Methods for detecting the hes1 marker of melanoma ctc using multiplexed immunofluorescence

Abstract

The application provides a method for detecting HES1 markers of melanoma CTCs by using multiplex immunofluorescence technology, and the method comprises the following steps: S1, collecting peripheral blood of a melanoma patient, and performing sorting and enrichment of CTCs through a microfluidic chip; S2, fixing and permeating after making all nucleated cells in the blood flatly and enriched on an adherent slide through a centrifuge; and sequentially performing co-incubation of HES1, HMB45 and CD45 antibodies on the screened melanoma CTCs through the multiplex immunofluorescence technology, and further analyzing the expression of the HES1 antibody and performing image analysis and quantification. The method is simple in operation, high in detection accuracy, can avoid false negatives and the like, and improves the screening accuracy of melanoma CTCs.

Description

Technical Field

[0001] This invention relates to the field of melanoma tumor suppressor gene HES1 technology, and in particular to a method for detecting HES1 markers in melanoma CTCs (Circulating Tumor Cells) using multiplex immunofluorescence technology. Background Technology

[0002] HES1 (hairy and enhancer of split homolog-1), a downstream target gene of the Notch signaling pathway, is abnormally highly expressed in various solid tumors (such as osteosarcoma, glioma, breast cancer, pancreatic cancer, and ovarian cancer), and is associated with tumor invasion and metastasis, seriously affecting the survival and prognosis of cancer patients. HES1 is generally considered a key oncogene downstream of Notch. Notch signaling is one of the key signaling pathways essential for normal vascular development, participating in multiple steps of tissue angiogenesis, including tip and stalk cell differentiation and proliferation, endothelial cell migration and adhesion, vascular remodeling, and arteriovenous differentiation, while also participating in the differentiation of mesenchymal stem cells into endothelial cells. Therefore, accurate detection and assessment of HES1 expression in melanoma patients is crucial.

[0003] Currently, there are three main methods commonly used to detect HES1 expression: (1) immunohistochemistry; (2) immunofluorescence; and (3) fluorescence in situ hybridization. In the field of tumor molecular pathology diagnosis, there are three main methods commonly used to detect HES1 expression: immunohistochemistry (IHC), immunofluorescence (IF), and fluorescence in situ hybridization (FISH) are three key complementary techniques. IHC is used to study the expression level, cellular localization, and correlation with pathological features of HES1 protein at the tissue level, but its multiplexing capability and quantitative accuracy are limited; IF, with its advantages of multicolor labeling and high resolution, is often used to study the nucleocytoplasmic localization of HES1, co-localization with other proteins, and signaling pathway dynamics at the single-cell level, but it has limitations such as fluorescence quenching and the need for dedicated imaging equipment; FISH is mainly used in tumor research to detect chromosomal abnormalities related to the HES1 gene at the genomic level, such as amplification or translocation, but the technology is complex and costly. In summary, IHC is suitable for routine protein screening, IF is suitable for multi-target protein co-localization studies, and FISH is the molecular "gold standard" for confirming specific gene abnormalities. The combined use of these three methods can achieve comprehensive analysis of biomarkers from the gene to the protein level, and has become an important strategy for precision medicine. However, for melanoma patients lacking surgical indications or with contraindications, the inability to resect the tumor tissue and the risk of tumor spread during fine-needle aspiration make it difficult to assess HES1 status.

[0004] Circulating tumor cells (CTCs) are malignant cells that detach from primary or metastatic lesions of solid tumors and enter the peripheral blood circulation. CTCs are a crucial intermediate step in the development of distant metastasis and a prerequisite for it, forming the molecular basis of "liquid biopsy." CTCs have significant biological and clinical value. First, the number of CTCs is significantly correlated with shorter patient survival, serving as an independent prognostic biomarker. Second, CTCs obtained through non-invasive blood collection can serve as samples for "real-time biopsy," overcoming the spatial and temporal limitations of tissue biopsy. Finally, in vitro culture or single-cell analysis of captured CTCs can reveal key gene mutations and signaling pathways driving metastasis, providing a direct model for studying tumor metastasis mechanisms and developing targeted therapies. Therefore, CTC detection and analysis technologies have become an important research direction in the field of precision cancer medicine.

