A biomarker and evaluation kit for evaluating cervical cancer lymph node metastasis and its application

Abstract

The present invention discloses a biomarker and an evaluation kit for evaluating cervical cancer lymph node metastasis and its application, and relates to the field of biological detection reagents. In the present invention, we have confirmed that the EndoMT pattern of tumor-associated lymphatic vessels in metastatic cervical cancer is significantly increased compared with non-metastatic cervical cancer. CAFs promote efficient lymph node metastasis of cervical cancer by mediating and activating the EndoMT pattern of tumor-associated lymphatic vessels. Therefore, the EndoMT pattern of tumor-associated lymphatic vessels can be used as a biomarker in the preparation of a cervical cancer lymph node metastasis evaluation kit.

Description

Technical Field

[0001] The present invention relates to the field of biological detection reagents, and in particular to a biomarker and an evaluation kit for evaluating cervical cancer lymph node metastasis and applications thereof. Background Art

[0002] Cervical cancer is the most common malignant tumor of the female reproductive system and the fourth leading cause of cancer death in women worldwide, seriously endangering the life and health of women worldwide. Although the combined prevention and treatment model of human papillomavirus (HPV) vaccine, cervical cytology screening and surgery has effectively improved the prognosis of patients with early cervical cancer, it is difficult to achieve complete cure of metastatic and recurrent cervical cancer, which is also the main risk factor for patient death. Lymph node metastasis (LNM) is the main metastasis mode of cervical cancer and an independent indicator affecting the treatment and prognosis of cervical cancer. At present, for patients with early cervical cancer (stage IA2-IB2), extensive hysterectomy and pelvic lymph node dissection are still the standard treatment procedures. However, this "one-size-fits-all" model brings a series of complications such as lymphedema, neurovascular injury, venous thrombosis and infection, which not only affect the treatment effect but also reduce the quality of life of patients. How to avoid excessive "lymph node dissection" and rationally implement systematic lymph node dissection guided by the treatment model of precision concept and individualized treatment has become a difficulty and hot spot in the current clinical treatment of cervical cancer.

[0003] Currently, the commonly used methods for evaluating pelvic lymph node metastasis in clinical practice include:

[0004] 1. Imaging examination: including CT, MRI, and PET-CT. The imaging shows the morphological characteristics of lymph nodes and directly reflects the status of lymph node metastasis. However, it has high requirements for equipment and technicians, is expensive, has low sensitivity and specificity, and cannot accurately distinguish between inflammatory lymph node enlargement and tiny metastatic lymph nodes.

[0005] 2. Sentinel lymph node biopsy: Using tracers or radionuclide tracing methods, the lymph nodes closest to the tumor are biopsied, but the detection rate is low, the process is cumbersome, the timeliness is not high, and it is limited by the integrity of lymphatic vessel function. It requires high technical level of researchers or surgeons, has a high false negative rate, and the radionuclide tracing method has problems such as radioactive contamination;

[0006] 3. Determination of tumor markers: squamous cell carcinoma antigen (SCC-Ag), D2-40, Vimentin, vascular endothelial growth factor (VEGFR), etc. However, there is no unified ideal cutoff value for SCC-AG to predict the risk of pelvic lymph node metastasis, and the sensitivity and specificity of existing markers for predicting pelvic lymph node metastasis are not high.

[0007] Although these methods have certain benefits for the preoperative evaluation of pelvic lymph node metastasis, they still have disadvantages such as large differences in sensitivity and specificity, low timeliness and economic practicality, and high false positive or false negative rates. Summary of the invention

[0008] The present invention provides a biomarker and an evaluation kit and application for evaluating cervical cancer lymph node metastasis. The present invention provides a new tumor marker, which evaluates the risk of cervical cancer lymph node metastasis by detecting and co-localizing lymphatic endothelial cell markers and stromal cell markers to help improve the accuracy of the detection results.

[0009] In order to solve the above technical problems, one of the purposes of the present invention is to provide the use of the EndoMT pattern of tumor-related lymphatic vessels as a biomarker in the preparation of a kit for evaluating cervical cancer lymphatic metastasis.

