Application of circulating tumor cells and circulating hybrid cells in cerebrospinal fluid as markers in meningeal metastasis

Abstract

The invention provides application of circulating tumor cells and circulating hybrid cells in cerebrospinal fluid as markers in meningeal metastasis, and belongs to the technical field of diagnostic markers. The CSF-CTCs and the CSF-CHCs in the cerebrospinal fluid are used as diagnostic markers, so that samples and detection substances are easy to obtain, and a stable and reliable basis is provided for early diagnosis, curative effect evaluation, relapse monitoring and survival prognosis evaluation of meningeal metastasis. The CSF-CTCs and the CSF-CHCs in the cerebrospinal fluid are jointly detected through multiple antibodies, an efficient, accurate and non-invasive novel method is provided for meningeal metastasis diagnosis, curative effect evaluation and relapse monitoring, and the method has remarkable clinical application advantages.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of diagnostic markers, and particularly relates to the use of circulating tumor cells and circulating hybrid cells in cerebrospinal fluid as markers in meningeal metastasis. BACKGROUND

[0002] Leptomeningeal metastasis (LM), also known as carcinomatous meningitis or cancerous meningitis, is a severe neurological complication of systemic malignancies, mainly occurring in patients with lung cancer, breast cancer and melanoma. According to different types of cancer, the estimated incidence of LM is about 5-20%, and with the progress of tumor diagnosis and treatment and survival management, the incidence of LM is gradually increasing. LM can cause a variety of clinical symptoms, including headache, nausea and vomiting, neurocognitive changes, gait difficulty, cranial nerve palsy, weakness, constipation, urinary retention, loss of consciousness and seizures. Although various treatment methods such as radiotherapy, chemotherapy, immunotherapy, targeted therapy and surgical intervention have been used after diagnosis, the prognosis of most cancer types after LM still remains poor, and if LM-specific treatment is not performed, the median survival period is usually only 6 to 8 weeks, while the survival period of patients can be prolonged under treatment for LM. It is crucial for the individualized treatment of LM to develop sensitive markers for early diagnosis and prediction of therapeutic efficacy.

[0003] Early diagnosis and monitoring of LM remains a challenge. Although a comprehensive neurological examination is recommended, it has poor sensitivity and specificity. Cerebrospinal fluid (CSF) cytology, which has high specificity, is often considered the gold standard for diagnosing LM, but due to the small number of malignant cells in the CSF and tumor cell heterogeneity, the sensitivity of cytology is limited, and if the initial CSF sample result is negative, a second CSF sample is usually required for analysis. The sensitivity and specificity of neuroimaging examinations such as magnetic resonance imaging (MRI) and computed tomography (CT) also vary greatly, depending on factors such as the patient's clinical symptoms, advances in imaging technology, but according to existing research, its sensitivity is estimated to be 66-98%, and its specificity is 77-97.5%. MRI may appear normal even when there are tumor cells in the CSF without measurable meningeal lesions. Invasive meningeal biopsy is complex and risky, and sometimes not feasible in LM patients, usually when CSF cytology is repeatedly negative, there is no history of cancer, or the imaging findings are inconsistent with the clinical symptoms, and treatment intervention is required. Therefore, for the detection and evaluation of LM metastasis, there are clinical problems of low sensitivity of cytology and imaging diagnosis and difficulty in evaluating efficacy. SUMMARY

[0004] The application provides a use of circulating tumor cells and circulating hybrid cells in cerebrospinal fluid as markers in meningeal metastasis, and the presence of circulating tumor cells (CTCs) and circulating hybrid cells (CHCs) in CSF is detected by an immunofluorescence method, so that the LM can be diagnosed or excluded, and the treatment effect and prognosis can be evaluated, and the accuracy is high and the sensitivity is good.

[0005] The application provides a use of a reagent for detecting circulating tumor cells and circulating hybrid cells in cerebrospinal fluid in preparation of an early screening tool for meningeal metastasis.

[0006] The application provides a use of a reagent for detecting circulating tumor cells and circulating hybrid cells in cerebrospinal fluid in preparation of a diagnosis tool for meningeal metastasis.

[0007] The application provides a use of a reagent for detecting circulating tumor cells and circulating hybrid cells in cerebrospinal fluid in preparation of an evaluation tool for treatment effect of meningeal metastasis.