[0005] With advancements in bioengineering technology, breakthroughs and progress have been made in the separation of causative clinical tumors (CTCs) using microfluidic chip technology. A representative chip technology in the industry is the CTC-ichip microfluidic technology developed by Professor Daniel Haber's team at Harvard Medical School. This technology employs deterministic lateral displacement, inertial focusing, and magnetophoresis techniques, achieving a sorting speed of up to 102. 7 Cells / second. By using two-stage magnetic leukocyte removal and antibody removal, a leukocyte removal rate of 3.8-log and a rare cell yield of 97% can be achieved, with a sample processing rate of 8 mL whole blood / hour. Furthermore, the spiral microfluidic chip (currently commercialized, such as Celutriator TX1) developed by Professor Lim Chwee Teck's team at the National University of Singapore, can rapidly (within 15 minutes) and efficiently enrich viable CTCs, while seamlessly integrating with CTC in vitro culture technology. This allows for the expansion of rare CTC cells into cell lines suitable for long-term in vitro culture and the establishment of CTC-derived xenograft (CDX) mouse models. It also ensures the needs of downstream single-cell omics and molecular biology validation, becoming a crucial technological support for elucidating the biological characteristics of CTCs and representing a significant breakthrough in the field.

[0006] The existing method of CTC counting as a clinical detection tool has several drawbacks: ① Complex technical methods: CTC detection techniques are diverse and complex, and differences between methods can lead to inconsistent results. Normal cells from peripheral blood may be introduced during blood sample collection, affecting the accuracy of the test. ② Need to improve the specificity and sensitivity of results: Some methods for isolating CTCs may produce false positives or false negatives, affecting diagnostic accuracy. CTC concentration may be related to tumor size, stage, and other factors, making a single counting method insufficient to fully reflect the true tumor situation. Furthermore, detection techniques may miss or confuse CTCs during identification and quantification. ③ Lack of standardization and clinical decision-making guidelines: The current lack of unified detection methods and standardized procedures makes it impossible to compare results between different laboratories. The lack of clinical decision-making guidelines based on CTC data presents challenges for physicians in clinical application. Further research and validation are needed to establish reliable clinical application guidelines and standards. These issues limit the widespread clinical application of CTC counting, primarily using it as a tool for auxiliary diagnosis and prognostic assessment. Summary of the Invention

[0007] To overcome the limitations of traditional CTCs, which are only used for counting and have outdated detection techniques, and the limited significance of HES1 identification in patients with advanced melanoma who have already experienced remote multi-organ metastasis, this invention provides a method for detecting HES1 markers in melanoma CTCs using multiplex immunofluorescence technology. This method is simple to operate, has high detection accuracy, avoids false negatives, and improves the accuracy of melanoma screening.

[0008] To address the aforementioned technical problems, this invention provides a method for detecting HES1 markers in melanoma CTCs using multiplex immunofluorescence technology, the method comprising the following steps:

[0009] S1. Collect peripheral blood from melanoma patients and sort and enrich CTCs using a microfluidic chip;

[0010] S2. Nucleated cells in the blood were spread and enriched onto an adhesive glass slide by centrifugation, then fixed and permeable. The selected melanoma CTCs were co-incubated with HES1, HMB45 and CD45 antibodies in sequence using multiplex immunofluorescence technology. The expression of HES1 antibody was further analyzed and the images were analyzed and quantified.

[0011] In more detail, step S1 involves collecting 5-10 mL of blood from a melanoma patient, sorting the blood using a microfluidic sorting instrument, and then further fixing the melanoma CTCs obtained through microfluidic chip sorting with 4% PFA and washing with PBS.

[0012] Furthermore, step S1 specifically includes the following steps:

[0013] S11. Collect 5-10 mL of venous blood from melanoma patients into EDTA anticoagulated vacuum blood collection tubes;

[0014] S12. Before blood sample sorting, mix at room temperature at low speed of 50 rpm for 1 hour.

[0015] S13. Use a microfluidic sorting instrument and follow the corresponding steps to obtain approximately 5 mL of cell suspension after sorting by the microfluidic chip.

[0016] S14. The cell suspension was centrifuged at 1410 rpm for 5 min at room temperature to obtain cell pellet;

[0017] S15. The cell pellet was resuspended in 1 mL of PBS and transferred to a 1.5 mL EP tube. The tube was centrifuged at 1410 rpm for 5 min at room temperature and washed twice.