[0010] In order to solve the above technical problems, the second purpose of the present invention is to provide a biomarker for evaluating cervical cancer lymph node metastasis, including the EndoMT pattern of tumor-related lymphatic vessels.

[0011] As a preferred embodiment, the EndoMT pattern of tumor-associated lymphatic vessels includes detection of interstitial cell markers in the lymphatic vessels, and the interstitial cell markers are at least one of α-SMA, Vimentin and FAP.

[0012] As a preferred embodiment, the mesenchymal cell marker is α-SMA.

[0013] In order to solve the above technical problems, the third object of the present invention is to provide a cervical cancer lymph node metastasis assessment kit, including a reagent for detecting the EndoMT pattern of tumor-related lymphatic vessels.

[0014] As a preferred embodiment, the EndoMT pattern of tumor-associated lymphatic vessels includes detection of interstitial cell markers in lymphatic vessels, and the reagent includes reagent A for detecting expression of interstitial cell markers in lymphatic vessels, and the interstitial cell marker is at least one of α-SMA, Vimentin and FAP.

[0015] As a preferred embodiment, the reagent A is an immunofluorescence detection reagent, and the immunofluorescence technique is used to perform co-localization detection on the expression of lymphatic endothelial cell markers and interstitial cell markers.

[0016] As a preferred embodiment, the detection reagent also includes a reagent B for detecting lymphatic endothelial cell markers.

[0017] Compared with the prior art, the embodiments of the present invention have the following beneficial effects:

[0018] The inventors have found that the occurrence of EndoMT in lymphatic endothelial cells is a prerequisite event for the generation of cervical cancer-related lymphangiogenesis. The density of lymphatic vessels and the content of CAFs are both proportional to the expression of interstitial cell markers in lymphatic vessels. CAFs can mediate and activate the generation of tumor-related lymphatic vessels, thereby inducing the formation of EndoMT patterns in lymphatic vessels and promoting efficient lymph node metastasis of cervical cancer. Therefore, the kit marks the EndoMT pattern of tumor-related lymphatic vessels by detecting and co-localizing lymphatic endothelial cell markers and interstitial cell markers, thereby assessing the risk of lymph node metastasis in patients with cervical cancer and establishing a complete individualized treatment plan for patients with early cervical cancer. It has the advantages of high sensitivity, high specificity, economy, convenience and high accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 :The immunohistochemical and immunofluorescence detection results of cervical cancer tissue samples in Example 1 of the present invention (Note: A-expression distribution of α-SMA and LYVE-1 in immunohistochemistry; B-expression distribution of α-SMA+LV in immunofluorescence; C-statistical results of CAFs and LDV in immunohistochemistry; D-statistical results of CAFs content and α-SMA+LV in immunofluorescence; E-statistical results of LDV and α-SMA+LV in immunofluorescence);

[0020] Figure 2 : Immunofluorescence and Western blot test results of different samples in Example 2 of the present invention (Note: A-immunofluorescence test results; B, C-Western blot test results);

[0021] Figure 3 : The test results of the in vitro permeation experiment, tube formation experiment and tumor cell transendothelial migration experiment of different samples in Example 3 of the present invention (Note: A-in vitro permeation experiment; B-tube formation experiment; C-tumor cell transendothelial migration experiment);

[0022] Figure 4 : Small animal imaging fluorescence detection results of different samples in Example 4 of the present invention;

[0023] Figure 5 : Immunohistochemistry and immunofluorescence detection results of different samples in Example 4 of the present invention (Note: A-left column 1 is the immunohistochemistry detection result, right columns 2-4 are the immunofluorescence detection results; B-statistical results of lymphatic vessel density; C-statistical results of tumor vascular infiltration ratio; D-statistical results of α-SMA+LV expression). DETAILED DESCRIPTION

[0024] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] Table 1 - Explanation of terms involved in the present invention

[0026] EndoMT Endothelial-mesenchymal transition LV Lymphatic vessels α-SMA+LV Lymphatic vessels express α-SMA CAFs Tumor-associated fibroblasts LVD Lymphatic vessel density NFs Normal fibroblasts HDLECs Lymphatic endothelial cells CSCC Cervical cancer Squamous cell carcinoma

[0027] Endothelial-mesenchymal transition (EndoMT) refers to a pathophysiological process in which endothelial cells lose their characteristic phenotype, function and morphology and gradually transform into mesenchymal cells under certain stimuli. It is the pathophysiological basis of the dynamic changes of lymphatic endothelial cells. Tumor-associated lymphangiogenesis is an early prerequisite for tumor metastasis. In the tumor microenvironment, lymphatic endothelial cells that undergo EndoMT often highly express mesenchymal cell markers such as α-SMA, FAP, and FSP-1, and show high mobility, high tube formation, and high permeability.