[0008] The application provides a use of a reagent for detecting circulating tumor cells and circulating hybrid cells in cerebrospinal fluid in preparation of an evaluation tool for prognosis of meningeal metastasis.

[0009] In a preferred mode of the application, the circulating tumor cells and the circulating hybrid cells in the cerebrospinal fluid are separated by using a CTC / CHC sorter.

[0010] In a preferred mode of the application, the circulating tumor cells and the circulating hybrid cells separated from the cerebrospinal fluid are identified and counted.

[0011] In a preferred mode of the application, the circulating tumor cells and the circulating hybrid cells separated from the cerebrospinal fluid indicate that tumor meningeal metastasis occurs.

[0012] In a preferred mode of the application, the number of the circulating tumor cells and the circulating hybrid cells separated from the cerebrospinal fluid continuously decreases, which indicates that the treatment is effective, and the number is unchanged or increased, which indicates that the treatment is not obvious or the tumor has drug resistance.

[0013] The application also provides a kit for early screening, diagnosis, treatment effect evaluation and / or prognosis evaluation of tumor meningeal metastasis, which comprises a CTC / CHC sorter, a plurality of fluorescently labeled antibody molecules, a PBS solution, a cell culture plate and reagents for cell plating.

[0014] In a preferred mode of the application, the antibody molecules comprise at least one of CD45, CK, EpCAM, BCL, GD2, PHOX2A and PHOX2B.

[0015] The fluorescent labeling comprises a cell nucleus dye and / or an antibody conjugated dye.

[0016] Beneficial effects: The present application takes CSF-CTCs and CSF-CHCs in cerebrospinal fluid as markers of LM, and realizes specific labeling of CSF-CTCs and CSF-CHCs in cerebrospinal fluid by using specific antibody combinations for different tumor types. The multi-antibody combined detection method provided by the present application effectively avoids misdiagnosis or missed diagnosis that may be caused by single antibody detection in the preliminary screening of meningeal metastasis, and also reduces the diagnostic bias caused by the different expression of protein molecules due to individual differences. Compared with the technology of detecting CSF-CTCs based on single antibody molecules, multi-antibody combined diagnosis significantly reduces the misdiagnosis rate and missed diagnosis rate in theory, and improves the accuracy and reliability of diagnosis. In the diagnosis of meningeal metastasis, the present application separates CSF-CTCs and CSF-CHCs by using a microfluidic cell sorter after collecting cerebrospinal fluid samples of patients. For patients suspected of meningeal metastasis, doctors can select the corresponding specific antibody combination by combining the tumor type, clinical manifestations and imaging results of the patient, and detect whether there are circulating tumor cells and circulating hybrid cells in the cerebrospinal fluid by immunofluorescence method, so as to realize the diagnosis or exclusion of meningeal metastasis. In the evaluation of the efficacy of intrathecal chemotherapy for meningeal metastasis, the cerebrospinal fluid samples of the patient during treatment are obtained, and specific antibodies corresponding to the tumor are selected, and the number change of CSF-CTCs and CSF-CHCs is detected to evaluate the treatment effect. If the number of CSF-CTCs and CSF-CHCs decreases significantly, it indicates that the treatment is effective; otherwise, if the number increases or remains at a high level for a long time, it indicates that the treatment is ineffective or drug resistance occurs, and the treatment plan needs to be adjusted in time to avoid delaying the treatment opportunity and to curb the deterioration of the disease.

[0017] The present application is based on a microfluidic cell sorting platform, which only needs to collect cerebrospinal fluid samples, significantly reducing the sampling difficulty of doctors, and reducing the pain and trauma of patients. Taking CSF-CTCs and CSF-CHCs in cerebrospinal fluid as diagnostic markers not only makes the sample and detection material easy to obtain, but also provides a stable and reliable basis for early diagnosis, efficacy evaluation, recurrence monitoring and survival prognosis evaluation of meningeal metastasis. CTCs and CHCs have important significance in the occurrence, development and invasion and metastasis of meningeal metastasis, and can provide dynamic and timely guidance for clinical decision-making, which has important clinical application value. In summary, the present application detects CSF-CTCs and CSF-CHCs in cerebrospinal fluid by multi-antibody combination, which provides a new method of high efficiency, precision and non-invasiveness for the diagnosis, efficacy evaluation and recurrence monitoring of meningeal metastasis, and has significant clinical application advantages. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1Figure A: Determination of the capture efficiency of the CFD-Chip for breast and lung cancer using MDA-MB-231 or A549 cells in a cascade filtration deterministic lateral displacement microfluidic chip (CFD-Chip); B: Average capture rate of MDA-MB-231 by the CFD-Chip in CSF samples was 84.1%; C: Average capture rate of A549 by the CFD-Chip in CSF samples was 85.65%;