[0018] S16. Resuspend the cell pellet in 1 mL of 4% PFA and fix at room temperature for 10 min.

[0019] S17. Centrifuge the cell suspension at 1410 rpm for 5 min at room temperature to obtain cell pellet.

[0020] S18. The cell pellet was resuspended in 1 mL of PBS, centrifuged at 1410 rpm for 5 min at room temperature, and washed twice.

[0021] S19. Finally, resuspend in 1 mL PBS and store briefly at 4°C for 1-2 weeks.

[0022] In more detail, step S2 involves: centrifuging the cells obtained in step S1 using a cell centrifuge smear machine, followed by a blocking and permeabilization step, then using 1% H2O2 to block endogenous peroxidase, and then sequentially performing primary antibody, secondary antibody, and dye incubation steps; if multiple targets are required, the cells can be eluted with antibody elution buffer and the primary antibody, secondary antibody, and dye incubation steps can be repeated, and finally, cell nuclear staining and mounting are performed.

[0023] Furthermore, step S2 specifically includes the following steps:

[0024] S21. Take an appropriate amount of cell suspension and centrifuge at 1000 rpm for 10 min at room temperature using a cell smear centrifuge to prepare cell smears.

[0025] S22. Use an immunohistochemical pen to delineate the cell range, add 300 μL of blocking-permeability mixture and treat for 20 min, then wash 3 times with 300 μL of PBS.

[0026] S23. Treat with 200 μL of 1% H2O2 for 1 h, then wash 3 times with 300 μL of PBS;

[0027] S24. Incubate with 200 μL of primary antibody at room temperature for 30 min, then wash three times with 300 μL of PBS;

[0028] S25. Incubate with 200 μL of secondary antibody at room temperature for 20 min, then wash three times with 300 μL of PBS.

[0029] S26. Incubate with 200 μL of dye at room temperature for 10 min, then wash three times with 300 μL of PBS.

[0030] S27. Incubate with 200 μL of antibody elution buffer at 37°C for 10 min, then wash once with 300 μL of PBS.

[0031] S28. Repeat steps S24-S27 until the last antibody has finished incubating.

[0032] S29. Incubate with 200 μL of DAPI at room temperature for 10 min, then wash three times with 300 μL of PBS.

[0033] S210, Seal the sheet with 50 μL of anti-quenching sealant;

[0034] S211. Use a digital pathology slide scanner (KFBIO KF-FL-120) to scan slides;

[0035] S212. The obtained scan results are analyzed using pathological analysis software (Pano Analyzer) to determine whether the cells conform to the CTC phenotype and to statistically analyze the positive and negative expression of HES1.

[0036] Furthermore, the blocking-permeability mixture in step S22 is a mixture of 1% BSA and 0.25% Triton X-100.

[0037] To elaborate further, the repetition of steps S24-S27 mentioned in step S28 refers to performing the steps twice.

[0038] Furthermore, steps S24-S27 mentioned in step S28 need to be performed twice more (i.e., S24-S27 are performed a total of three times). In the primary antibody incubation step in step S24, the primary antibody used for the first incubation is Mouse anti-Human Melanosome antibody, diluted 1:250 with 1% BSA as the blocking solution, for example, the manufacturer and catalog number is DAKO M0634; the primary antibody used for the second incubation is Mouse anti-Human CD45 antibody, diluted 1:1000 with 1% BSA as the blocking solution, for example, the manufacturer and catalog number is abcam ab8216; the primary antibody used for the third incubation is Rabbit anti-Human HES1 Rabbit Monoclonal Antibody, diluted 1:4000 with 1% BSA as the blocking solution, for example, the manufacturer and catalog number is abcam ab8216 CST 11988.

[0039] Among them, Mouse anti-Human Melanosome antibody (or HMB-45) is named as follows: Mouse anti-human melanosome HMB-45 antibody, where HMB-45 is the clone number of this antibody; Mouse anti-Human CD45 antibody is named as follows: Mouse anti-human CD45 antibody, for example, the antibody with clone number MEM-28 can be used, specifically referring to anti-CD45 monoclonal antibody produced by a specific hybridoma cell line; Rabbit anti-Human HES1 Rabbit Monoclonal Antibody is named as follows: Rabbit anti-human HES1 rabbit monoclonal antibody, for example, the antibody with clone number D6P2U can be used.