[0028] Based on this, the inventors detected the occurrence of EndoMT (α-SMA+LV) in lymphatic endothelial cells in cervical cancer tissues and verified its correlation with lymph node metastasis. They clarified that EndoMT is an effective marker for predicting lymph node metastasis. Based on the expression level of α-SMA+LV, it can be used to assess the risk of lymph node metastasis in patients with cervical cancer. They developed EndoMT as a biomarker for assessing lymph node metastasis of cervical cancer, laying a theoretical foundation for improving the sensitivity and specificity of lymph node metastasis detection, assisting in assessing the risk of lymph node metastasis of cervical cancer, and providing guidance for the establishment of personalized treatment plans for patients with early cervical cancer. This has important clinical and scientific significance for improving the precise staging and personalized treatment of cervical cancer.

[0029] Embodiment 1

[0030] This embodiment provides a detection method for evaluating the risk of cervical cancer lymph node metastasis, comprising the following steps:

[0031] 1. Detect the content of tumor-associated fibroblasts (CAFs) and lymphatic vessel density (LVD) in cervical cancer tissue samples;

[0032] 1.1. 55 cases of cervical squamous cell carcinoma (CSCC) tissue pathological sections were selected for immunohistochemical staining of serial tissue sections, of which 20 cases were CSCC with lymph node metastasis. LNM , 35 cases of CSCC without lymph node metastasis non-LNM ;

[0033] 1.2. Serial immunohistochemical staining of 55 CSCC tissue pathological sections was performed, with α-smooth muscle actin (α-SMA) marking CAFs and lymphatic vessels marked by lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1);

[0034] Test results such as Figure 1 As shown in Figure A, in the area where CAFs are enriched, the lymphatic vessel density (LVD) showed a significant increase. Figure 1 The statistical results of Figure C show that the CAFs content is positively correlated with LVD. Since normal lymphatic vessels (LV) do not express α-SMA, only when the lymphatic vessels (LV) express α-SMA (α-SMA+LV) can it be indicated that endothelial-mesenchymal transformation (EndoMT) has occurred. However, the immunohistochemical staining technology of continuous tissue sections cannot co-localize α-SMA and LDV to determine the relationship between the two. Therefore, this example continues to explore the relationship between the content of tumor-associated fibroblasts (CAFs) and endothelial-mesenchymal transformation (EndoMT) of lymphatic vessels (LV).

[0035] 2. Opal 4-kit multicolor immunofluorescence staining was performed on 55 CSCC tissue pathological sections to more intuitively observe the expression of α-SMA (α-SMA+LV) in lymphatic vessels (LV) in tumor-associated fibroblasts (CAFs)-enriched areas of cervical cancer tissue samples;

[0036] Test results such as Figure 1 As shown in Figure B, CSCC LNM Some lymphatic vessels (LV) in the samples highly expressed α-SMA, indicating that endothelial-mesenchymal transformation (EndoMT) occurred (equivalent to α-SMA+LV), while in CSCC non-LNM No expression of α-SMA was found in the sample lymphatic vessels (LV); statistical analysis of α-SMA+LV and CAFs in the above 55 cervical cancer tissue samples showed that Figure 1 As shown in D, the CAFs content was significantly positively correlated with the expression of α-SMA+LV (r=0.8053; P<0.0001). Statistical analysis of α-SMA+LV and LDV in the above 55 cervical cancer tissue samples showed that Figure 1 As shown in Figure E, α-SMA+LV was significantly positively correlated with LVD (r=0.354; P=0.0069); these data confirmed that EndoMT (equivalent to α-SMA+LV) occurred in lymphatic endothelial cells in the CAFs-rich areas of cervical cancer tissues and was closely related to local cervical cancer lymph node metastasis.

