Biomarker for predicting skin melanoma metastasis and application thereof

Abstract

The invention discloses a biomarker for predicting skin melanoma metastasis and application thereof, and relates to the technical field of biological medicine. The biomarkers are mitochondrial dysfunction related proteins in macrophages in skin melanoma tissues and comprise GLUL and C3, the GLUL and the C3 are taken as the biomarkers, and the expression level of the GLUL and the C3 in the macrophages is detected through immunohistochemical staining, so that the metastasis prediction of the skin melanoma is realized. In the dyeing process, the macrophages are marked with CD163, then GLUL and C3 are specifically detected, an AEC red color developing agent is adopted, and a detection sample is a paraffin section of a primary focus and / or a metastatic focus of an SKCM patient. According to the invention, GLUL and C3 are determined as metastasis prediction biomarkers for the first time, and a specific immunohistochemical detection scheme is matched, so that double values of accurate prediction of metastasis risk and treatment guidance are realized, and a new technical path is provided for prognosis evaluation and individualized treatment of skin melanoma.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a biomarker for predicting the metastasis of cutaneous melanoma and its application. Background Technology

[0002] Skin cutaneous melanoma (SKCM) is a highly malignant tumor originating from epidermal melanocytes, characterized by its high malignancy, aggressiveness, high mortality, and poor prognosis. In clinical treatment, SKCM readily develops primary or acquired drug resistance, significantly limiting the efficacy of existing treatments and becoming a core bottleneck in clinical efforts to overcome this disease. Therefore, in-depth exploration of the potential molecular mechanisms of SKCM metastasis and the identification of clinically valuable therapeutic targets are of great significance and urgent importance for improving the prognosis of SKCM patients and overcoming the treatment challenges.

[0003] Existing research indicates that mitochondrial dysfunction is closely related to the occurrence, development, and metastasis of various malignant tumors, including melanoma, lung cancer, glioblastoma, neuroblastoma, and ovarian cancer. In the pathological progression of SKCM, the mitochondrial stress response and metabolic reprogramming of melanoma cells can promote tumor cell proliferation, invasion, metastasis, and resistance to targeted therapy by regulating energy supply and oxidative stress levels. Simultaneously, abnormal changes in the mitochondrial functional state of macrophages, the most abundant immune cells in the SKCM tumor microenvironment (TME), can also indirectly affect SKCM progression by regulating immune microenvironment homeostasis, suggesting that mitochondrial-related pathways may be a key link in the pathological regulation of SKCM.

[0004] Glutamate-ammonia ligase (GLUL), also known as glutamine synthetase, is a key metabolic enzyme that catalyzes the synthesis of glutamine from glutamate and ammonia in a ATP-dependent manner. Glutamine, as a core precursor in the mitochondrial tricarboxylic acid cycle and various biosynthetic reactions, provides ample energy and nutrient substrates for the rapid proliferation of tumor cells and is a crucial regulatory node in tumor metabolic reprogramming. Studies have confirmed that GLUL is abnormally highly expressed in various malignant tumors, including liver cancer, lung cancer, breast cancer, pancreatic cancer, ovarian cancer, and colon cancer, and promotes tumor progression by regulating the glutamine metabolic pathway. However, its role in SKCM and its association with mitochondrial dysfunction remain unclear.

[0005] Complement 3 (C3), a β2-globulin synthesized by the liver, is a core component of the complement system and is widely involved in the activation of both the classical and alternative complement pathways. Recent studies have found that complement-derived effector molecules can participate in tumor pathological processes by regulating multiple aspects, including tumor cell anchoring and proliferation, tumor-associated angiogenesis, matrix remodeling, cell migration, and invasion and metastasis. Evidence suggests that C3 is upregulated in colorectal cancer, gastric cancer, and squamous cell carcinoma of the skin, and can accelerate tumor growth and metastasis. Furthermore, research indicates that C3 may participate in the regulation of the mitochondrial electron transport chain and play a role in cellular oxidative phosphorylation; however, its association with SKCM and macrophage mitochondrial dysfunction remains to be elucidated.

[0006] Currently, research on the correlation between mitochondrial dysfunction-related proteins (such as GLUL and C3) in macrophages and the development of SKCM remains scarce. Existing reports are mostly limited to bioinformatics analysis, lacking solid experimental data to further verify and elucidate their regulatory mechanisms, interactions, and clinical significance. Therefore, in-depth exploration of the role and mechanism of mitochondrial dysfunction mediated by proteins such as GLUL and C3 in macrophages in SKCM can provide a theoretical basis for establishing biomarkers for predicting melanoma metastasis and for discovering new therapeutic targets for SKCM, possessing significant scientific research value and clinical application prospects. Summary of the Invention

[0007] To address the problems mentioned in the background section, this invention provides a biomarker for predicting the metastasis of cutaneous melanoma and its application.

[0008] To achieve the above objectives, the present invention provides the following technical solution: biomarkers for predicting the metastasis of cutaneous melanoma, wherein the biomarkers are mitochondrial dysfunction-related proteins, including GLUL and C3. Specifically, these are mitochondrial dysfunction-related proteins within macrophages in cutaneous melanoma tissue.