[0040] Furthermore, the primary antibody incubation, secondary antibody incubation, and dye incubation steps S24-S27 mentioned in step S28 need to be performed twice more. In the primary antibody incubation step S24, the primary antibody HES1 is incubated overnight at 4°C, and the primary antibodies HMB45 and CD45 are incubated at room temperature for 30 minutes.

[0041] Furthermore, the primary antibody incubation, secondary antibody incubation, and dye incubation steps S24-S27 mentioned in step S28 need to be performed twice more. In the secondary antibody incubation step S25, HRP-labeled goat anti-rabbit mixed secondary antibody is used for all three steps without dilution.

[0042] Furthermore, steps S24-S27 mentioned in step S28 need to be performed twice more (i.e., S24-S27 are performed a total of three times). In step S27, the dye incubation step involves the following steps: the first incubation uses dye 520 to label the primary antibody HMB-45, and the dye is diluted 1:200 with signal amplification solution; the second incubation uses dye 650 to label the primary antibody CD45, and the dye is diluted 1:200 with signal amplification solution; the third incubation uses dye 570 to label the primary antibody HES1, and the dye is diluted 1:200 with signal amplification solution.

[0043] Furthermore, in step S212 software analysis, when the immunofluorescence expression index of cells meets the condition of HMB45+ / CD45- / DAPI+, it can be identified as melanoma CTC. On this basis, the expression ratio of HES1 positive expression CTC and HES1 negative expression CTC is statistically analyzed and matched with the tumor metastasis of melanoma patients to analyze whether the two are correlated.

[0044] Preferably, in step S21, 300-500 μL of cell suspension is taken.

[0045] Preferably, step S22 is as follows: use a dedicated multiplex immunohistochemistry pen to delineate the cell range, place the slide in a humidified chamber, add 300 μL of 1% BSA and 0.25% Triton X-100 blocking and permeabilizing mixture to each slide, and place it on a decolorizing shaker at 55 rpm for 20 min.

[0046] Place the sample in a staining jar, put it on a decolorizing shaker at 55 rpm, wash with 300 μL of PBS for 3 min three times, and finally shake off the excess liquid.

[0047] Preferably, step S23 is as follows: place the slide in a humidified chamber, add 200 μL of 1% H2O2 diluted with PBS to each slide, and treat it on a decolorizing shaker at 55 rpm for 1 h.

[0048] Place the sample in a staining jar, put it on a decolorizing shaker at 55 rpm, wash with 300 μL of PBS for 3 min three times, and finally shake off the excess liquid.

[0049] Preferably, step S24 is as follows: place the slide in a humidified chamber, add 200 μL of primary antibody to each slide, and incubate at room temperature for 30 min;

[0050] Place the sample in a staining jar, put it on a decolorizing shaker at 55 rpm, wash with 300 μL of PBS for 3 min three times, and finally shake off the excess liquid.

[0051] Preferably, step S25 is as follows: place the slide in a humidified chamber, add 200 μL of secondary antibody to each slide, and incubate at room temperature for 20 min.

[0052] Place the sample in a staining jar, put it on a decolorizing shaker at 55 rpm, wash with 300 μL of PBS for 3 min three times, and finally shake off the excess liquid.

[0053] Preferably, step S26 is as follows: place the slide in a humidified box to avoid light, add 200 μL of dye to each slide, and incubate at room temperature for 20 min;

[0054] Place the sample in a staining jar and operate in the dark. Place it on a decolorizing shaker at 55 rpm and wash three times with 300 μL of PBS for 3 minutes each time. Finally, remove excess liquid.

[0055] Preferably, step S27 is as follows: place the slide in a humidified box to protect it from light, add 200 μL of antibody elution buffer to each slide, and incubate at 37°C for 10 min.

[0056] Place the sample in a staining jar and protect it from light. Place it on a destaining shaker at 55 rpm and wash with 300 μL of PBS for 3 min each time.

[0057] Preferably, the repeated steps S24-S27 mentioned in step S28 means performing them twice more. After the first dye incubation in step S27, all subsequent steps S27-S210 should be performed in the dark.

[0058] Step S29 is as follows: Place the slide in a humidified chamber, add 200 μL of DAPI to each slide, and incubate at room temperature for 10 min.