[0019] Figure 2 Figure of CSF-CTCs and CSF-CHCs detection results, Figure A: DAPI+ / CD45+ / CK- immunofluorescence staining for detection of white blood cells (WBCs); CSF-CTCs were identified by DAPI+ / CD45- / CK+ immunofluorescence staining, Scale bar: 10 μm; B: DAPI+ / CD45+ / CK+ immunofluorescence staining for detection of CSF-CHCs, Scale bar: 10 μm;

[0020] Figure 3 Figure of the operation process;

[0021] Figure 4 Diagnostic value of CSF-CTCs and CSF-CHCs and CK+ cells in CSF for LM, Figure A-C: Box plots showing that CSF-CTCs and CSF-CHCs and CK+ cells were significantly higher in LM patients compared to patients with non-tumorous neurological diseases; D-F: ROC analysis showed the diagnostic value of CSF-CTCs and CSF-CHCs and CK+ cell counts in the diagnosis of LM, AUC and optimal threshold are shown in the figure; G: Table summarizes the diagnostic indicators of each biomarker, including sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV); H: There was a significant positive correlation between CSF-CTC count and CSF-CHC count (Spearman test, p = 0.0004);

[0022] Figure 5 Figure of dynamic monitoring of ITC effect by CSF-CTC and CSF-CHC detection, Figure A: Line chart showing the change of CTC count per 5 ml of CSF in LM patients during ITC; B: Change of CHC count per 5 ml of CSF during ITC; C: Change of CK+ cell (CTC & CHC) count per 5 ml of CSF during ITC. DETAILED DESCRIPTION

[0023] The application provides a reagent for detecting circulating tumor cells and circulating hybrid cells in cerebrospinal fluid in the preparation of a tool for early screening of meningeal metastasis.

[0024] This study is the first to use CSF-CTCs and CSF-CHCs in CSF as biomarkers for LM. Circulating tumor cells (CTCs) are tumor seeds that spread through the blood, while circulating hybrid cells (CHCs) exhibit characteristics of both tumor and immune cells and play a key role in tumor drug resistance, immune escape, metastasis, and recurrence. The biomarkers described in this study can be used in clinical liquid biopsies to diagnose meningeal metastases.

[0025] The present invention utilizes a CTC / CHC sorter to separate CSF-CTCs and CSF-CHCs from cerebrospinal fluid. The present invention is based on the principle of determining lateral displacement using a microfluidic chip, and is capable of label-free separation of CSF-CTCs and CSF-CHCs. The separated cell suspension is highly active and can be directly applied to immunofluorescence, PCR, high-throughput single-cell transcriptome sequencing, and cell culture. The microfluidic chip described in the present invention has been disclosed in Chinese patent CN107723207A. The present invention is capable of automated on-machine separation of samples and can detect cerebrospinal fluid samples of various tumor types, including neuroblastoma, lung cancer, breast cancer, and melanoma. The present invention selects a combination of multiple marker antibodies, such as using BCL, GD2, PHOX2A, and PHOX2B antibodies simultaneously to detect specific tumor marker antigens when detecting neuroblastoma, which can reduce the adverse effects of antibody cross-reaction or tumor cell heterogeneity, effectively improving the detection rate and reducing the missed diagnosis rate from the root.

[0026] In this study, CSF-CHCs were not detected in CSF samples from non-cancer patients, while very small amounts of CSF-CTCs were detected in very rare non-cancer patient CSF samples. However, both CSF-CTCs and CSF-CHCs were effectively detected in CSF samples from cancer patients with LM metastasis. Therefore, by isolating, identifying, and counting the target markers CSF-CTCs and CSF-CHCs in patient CSF samples, doctors can assist in early screening and assessment of meningeal metastasis in cancer patients, promptly identifying potential micromeningeal metastases and enabling timely intervention and treatment, thereby reducing missed or misdiagnosed cancers.

[0027] The present invention provides an application of a reagent for detecting circulating tumor cells and circulating heterozygous cells in cerebrospinal fluid in the preparation of a diagnostic tool for meningeal metastasis.

