Prediction of prognostic survival of tumor patients by expression of lymph node mast cells

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of expression of lymph node mast cells to prediction of prognosis survival of tumor patients. The tumor is breast cancer and / or head and neck squamous cell carcinoma. The inventor finds that high-level expression of mast cells in metastatic lymph nodes is related to prognosis survival of tumor patients by exploring functions of the mast cells in the metastatic lymph nodes, and the mast cells can be used as biomarkers for prognosis survival judgment of the tumor patients, so that more valuable information is provided for clinical treatment.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology, and particularly relates to the application of lymph node mast cells in predicting the prognosis and survival of tumor patients. Background Art

[0002] Metastasis is the main cause of death in cancer patients, including hematogenous metastasis, direct dissemination and lymphatic metastasis. Lymph nodes are the first micrometastatic site for tumor cells to enter the circulation after migration. Lymph node involvement is usually seen before organ metastasis and is the most important prognostic factor for most solid tumors. In most solid tumors, lymph nodes are relatively easy to obtain and have abundant tissues, which is an important indicator for predicting tumor prognosis. Compared with hematogenous tumor cells, lymph node-colonized cancer cells show stronger immune tolerance, ferroptosis tolerance and metastatic ability. However, most studies have not considered the possible role of lymph node-colonized cancer cells themselves in tumor dissemination. Myeloid cell infiltration into the tumor microenvironment is a major regulator of tumor progression, and mast cells (MCs) are a major subpopulation of myeloid cells. Analysis of previous single-cell RNA-seq data found that mast cells are significantly enriched in metastatic lymph nodes, and MCs play a key role in multiple primary tumors, but their function in metastatic lymph nodes (LNs) is still unclear. Therefore, it is particularly important to reveal the function of mast cells in lymph nodes and predict the prognosis and survival of tumor patients. Summary of the invention

[0003] In response to the above problems existing in the prior art, the inventors explored the function of mast cells in metastatic lymph nodes and found that the high level of expression of mast cells in metastatic lymph nodes is related to the prognosis and survival of tumor patients. It can be used as a biomarker to judge the prognosis and survival of tumor patients, thereby providing more valuable information for clinical treatment.

[0004] The technical solution of the present invention is: The present invention provides a biomarker for tumor prognosis judgment, wherein the biomarker is lymph node mast cells, and the tumor is breast cancer and / or head and neck squamous cell carcinoma.

[0005] The present invention also provides a kit for judging tumor prognosis, wherein the kit comprises a reagent for detecting the expression of lymph node mast cells.

[0006] The present invention also provides the use of a reagent for detecting the expression level of lymph node mast cells in preparing a tool for tumor prognosis judgment.

[0007] Furthermore, the tumor prognosis judgment includes the following steps: (1) Collect samples from test subjects and control samples; (2) detecting and comparing the expression levels of lymph node mast cells in the test subject samples and control samples; If the expression level of lymph node mast cells in the sample of the test subject is increased compared with the expression level of lymph node mast cells in the control sample, the test subject is diagnosed as being at risk of distant tumor metastasis or having a poor prognosis; If the expression level of lymph node mast cells in the sample of the test subject is lower than that in the control sample, the test subject is diagnosed as having a lower risk of distant tumor metastasis or a good prognosis.

[0008] Furthermore, the sample is a paraffin-embedded lymph node sample.

[0009] The subject includes a mammal, preferably a primate mammal, and more preferably a human.

[0010] The reagent for detecting the expression of mast cells in the sample of the subject to be tested is not particularly limited, and is a reagent for detecting the expression of mast cells in the sample of the subject that is well known and easily available to those skilled in the art. For example, the reagent for detecting the expression of mast cells in the sample of the subject may include corresponding reagents for immunofluorescence, immunohistochemistry, etc., and the expression level of lymph node mast cells in the sample is detected by immunohistochemistry or immunofluorescence labeling.

[0011] Furthermore, MCs in lymph node samples were detected by immunofluorescence and immunohistochemistry techniques. The fluorescence value was detected by Image J, and the expression of MCs was grouped by X-tile to determine the prognosis.