[0059] Place the sample in a staining jar, put it on a decolorizing shaker at 55 rpm, wash with 300 μL of PBS for 3 min three times, and finally shake off the excess liquid.

[0060] The beneficial effects of this invention are:

[0061] The method of this invention is simple to operate, has high detection accuracy, and can avoid false negatives, thereby improving the accuracy of melanoma screening. Specifically:

[0062] Simple operation: Multiplex immunofluorescence staining is performed on CTC cells obtained by microfluidics. Classic and specific staining markers (HMB45 melanoma marker combined with CD45 leukocyte marker) are selected for labeling and staining. A uniform standard threshold is set using image analysis software to screen tumor cells that express HMB45+CD45- fluorescence. This ensures the positive expression rate of tumor cells while excluding the interference of leukocytes, further improving the accuracy of detection and eliminating false positives.

[0063] The limitations of traditional monochromatic immunofluorescence (IF) labeling are mainly reflected in the following aspects: firstly, it cannot simultaneously detect multiple target proteins on the same tissue section, limiting the study of complex cellular microenvironments, cell interactions, and spatial relationships; secondly, the interpretation of results is limited to a single signal, and cross-reactivity and non-specific staining interference may occur; furthermore, multiple repeated staining is required to analyze multiple markers, which is time-consuming and easily introduces section differences, and the signal intensity is usually semi-quantitative, lacking the fine quantification capabilities of multiplex methods. These problems all significantly affect the accuracy, reproducibility, and analytical difficulty of the results.

[0064] Multiplex immunofluorescence staining is an advanced tissue analysis method that allows for the simultaneous labeling and detection of multiple (typically eight or more) protein biomarkers on the same tissue section. By using fluorescent probes of different colors to visualize their distribution and interactions within cells, it greatly enhances insights into complex biological phenomena such as the tumor microenvironment. Through the coupling of unique DNA barcodes with antibodies and the use of iterative signal amplification and detection, it overcomes the trade-off between sensitivity and throughput in traditional methods, achieving high-throughput, high-resolution, multi-marker detection, making it an important tool for translational medicine research.

[0065] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Attached Figure Description

[0066] Figure 1 These are multiple immunofluorescence staining images of peripheral blood CTCs from melanoma patients (pictures selected under low magnification).

[0067] Figure 2 This is a schematic diagram (pictured under high magnification) illustrating the relationship between HES1 in melanoma patients and clinical prognosis, based on the present invention. Detailed Implementation

[0068] The following specific embodiments illustrate the detailed implementation of the present invention. Those skilled in the art can easily understand the advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented in other different ways, that is, different modifications and changes can be made without departing from the scope disclosed in the present invention.

[0069] In this context, HMB45+ indicates the detection of the HMB45 antigen in the cell sample. HMB45 is a monoclonal antibody that specifically recognizes antigens related to melanocyte differentiation and is commonly used to diagnose tumors of melanocyte origin. CD45 is a leukocyte common antigen (LCA), a protein expressed on the surface of almost all mature and immature leukocytes. It is a universal marker of immune cells used to distinguish cells of hematopoietic origin and is crucial in the diagnosis and research of blood cancers such as lymphoma and leukemia. Therefore, CD45- indicates non-leukocytes; HES1+ indicates a positive HES1 test; and DAPI+ indicates positive DAPI staining in the cells, which can be used to determine the number of cell nuclei and represent cell viability. DAPI+ is a common representation method in flow cytometry or immunofluorescence detection, used to identify DAPI-positive cells or regions in a sample, i.e., samples where the cell nuclei are effectively stained by DAPI dye.

[0070] Example: A method for detecting HES1 markers in melanoma CTCs using multiplex immunofluorescence technology. This method was used to isolate and identify peripheral blood circulating tumor cells (PCCs) from 15 melanoma patients (divided into 5 cases without remote metastasis and 10 cases with remote metastasis), and to detect their HES1 expression.

[0071] S1. Microfluidic chip devices were used to separate and obtain circulating tumor cells from the peripheral blood of melanoma patients.