[0028] The present invention combines CSF-CTCs and CSF-CHCs detection to predict tumor meningeal metastasis, which is conducive to timely identification of tumor meningeal micrometastasis, rapid improvement of LM-related symptoms and inhibition of potential progression, ultimately ensuring and improving the patient's quality of life.

[0029] The application provides application of a reagent for detecting circulating tumor cells and circulating hybrid cells in cerebrospinal fluid in preparation of an evaluation tool for treatment effect of meningeal metastasis.

[0030] In the application, ITC (such as intrathecal injection of chemotherapeutic drugs such as pemetrexed, methotrexate, cytarabine and dexamethasone) is the main treatment method for LM patients at present, but the curative effect is difficult to dynamically monitor, and drug resistance often occurs. Dynamic monitoring of curative effect and timely discovery of potential drug resistance are crucial for adjusting the chemotherapy regimen and realizing personalized treatment, and are crucial for improving the survival and quality of life of LM patients. The ITC curative effect evaluation based on CSF-CTCs and CSF-CHCs can timely discover the occurrence of LM chemotherapy drug resistance, and considering adjusting the treatment regimen is beneficial to improving the treatment effect of patients, further exploring the tumor LM drug resistance molecular mechanism mediated by CSF-CHCs can provide a theoretical basis for targeted drug design. If the number of CSF-CTCs and CSF-CHCs decreases significantly, it indicates that the treatment is effective; on the contrary, if the number increases or remains at a high level for a long time, it indicates that the curative effect is poor or drug resistance occurs, and the treatment regimen needs to be adjusted in time to avoid delaying the treatment opportunity and to curb the deterioration of the disease.

[0031] The application provides application of a reagent for detecting circulating tumor cells and circulating hybrid cells in cerebrospinal fluid in preparation of an evaluation tool for meningeal metastasis prognosis.

[0032] In a preferred mode of the application, circulating tumor cells and circulating hybrid cells in cerebrospinal fluid are separated by using a CTC / CHC sorter. The microfluidic cell sorter used in the application uses a non-invasive liquid biopsy diagnostic technology, selects CSF-CTCs and CSF-CHCs of patients as a new type of diagnostic marker, and is applied to early screening of tumor meningeal metastasis and dynamic curative effect evaluation of meningeal metastasis ITC, realizes dynamic monitoring in the occurrence, development and change process of meningeal metastasis, and is beneficial to assisting clinicians to formulate individualized treatment plans, timely intervention from each stage of the occurrence and development of tumors, block the progress and deterioration of tumors, and comprehensively improve the long-term survival rate of patients.

[0033] The CSF-CTCs and CSF-CHCs separation technology adopted in the application follows the microfluidic cell sorting patent product (CN107723207A), is based on the microfluidic determination lateral displacement principle; can label-free separate CSF-CTCs and CSF-CHCs, and the cell suspension obtained by separation has high activity, and can be directly applied to immunofluorescence, PCR, high-throughput single cell transcriptome sequencing and cell culture.Can realize the automatic separation of sample on machine, and can detect the cerebrospinal fluid samples of various tumor types including lung cancer, breast cancer and melanoma and the like.A variety of marker antibody combinations are selected to reduce the adverse effects of antibody cross-reaction or tumor cell heterogeneity, effectively improve the detection rate and reduce the missed diagnosis rate.

[0034] The application also provides a kit for early screening, diagnosis, treatment effect evaluation and / or prognosis evaluation of tumor meningeal metastasis, comprising a CTC / CHC sorter, a plurality of fluorescently labeled antibody molecules, a PBS solution, a cell culture plate and reagents for cell plating.

[0035] The kit comprises a CTC / CHC sorter, different fluorescently labeled antibody molecules CD45 / PHOX2B / GD2, a PB sample diluent, an adhesion liquid for cell plating, a PBS solution for washing the culture plate and a cell fixation reagent 4% paraformaldehyde fixation solution.

[0036] In a preferred mode of the application, the antibody molecules include CD45, CK, EpCAM, BCL, GD2, PHOX2A and PHOX2B; and the fluorescent labels include cell nucleus dyes DAPI and Hoechst and antibody conjugated dyes such as FITC and PE.

[0037] In order to further illustrate the application, the use of circulating tumor cells and circulating hybrid cells in cerebrospinal fluid as markers in meningeal metastasis provided by the application is described in detail below in conjunction with examples, but they should not be understood as limiting the scope of protection of the application.