[0037] Embodiment 2

[0038] This example verifies the experiment that CAFs supernatant promotes endothelial-mesenchymal transition (EndoMT) of HDLECs.

[0039] From the experimental results of the above-mentioned Example 1, it can be seen that CAFs are closely related to the occurrence of EndoMT in lymphatic endothelial cells (HDLECs). In order to explore the role of CAFs in inducing EndoMT in lymphatic endothelial cells (HDLECs) and the relationship between cells, fibroblasts (CAFs) derived from cervical cancer tissue were extracted in this example, and fibroblasts (NFs) derived from normal cervical tissue were used as the control group, and blank culture medium was set as the blank group. The culture supernatant of different fibroblasts was collected as the conditioned medium (CM) of lymphatic endothelial cells (HDLECs), which were divided into Blank group, NFs-CM control group and CAFs-CM experimental group in turn to evaluate the phenotypic changes of HDLECs under different culture conditions.

[0040] like Figure 2 The morphological results obtained by immunofluorescence detection in Figure A show that compared with the Blank group and the NFs-CM control group, the CAFs-CM experimental group can significantly induce the cell morphology of HDLECs to change from the characteristic cobblestone shape to the fibroblast-like spindle shape. Figure 2 Western blot experiments in Figures B and C showed that the lymphatic endothelial cells (HDLECs) in the CAFs-CM experimental group lowly expressed VE-cadherin and highly expressed α-SMA, Vimentin, and FAP (mesenchymal cell markers).

[0041] The above experimental data results show that CAFs can induce endothelial-mesenchymal transition (EndoMT) of HDLECs.

[0042] Embodiment 3

[0043] This example verifies that EndoMT in HDLECs induces abnormal lymphangiogenesis.

[0044] From the experimental results of Example 2 above, it can be inferred that EndoMT has an important impact on the function of HDLECs by inducing skeleton rearrangement and reducing the expression of VE-cadherin. Therefore, this example further detects whether the HDLECs that undergo EndoMT are accompanied by functional changes, and the role of these functional changes in tumor metastasis.

[0045] 1. The Blank group, NFs-CM control group and CAFs-CM experimental group samples obtained using different culture media in Example 2 were subjected to in vitro permeation experiments and tube formation experiments to monitor the functional changes of HDLECs induced by CAFs supernatant.

[0046] like Figure 3 The results in Figures A and B in the middle showed that compared with the Blank group and the NFs-CM treatment group, the migration and tube-forming ability of lymphatic endothelial cells (HDLECs) in the CAFs-CM experimental group was significantly enhanced.

[0047] 2. VE-cadherin is an important adhesion protein that maintains the barrier function of endothelial cells. Its reduced expression will induce high permeability changes in the endothelial barrier. In order to simulate the intravasation / extravasation process of tumor cells in vitro, lymphatic endothelial cells (HDLECs) were co-cultured with SiHa-mCherry to conduct a tumor cell transendothelial migration experiment.

[0048] In this example, fibroblasts (CAFs) derived from cervical cancer tissue were extracted as the CAFs-CM experimental group, and fibroblasts (NFs) derived from normal cervical tissue were used as the NFs-CM control group. A blank culture medium was set as the Blank group, and the culture supernatants of different fibroblasts were collected. The monolayer of HDLECs plated on the upper chamber of the transwell was treated with the culture supernatant in advance. After 2 days, the endothelial cells were fused to form a lymphatic endothelial monolayer, and then SiHa-mCherry cells were seeded on the endothelial monolayer in the upper chamber of the transwell. After 2 days of contact co-culture, the SiHa-mCherry cells that penetrated the endothelial monolayer HDLECs to reach the lower chamber were observed and counted under a fluorescence microscope to evaluate the permeability of the lymphatic endothelium.