[0072] Peripheral blood (5 ml) was collected from each of 15 melanoma patients. Based on the presence or absence of metastasis, the patients were divided into non-metastatic (Non-Mets) and metastatic (Mets) groups. The peripheral blood was rotated and mixed at room temperature for approximately 1 hour. CTC samples were then obtained using a microfluidic sorting instrument. Under a microscope, CTC cells with a diameter of 10-20 μm could be observed, specifically following steps S11 to S19 of the invention description. The sorted cells were then further processed as follows: directly for mIF (multiplex immunofluorescence) staining: The extremely small number of CTC cells obtained through microfluidic sorting, besides being used for in vitro culture to construct cell lines, are the key cellular tool required for this application. The resulting mIF staining step is a method that this application requires special protection for, specifically step S2 below.

[0073] S2. Identify circulating tumor cells in the peripheral blood of melanoma patients using multiplex immunofluorescence techniques.

[0074] The fixed cell suspension was centrifuged to prepare cell slides. Subsequent procedures included: blocking and permeabilization for 20 min, followed by washing three times with PBS; eliminating endogenous peroxidase with 1% H2O2 for 1 h, followed by washing three times with PBS; primary antibody incubation for 30 min, followed by washing three times with PBS; secondary antibody incubation for 20 min, followed by washing three times with PBS; dye incubation for 10 min, followed by washing three times with PBS; antibody elution buffer incubation for 10 min, followed by washing once with PBS. This primary antibody-secondary antibody-dye-antibody stripping process was repeated until the last antibody was used. Finally, DAPI incubation was performed for 10 min, followed by washing three times with PBS. The slides were then mounted with 50 μL of aqueous anti-quenching blocking agent, avoiding light as much as possible. The slides were scanned using a digital pathology slide scanner (KFBIO KF-FL-120). The scanned images were analyzed using pathology analysis software (Pano Analyzer). Detailed steps are described in steps S21 to S212 of the instructions.

[0075] Results: CTCs (HMB45+CD45-) were detected in the peripheral blood of all 15 melanoma patients. This indicates that CTC cells can be reliably isolated and obtained using a microfluidic sorting instrument. Furthermore, this method can be applied to multiplex immunofluorescence staining analysis, laying the foundation for future clinical diagnostic development and applications.

[0076] Figure 1The image shows multiplex immunofluorescence staining images (low magnification) of peripheral blood CTCs from melanoma patients. The patients were divided into two groups: non-metastatic and metastatic. The images show that melanoma cells have large, irregularly shaped, multinucleated nuclei with a high nucleocytoplasmic ratio. This is consistent with the typical characteristics of melanoma cells (HMB45+CD45-), indicating that healthy CTCs can be obtained after microfluidic sorting, which is beneficial for downstream staining analysis.

[0077] Figure 2 The diagram illustrates the relationship between HES1 and clinical prognosis in melanoma patients, as presented in this invention. This project included 15 melanoma patients, divided into non-metastatic (n=5) and metastatic (n=10) groups. The metastatic group was further divided into lymph node metastasis (n=5) and distant metastasis (n=5). mIF staining was performed using HMB45 (melanoma marker, cytoplasmic expression, green), CD45 (leukocyte marker, cell membrane expression, yellow), HES1 (a key detection indicator in this application, nuclear expression, orange), and DAPI (nuclear expression, blue). A complete cell merge map was then generated. See [link to mIF staining]. Figure 1 This detection method assesses the expression of HES1 in blood-derived central cytokines (CTCs) and determines its correlation with patient clinical prognosis. The left-hand r-plot primarily illustrates that HES1 expression in blood CTCs of melanoma patients without distant metastasis is significantly higher (HES1+CTC PT#5, where PT#5 represents patient 5), compared to patients with distant metastasis (HES1-CTC PT#1, where PT#1 represents patient 1). The right-hand S-plot shows the percentage of HMB45+CD45-HES1+ CTCs, indicating that the rate of HES1 positivity in blood CTCs is significantly lower in patients with focal and distant metastases compared to those without metastasis. This demonstrates that HES1 can serve as a staining indicator for assessing patient clinical prognosis, and multiplex immunofluorescence staining allows for precise determination of multiple indicators on a single slide.

[0078] Furthermore, by performing multiplex immunofluorescence histochemical staining on CTCs obtained after blood sorting using microfluidics, we can further analyze the staining results in detail using Pano Analyzer software (such as the pathology analysis software from Pano Biotechnology (Beijing) Co., Ltd.). This method is based on the principle of fluorescence density value analysis. Using this software for analysis enables automated, high-precision, and reproducible quantification, transforming traditional pathological images into structured data for phenotypic colocalization analysis that can be directly used in statistical analysis software, empowering precision medicine and translational research.