[0012] Compared with the prior art, the present invention has the following beneficial effects: The inventors have found through molecular biological studies of breast cancer, head and neck squamous cell carcinoma tissue specimens and in vitro cell models in clinical work that mast cells are significantly enriched in tumor metastatic lymph nodes, which enhances the ability of tumors to metastasize to distant organs and inhibits the disease-free survival rate and prognosis of tumor patients. Based on this, we speculate that the expression of mast cells in the lymph nodes of tumor patients can be used as an important biomarker for predicting tumor progression and patient survival. The special functional state of mast cells may affect the patient's prognosis by affecting the tumor microenvironment and the biological behavior of tumor cells. Studies on the function of mast cells in lymph nodes of tumor patients have found that, on the one hand, the molecular mechanism of the interaction between mast cells and tumor cells can be identified through in-depth research, providing a scientific basis for the development of new tumor prognosis and treatment strategies. On the other hand, the prognosis of tumor patients can also be accurately assessed, which helps to achieve personalized medicine and precision treatment, optimize clinical treatment plans, and improve the quality of life and survival rate of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 It is a diagram showing that mast cells are highly expressed in lymph nodes metastasized from breast cancer and head and neck squamous cell carcinoma in Example 1; Figure 2 The results of the analysis of the relationship between the expression of mast cells in lymph nodes and the prognosis of patients with breast cancer and head and neck squamous cell carcinoma in Example 2; Figure 3 This is a graphical representation of the fact that mast cell knockout in Example 3 inhibits distant metastasis of breast cancer and head and neck squamous cell carcinoma cells. DETAILED DESCRIPTION

[0014] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0015] In the present invention, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0016] Example 1 Study on the relationship between the expression level of mast cells in lymph nodes and the prognosis of patients in breast cancer and head and neck squamous cell carcinoma (HNSCC) Detection of mast cell expression in non-metastatic and metastatic lymph nodes in breast cancer and head and neck squamous cell carcinoma 1.1 Immunofluorescence and immunohistochemistry For paraffin-embedded tissues fixed with 4% formaldehyde overnight, they were cut into 4-6 μm serial sections and antigens were retrieved using EDTA buffer (pH 8.0) for 3 minutes. The sections were then blocked in 5% BSA for 20 minutes. The primary antibodies were diluted in PBS with 1% BSA and incubated with the samples overnight at 4°C. The antibody information is as follows: TPSAB1 (1:100) and cytokeratin (1:100). The next day, the samples were washed three times in PBS. The corresponding secondary antibodies conjugated with Alexa Fluor (dilution of 1:300) and DAPI prepared in PBS were incubated with the samples for 2 hours at room temperature. After washing three times in PBS, the samples were treated with ProLong antifade reagent and coverslips, and images were acquired by laser scanning confocal microscopy.

[0017] Paraffin-embedded samples were processed as above and incubated with tryptase antibody at 4°C overnight. The next day, after washing three times, the specimens were stained with anti-mouse / rabbit IHC secondary antibody kit. IHC Profiler was used as an ImageJ plugin to examine the optical density vectors of DAB and hematoxylin. Low and high expression of MCs were judged based on immunohistochemistry (IHC) score and fluorescence intensity, and the optimal cutoff point was determined with the minimum P value using X-tile statistical software. Medium and strong represent these cells as MCs.

[0018] The results are as follows Figure 1 As shown, immunofluorescence and immunohistochemistry revealed more mast cell infiltration in metastatic lymph nodes compared with non-metastatic lymph nodes.

[0019] Example 2 Relationship between mast cell expression and prognosis in patients with breast cancer and head and neck squamous cell carcinoma With the approval of the Research Ethics Committee of Sun Yat-sen University and the informed consent of the patients, paraffin-embedded lymph node samples from 1515 patients with breast cancer and 614 patients with head and neck squamous cell carcinoma were collected. These samples had complete survival prognosis information and had been registered accordingly. MCs in lymph node samples were detected by immunofluorescence and immunohistochemistry. The fluorescence value was detected by Image J. The low and high expression of MCs were based on the immunohistochemistry (IHC) score and fluorescence intensity. The X-tile statistical software was used to determine the optimal cutoff point by the minimum P value. The Kaplan-Meier survival curve was drawn, and the survival curves of patients with different MCs density levels were compared by the log-rank (Mantel-Cox) test. The results are shown in Figure 2. Figure 2 As shown, patients with breast cancer or head and neck squamous cell carcinoma with a high density of MCs had worse progression-free survival (PFS) and overall survival (OS), with significance defined as a P value less than 0.05. Therefore, high expression of mast cells leads to poor prognosis in patients with breast cancer and head and neck squamous cell carcinoma.