[0049] like Figure 3 The results in Figure C show that compared with the Blank group and the NFs-CM control group, the number of penetrating SiHa-mCherry cells in the CAFs-CM experimental group was significantly increased (changes in penetrating cells: Blank vs. NFs-CM vs. CAFs-CM = 26±4 vs. 39±5 vs. 112±5).

[0050] The above data indicate that CAFs induce abnormal tube formation of HDLECs while initiating EndoMT of HDLECs. The tube lumen generated under this condition is usually highly permeable, and its easy penetration of tumor cells increases the risk of tumor lymph node metastasis.

[0051] Embodiment 4

[0052] This example conducts in vivo experiments to verify that CAFs promote EndoMT and lymph node metastasis of HDLECs.

[0053] 1. In this example, Siha-mCherry was inoculated into the paw pads of nude mice. 3 Human cervical cancer tissue-derived fibroblasts (CAFs) were extracted as the CAFs-CM experimental group, and human normal cervical tissue-derived fibroblasts (NFs) were used as the NFs-CM control group. A blank culture medium was set up as the Blank group. The culture supernatants of different fibroblasts were collected and 50 μL of culture supernatants were injected into the paw pads. The injection cycle was 2 times / week × 2 weeks. During this period, the popliteal lymph node metastasis was observed using a small animal imaging device.

[0054] like Figure 4 The results showed that after 4 weeks of the experimental period, the rate of lymph node metastasis in the CAFs-CM experimental group was significantly increased compared with the Blank group and the NFs-CM control group.

[0055] 2. In this example, the tumor in situ tissue cultured in the above steps is further removed, fixed and sliced, and the lymphatic vessel density (LDV) of the tumor in situ tissue is detected by immunohistochemistry, and the lymphatic vessels are marked with lymphatic vessel endothelial hyaluronic acid receptor-1 (LYVE-1). Immunofluorescence technology is used to detect the occurrence of Endo-MT in the lymphatic endothelial cells (HDLECs) of the tumor in situ tissue, that is, the expression of α-SMA (α-SMA+LV) in the lymphatic vessels (LV) is detected.

[0056] like Figure 5 As shown, compared with the NFs-CM control group, the ratio of α-SMA+LVs in the CAFs-CM experimental group ( Figure 5 Middle A and Figure 5 D) and lymphatic vessel density (LVD) increased significantly ( Figure 5 Meanwhile, the CAFs-CM experimental group was more prone to tumor vascular invasion ( Figure 5 Middle A Left and Figure 5 Middle C).

[0057] The above data results indicate that CAFs promote tumor-associated lymphangiogenesis by inducing EndoMT in HDLECs, thereby accelerating lymph node metastasis of cervical cancer cells.

[0058] The specific embodiments described above further illustrate the purpose, technical solutions and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. It is particularly pointed out that for those skilled in the art, any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of protection of the present invention.

Claims

1. Application of the EndoMT pattern of tumor-associated lymphatic vessels as a biomarker in the preparation of a kit for evaluating cervical cancer lymph node metastasis, characterized in that: The kit includes a reagent for detecting the EndoMT pattern of tumor-related lymphatic vessels, wherein the EndoMT pattern of tumor-related lymphatic vessels includes detecting interstitial cell markers in lymphatic vessels, wherein the interstitial cell markers are at least one of α-SMA, Vimentin and FAP, and the reagent uses immunofluorescence technology to perform co-localization detection on the expression of lymphatic vessel endothelial cell markers and interstitial cell markers.

2. The use of the EndoMT pattern of tumor-associated lymphatic vessels as a biomarker in the preparation of a kit for evaluating cervical cancer lymph node metastasis as claimed in claim 1, characterized in that: The interstitial cell marker is α-SMA.

3. Use of the EndoMT pattern of tumor-associated lymphatic vessels as claimed in claim 1 as a biomarker in the preparation of a kit for evaluating cervical cancer lymph node metastasis, characterized in that: The reagents include a reagent A for detecting the expression of an interstitial cell marker in lymphatic vessels.

4. The use of the EndoMT pattern of tumor-associated lymphatic vessels as a biomarker in the preparation of a kit for evaluating cervical cancer lymph node metastasis as claimed in claim 1, characterized in that: The reagents include a reagent B for detecting a lymphatic endothelial cell marker.

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