[0038] Example 1

[0039] The sorting platform based on the cascade filtration deterministic lateral displacement microfluidic chip (CFD-Chip) is used to separate CSF-CTCs and CSF-CHCs from the CSF of 27 cancer patients suspected of LM (ethical number: Xinxiang Medical College First Affiliated Hospital EC-024-415):

[0040] (1) Patient sampling: lumbar puncture is used to collect cerebrospinal fluid of patients, and 5 mL of cerebrospinal fluid sample is taken for separation and identification of CSF-CTCs and CSF-CHCs.

[0041] (2) Patient sample separation:

[0042] Fresh CSF sample was diluted with PBS solution at a dilution ratio of 1:1;

[0043] The diluted sample was used to enrich CSF-CTCs and CSF-CHCs by using ZigZag microfluidic sorter and CFD-Chip microfluidic chip;

[0044] The obtained cells were collected and centrifuged at 1200 rpm for 5 min at 4°C, and the supernatant was discarded;

[0045] The cells were plated in a 96-well cell culture plate and fixed with 4% tissue fixative after adhering to the bottom of the plate at room temperature;

[0046] According to the tumor type, fluorescently labeled CD45 antibody and tumor Marker antibody Ab1 / Ab2 were added, and incubated overnight at 4°C in the dark; DAPI was used to stain the nucleus for 10 min.

[0047] (3) Marker detection and identification:

[0048] Under fluorescence microscopy, CSF-CTCs had tumor cell nucleus morphology (significant nuclear atypia, irregular nuclear morphology, and multiple mitotic figures), tumor Marker fluorescent staining was positive, and CD45 immunolabeling was negative. CSF-CHCs had tumor cell nucleus morphology, tumor Marker fluorescent staining was positive, and CD45 immunolabeling was positive. The concentration of CSF-CTCs and CSF-CHCs in the patient's CSF sample was quantitatively calculated according to the plating amount.

[0049] (4) Report evaluation:

[0050] According to the presence and number of target markers CSF-CTCs and CSF-CHCs observed by immunofluorescence, the condition of the subject was evaluated.

[0051] 1. In the early screening stage of tumor meningeal metastasis, if CSF-CTCs and CSF-CHCs exceed the threshold of 0.5 per 5 mL, combined with the patient's clinical neurological features and imaging cytology results, the diagnosis of tumor meningeal metastasis is made;

[0052] The results show that the microfluidic sorting platform has good capture efficiency for CSF tumor cells, with an average recovery rate of 84.1% and 85.65% for MDA-MB-231 and A549 cells, respectively. Figure 1). The captured cells are labeled by immunofluorescence technology with corresponding antibody combination for different suspected tumor types, and then identified by fluorescence microscopy. The CSF-CTCs have tumor cell nuclear morphology, tumor marker fluorescence staining positive, and CD45 immunolabeling negative. The CSF-CHCs have tumor cell nuclear morphology, tumor marker fluorescence staining positive, and CD45 immunolabeling positive Figure 2

[0053] No CSF-CHCs can be detected in non-tumor CSF samples, and a small amount of CSF-CTCs can be detected in a few non-tumor CSF samples, while CSF-CTCs and CSF-CHCs can be effectively detected in CSF samples of tumor patients with LM metastasis. Therefore, by separating and identifying the target markers CSF-CTCs and CSF-CHCs in the CSF samples of patients, doctors can assist in early screening and evaluation of whether cancer patients have meningeal metastasis, timely detect potential micro-meningeal metastasis, and intervene in treatment in time, reducing the occurrence of clinical cancer misdiagnosis or misdiagnosis. Through CSF cytology or imaging combined with clinical symptoms, 25 patients were diagnosed as LM, and another 2 patients were not diagnosed as LM in CSF cytology and imaging, but their clinical symptoms were consistent with LM, and CSF-CTCs and CSF-CHCs were positive. At present, the sensitivity of cerebrospinal fluid cytology is insufficient, and the sensitivity of MRI and CT imaging is insufficient, which cannot detect early meningeal metastasis and delay treatment. The combined detection of CSF-CTCs and CSF-CHCs for predicting tumor meningeal metastasis is beneficial to timely identify tumor meningeal micro-metastasis, with a sensitivity of 92.00% and a specificity of 95.45%. It is beneficial to rapidly diagnose LM-related symptoms and inhibit potential progression, and ultimately ensure and improve the quality of life of patients (such as Figure 3 , Figure 4 as shown).