[0079] Quantitative analysis was performed using Pano Analyzer software:

[0080] (1) Label the channels: blue for DAPI, green for HMB45, orange for HES1, and yellow for CD45.

[0081] (2) Define the layer outline to delineate the area where cells are distributed;

[0082] (3) Perform tissue segmentation and mark the blue + green cells as tumor areas;

[0083] (4) Perform cell identification, adjust the pixel size to 0.5-1, the precision parameter to 1-1.5, the signal strength to 10, and leave the rest as default;

[0084] (5) Perform phenotypic analysis: Input the "tissue category", "tissue segmentation result" and "cell identification result" of the successful execution, adjust the threshold of each channel to prevent overexposure, set the target phenotype HMB45+ CD45-HES1+ parameters as weak positive, negative and weak positive respectively, and finally generate a configuration file for execution;

[0085] (6) After generating the “Phenotype Results”, you can use the “Result Mask” module in the “Phenotype Analysis” interface to find the specific target cells of HMB45+CD45-HES1+DAPI+.

[0086] Note: HMB45+CD45-HES1+DAPI+ are related by "and", and in biology, they are usually listed together without any breaks.

[0087] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structure made using the contents of the present invention specification and drawings, or directly or indirectly applied to other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for detecting HES1 markers in melanoma CTCs using multiplex immunofluorescence technology, characterized in that: The method includes the following steps: S1. Collect peripheral blood from melanoma patients and sort and enrich CTCs using a microfluidic chip; S2. Nucleated cells in the blood were spread and enriched onto an adhesive glass slide by centrifugation, then fixed and permeable. The selected melanoma CTCs were co-incubated with HES1, HMB45 and CD45 antibodies in sequence using multiplex immunofluorescence technology. The expression of HES1 antibody was further analyzed and the images were analyzed and quantified.

2. The method for detecting HES1 markers in melanoma CTCs using multiplex immunofluorescence technology according to claim 1, characterized in that: Step S1 specifically involves collecting 5-10 mL of blood from melanoma patients, sorting the blood using a microfluidic sorting instrument, and further fixing the melanoma CTCs obtained through microfluidic chip sorting with 4% PFA and washing with PBS.

3. The method for detecting HES1 markers in melanoma CTCs using multiplex immunofluorescence technology according to claim 2, characterized in that: Step S1 specifically includes the following steps: S11. Collect 5-10 mL of venous blood from melanoma patients into EDTA anticoagulated vacuum blood collection tubes; S12. Before blood sample sorting, mix at room temperature at low speed of 50 rpm for 1 hour. S13. Use a microfluidic sorting instrument and follow the corresponding steps to obtain approximately 5 mL of cell suspension after sorting by the microfluidic chip. S14. The cell suspension was centrifuged at 1410 rpm for 5 min at room temperature to obtain cell pellet; S15. The cell pellet was resuspended in 1 mL of PBS and transferred to a 1.5 mL EP tube. The tube was centrifuged at 1410 rpm for 5 min at room temperature and washed twice. S16. The cell pellet was resuspended in 1 mL of 4% PFA and fixed at room temperature for 10 min. S17. Centrifuge the cell suspension at 1410 rpm for 5 min at room temperature to obtain cell pellet. S18. The cell pellet was resuspended in 1 mL of PBS, centrifuged at 1410 rpm for 5 min at room temperature, and washed twice. S19. Finally, resuspend in 1 mL PBS and store briefly at 4°C for 1-2 weeks.

4. The method for detecting HES1 markers in melanoma CTCs using multiplex immunofluorescence technology according to claim 1, characterized in that: Step S2 specifically involves: using a cell centrifuge to prepare slides of the cells obtained in step S1, followed by a blocking and permeabilization step, and then using 1% H2O2 to block endogenous peroxidase. After that, the primary antibody, secondary antibody, and dye incubation steps are performed in sequence. If multiple targets are required, the primary antibody, secondary antibody, and dye incubation steps can be repeated after elution with antibody elution buffer. Finally, the cell nuclei are stained and the slides are mounted.

