TEM8.

3. The use according to claim 2, characterized in that The unique characteristic peptide segment is specific to the tumor stem cell vascular mimicry marker protein TEM8.

4. The use according to claim 1, wherein TEM8 protein is expressed in tissues.

Images