[0054] 2. For the efficacy evaluation of intrathecal chemotherapy for meningeal metastasis, for patients in treatment, a sustained decrease in markers indicates that the treatment regimen is effective, and a constant or increased marker indicates that the treatment effect is not obvious or the tumor has developed drug resistance, and the treatment regimen needs to be adjusted in time according to the condition;

[0055] ​ITC (such as chemotherapeutic drugs like pemetrexed, methotrexate, cytarabine and dexamethasone, etc. intrathecal injection) is the main treatment method for LM patients at present, but its curative effect is difficult to monitor dynamically, and drug resistance often occurs. Dynamic monitoring of curative effect and timely detection of potential drug resistance are crucial for adjusting chemotherapy regimen and achieving personalized treatment, and are crucial for improving the survival and quality of life of LM patients. Traditional methods such as MRI and CSF cytology have limitations due to insufficient sensitivity in dynamic evaluation of ITC efficacy. In most patients, the CSF-CTCs count generally decreases in the initial treatment cycle, which indicates that ITC effectively inhibits LM. However, during the entire treatment process, the inventors observed an increase in the fluctuation of CSF-CHCs count, which may indicate the adaptive evolution of tumor LM to chemotherapy. In addition, the total count of CSF CK+ cells (CTC & CHC) decreased in the early treatment cycle, but rebounded in the subsequent treatment cycle. This rebound may mean resistance to the initial ITC regimen, indicating that tumor progression and drug resistance may be occurring, indicating the need to adjust the ITC regimen in a timely manner to improve treatment effect and overcome newly emerging drug resistance Figure 5 Based on the ITC efficacy evaluation of CSF-CTCs and CSF-CHCs, timely detection of the occurrence of LM chemotherapy resistance and consideration of adjusting the treatment regimen are beneficial to improve the treatment effect of patients, and further exploration of the molecular mechanism of tumor LM resistance mediated by CSF-CHCs can provide a theoretical basis for targeted drug design.

[0056] 3. For meningeal metastasis prognosis evaluation, sustained increase of the marker indicates that the patient has a high risk coefficient and poor prognosis.

[0057] Although the above embodiments make a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and people can also obtain other embodiments according to the present embodiments without creativity, which are within the protection scope of the present application.

Claims

1. Application of a reagent for detecting circulating tumor cells and circulating hybrid cells in cerebrospinal fluid in the preparation of an early screening tool for meningeal metastasis.

2. Use of a reagent for detecting circulating tumor cells and circulating hybrid cells in cerebrospinal fluid in the preparation of a diagnostic tool for meningeal metastasis.

3. Application of a reagent for detecting circulating tumor cells and circulating hybrid cells in cerebrospinal fluid in the preparation of an evaluation tool for the treatment effect of meningeal metastasis.

4. Application of a reagent for detecting circulating tumor cells and circulating hybrid cells in cerebrospinal fluid in the preparation of a prognostic assessment tool for meningeal metastasis.

5. The use according to any one of claims 1 to 4, characterized in that: It includes the use of a CTC / CHC sorter to separate circulating tumor cells and circulating hybrid cells in cerebrospinal fluid.

6. The application according to claim 5, characterized in that It also includes the identification and enumeration of circulating tumor cells and circulating hybrid cells isolated from cerebrospinal fluid.

7. The application according to claim 6, characterized in that The circulating tumor cells and circulating hybrid cells were isolated from the cerebrospinal fluid, indicating that tumor meningeal metastasis occurred.

8. The use according to claim 6, characterized in that A continuous decrease in the number of circulating tumor cells and circulating heterozygous cells isolated from the cerebrospinal fluid indicates that the treatment is effective; an unchanged or increased number indicates that the treatment is not obvious or the tumor has undergone a drug resistance transformation.

9. A kit for early screening, diagnosis, treatment effect evaluation and / or prognosis assessment of tumor meningeal metastasis, characterized in that: Includes a CTC / CHC sorter, several fluorescently labeled antibody molecules, PBS solution, cell culture plates, and reagents used for cell plating.

10. The kit according to claim 9, characterized in that The antibody molecules include at least one of the following: CD45, CK, EpCAM, BCL, GD2, PHOX2A and PHOX2B; The fluorescent markers include nuclear dyes and / or antibody-coupled dyes.

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