5. The method for detecting HES1 markers in melanoma CTCs using multiplex immunofluorescence technology according to claim 1 or 4, characterized in that: Step S2 specifically includes the following steps: S21. Take an appropriate amount of cell suspension and centrifuge at 1000 rpm for 10 min at room temperature using a cell smear centrifuge to prepare cell smears. S22. Use an immunohistochemical pen to delineate the cell range, add 300 μL of blocking-permeability mixture and treat for 20 min, then wash 3 times with 300 μL of PBS. S23. Treat with 200 μL of 1% H2O2 for 1 h, then wash 3 times with 300 μL of PBS; S24. Incubate with 200 μL of primary antibody at room temperature for 30 min, then wash three times with 300 μL of PBS; S25. Incubate with 200 μL of secondary antibody at room temperature for 20 min, then wash three times with 300 μL of PBS. S26. Incubate with 200 μL of dye at room temperature for 10 min, then wash three times with 300 μL of PBS. S27. Incubate with 200 μL of antibody elution buffer at 37°C for 10 min, then wash once with 300 μL of PBS. S28. Repeat steps S24-S27 until the last antibody has finished incubating; S29. Incubate with 200 μL of DAPI at room temperature for 10 min, then wash three times with 300 μL of PBS. S210, Seal the sheet with 50 μL of anti-quenching sealant; S211. Use a digital pathology slide scanner to scan slides; S212. Analyze the obtained scan results using pathological analysis software to determine whether the cells conform to the CTC phenotype and to statistically analyze the positive and negative expression of HES1.

6. The method for detecting HES1 markers in melanoma CTCs using multiplex immunofluorescence technology according to claim 5, characterized in that: The blocking-permeability mixture in step S22 is a mixture of 1% BSA and 0.25% Triton X-100.

7. The method for detecting HES1 markers in melanoma CTCs using multiplex immunofluorescence technology according to claim 5, characterized in that: The repetition of steps S24-S27 mentioned in step S28 means performing the steps twice more.

8. The method for detecting HES1 markers in melanoma CTCs using multiplex immunofluorescence technology according to claim 7, characterized in that: In step S24, the primary antibody used for the first incubation was Mouse anti-Human Melanosome antibody, diluted 1:250 with 1% BSA as the blocking buffer; the primary antibody used for the second incubation was Mouse anti-Human CD45 antibody, diluted 1:1000 with 1% BSA as the blocking buffer; and the primary antibody used for the third incubation was Rabbit anti-Human HES1 Rabbit Monoclonal Antibody, diluted 1:4000 with 1% BSA as the blocking buffer.

9. The method for detecting HES1 markers in melanoma CTCs using multiplex immunofluorescence technology according to claim 7, characterized in that: In step S25, the secondary antibody incubation step, HRP-labeled goat anti-rabbit mixed secondary antibody was used three times without dilution.

10. The method for detecting HES1 markers in melanoma CTCs using multiplex immunofluorescence technology according to claim 5, characterized in that: In step S24, the primary antibody incubation step, primary antibody HES1 is incubated overnight at 4°C, and primary antibodies HMB45 and CD45 are incubated at room temperature for 30 minutes.

11. The method for detecting HES1 markers in melanoma CTCs using multiplex immunofluorescence technology according to claim 7, characterized in that: In step S27, the dye incubation step, the first incubation uses dye 520 to label the primary antibody Mouse anti Human Melanosome antibody, and the dye is diluted 1:200 with signal amplification buffer; the second incubation uses dye 650 to label the primary antibody Mouse anti Human CD45 antibody, and the dye is diluted 1:200 with signal amplification buffer; the third incubation uses dye 570 to label the primary antibody Rabbit anti Human HES1 Rabbit Monoclonal Antibody, and the dye is diluted 1:200 with signal amplification buffer.

12. The method for detecting HES1 markers in melanoma CTCs using multiplex immunofluorescence technology according to claim 5, characterized in that: In step S212 software analysis, when the immunofluorescence expression index of cells meets the condition of HMB45+ / CD45- / DAPI+, it can be identified as melanoma CTC. Further, based on this, the expression ratio of HES1 positive expression CTC and HES1 negative expression CTC is statistically analyzed and matched with the tumor metastasis of melanoma patients to analyze whether the two are correlated.

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