[0020] Example 3 Knockout of mast cells in mice interferes with distant organ metastasis of tumor cells 3.1 Establishment of an in vivo metastasis model of breast cancer cell line EO771 in mice EO771 cells were obtained from ATCC (American Type Culture Collection). Cells were grown in RPMI 1640 (LifeTechnologies / Gibco) supplemented with 10% heat-inactivated FBS and 1% penicillin-streptomycin. Cells were maintained in an incubator at 37°C and 5% CO2 and passaged every 2 to 3 days at 60-90% confluence. Cells were routinely tested and verified to be free of mycoplasma contamination. EO771 cells (5 × 10 5) were injected into female wild-type or Kit W-sh The mice were euthanized within 5 weeks after tumor cell implantation, and the tumor-draining lymph nodes (TDLNs) and lungs were removed.

[0021] 3.2 In vivo animal imaging (IVIS) The lentivirus carrying the luciferase plasmid was pre-introduced into EO771 cells, and then EO771 cells (5×10 5 ) were injected into female wild-type or Kit W-sh The fat pads of mice were euthanized within 5 weeks after tumor cell implantation, and the tumor-draining lymph nodes and lungs were removed and soaked with D-luciferin for 10 minutes. The tissues were then imaged using an IVIS Lumina imager (Xenogen IVIS Lumina System, Caliper Life Sciences). Bioluminescent flux analysis was performed using Living Image software version 3.0 (Caliper Life Sciences).

[0022] 3.3 H&E staining Lung samples were stained with a hematoxylin and eosin staining kit according to the manufacturer's instructions. Lung tissue samples were fixed in 4% formaldehyde for 24 hours, embedded in paraffin, and cut into 4-6 μm thick sections on a microtome. The sections were stained with hematoxylin staining solution for 5 minutes and then washed with water, followed by staining with eosin staining solution for 2 minutes, then washed with water and dehydrated. The stained sections were covered with mounting media and coverslips, observed under a microscope, and images were obtained.

[0023] The results are as follows Figure 3 As shown, live imaging and H&E staining showed that MC-deficient Kit W-sh Compared with wild-type mice, the tumor metastasis in the lymph nodes of WT mice was significantly reduced, and the lack of MCs could specifically inhibit the further spread of LNCs.

[0024] These results suggest that mast cell infiltration in metastatic lymph nodes promotes disease progression in patients with breast cancer or head and neck squamous cell carcinoma.

[0025] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0026] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A biomarker for tumor prognosis judgment, characterized in that: The biomarker is lymph node mast cells, and the tumor is breast cancer and / or head and neck squamous cell carcinoma.

2. A kit for determining tumor prognosis, characterized in that: The kit contains reagents for detecting lymph node mast cell expression.

3. Use of a reagent for detecting the expression level of lymph node mast cells in the preparation of a tool for tumor prognosis judgment.

4. The use according to claim 3, characterized in that The tumor prognosis judgment comprises the following steps: (1) Collect samples from test subjects and control samples; (2) detecting and comparing the expression levels of lymph node mast cells in the test subject samples and control samples; If the expression level of lymph node mast cells in the sample of the test subject is increased compared with the expression level of lymph node mast cells in the control sample, the test subject is diagnosed as being at risk of distant tumor metastasis or having a poor prognosis; If the expression level of lymph node mast cells in the sample of the test subject is lower than that in the control sample, the test subject is diagnosed as having a lower risk of distant tumor metastasis or a good prognosis.

5. The use according to claim 4, characterized in that The sample is a paraffin-embedded lymph node sample.

6. The use according to claim 4, characterized in that The subject is a mammal.

7. The use according to claim 4, characterized in that In the step (2), the expression level of lymph node mast cells in the sample is detected by immunohistochemistry and immunofluorescence labeling.

8. The use according to claim 7, characterized in that In the step (2), MCs in the lymph node sample are detected by immunofluorescence and immunohistochemistry techniques, the fluorescence value is detected according to Image J, and the expression of MCs is grouped by X-tile.

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