[0009] This invention also claims protection for the use of the biomarkers described above in the preparation of biological products for predicting the metastasis of cutaneous melanoma.

[0010] Furthermore, the biological products include reagents, reagent kits, or chips.

[0011] Furthermore, the prediction process includes using GLUL and C3 as biomarkers to predict the metastasis of cutaneous melanoma by detecting the expression levels of GLUL and C3 in macrophages.

[0012] Furthermore, the metastasis prediction refers to the upregulation of GLUL and C3 expression (relative to the upregulation of corresponding protein expression in normal skin tissue macrophages), indicating a risk of metastasis in cutaneous melanoma.

[0013] Furthermore, the expression levels of GLUL and C3 in the biopharmaceutical for predicting melanoma metastasis were detected by immunohistochemical staining.

[0014] Furthermore, in the immunohistochemical staining, macrophages are first labeled using CD163 immunohistochemical staining, and then GLUL and C3 are specifically stained using immunohistochemical staining. The specific antibodies are labeled with a chromogenic agent to reveal the antigen-antibody binding sites, and the expression levels of GLUL and C3 in the sample are analyzed to target the expression of GLUL and C3 within macrophages in skin melanoma tissue. This includes qualitative, localization, and quantitative analysis.

[0015] Furthermore, in the immunohistochemical staining, the object of detection and analysis is paraffin section samples of primary and / or metastatic lesions of cutaneous melanoma.

[0016] Furthermore, the colorimetric agent is AEC red colorimetric agent.

[0017] This invention also claims protection for the use of the biomarkers described above in the preparation of therapeutic drugs for combating melanoma metastasis.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention clarifies that GLUL and C3 are mitochondrial dysfunction-related proteins used for SKCM metastasis prediction, and focuses on the detection of target proteins in macrophages, which makes up for the current lack of objective indicators of high SKCM metastasis risk. It is highly targeted and can accurately distinguish between metastasis risk and non-metastasis risk samples, and greatly reduces the impact of interference factors on the prediction results.

[0019] (2) This invention uses immunohistochemical staining technology, combined with the CD163 labeling macrophage targeting strategy and AEC red chromogenic agent. The operation process is standardized and easy to repeat. The test sample is a routinely stored paraffin section in clinical practice, which is easy to obtain. The operation is simple and low-cost, and it is suitable for large-scale clinical screening and prognostic assessment scenarios.

[0020] (3) This invention anchors the mechanism of macrophage mitochondrial dysfunction. By combining the detection of GLUL and C3, it can not only provide more accurate early warning of metastasis risk compared to single detection markers, but also reveal the functional status of macrophages in the tumor microenvironment. This provides a basis for the development of individualized immunotherapy plans that target macrophage regulation, and realizes integrated guidance of "prediction-treatment". Attached Figure Description

[0021] Figure 1 GLUL score in SKCM cytoplasmic staining.

[0022] Figure 2 :C3 cytoplasmic staining score in SKCM.

[0023] Figure 3 Expression of CD163 and GLUL in the same field of vision of the SKCM organization.

[0024] Figure 4 Expression of CD163 and C3 in the same field of view of the SKCM organization.

[0025] Figure 5 Expression of GLUL in macrophages of primary and recurrent metastatic lesions of SKCM.

[0026] Figure 6 Expression of C3 in macrophages of primary and recurrent metastatic lesions of SKCM.

[0027] Figure 7 KM curves showing the relationship between GLUL and DFS in SKCM patients.

[0028] Figure 8 KM curves showing the relationship between C3 and DFS in SKCM patients. Detailed Implementation

[0029] The following section, with specific implementation methods, elaborates in detail on biomarkers for predicting the metastasis of cutaneous melanoma and their applications.

[0030] Immunohistochemistry was used to detect the expression of GLUL, C3, and CD163 in macrophages within the primary SKCM lesion and in the tumor infiltration front.

[0031] Experimental materials I. Paraffin-embedded tissue specimens from SKCM patients Clinical data, primary lesions, and paraffin-embedded specimens of recurrent and metastatic lesions were collected from 62 patients diagnosed with SKCM at the Department of Pathology, Second Affiliated Hospital of Dalian Medical University from January 2018 to December 2023.

[0032] A total of 62 cases were included, with 3 patients having both primary and metastatic lesions. A total of 43 primary lesion specimens and 22 recurrent and metastatic lesion specimens were collected (including 3 recurrent specimens, 3 specimens from other skin sites, 9 lymph node metastases, 3 liver metastases, 1 lung metastases, and 3 brain metastases). Clinical information (gender, age, follow-up status, etc.) and clinicopathological characteristics (tumor location, histological type, presence or absence of ulceration, presence or absence of metastasis, Breslow thickness, Clark grade, T stage, degree of lymphocyte infiltration, mitotic count, etc.) were collected from all patients. The study was approved by the Ethics Committee of the Second Affiliated Hospital of Dalian Medical University, and all patients signed informed consent forms.

[0033] Inclusion criteria: (1) Patients diagnosed with primary and recurrent metastatic SKCM by histopathological examination; (2) Patients with complete clinical and follow-up data; (3) Patients with well-preserved paraffin specimens after surgery.

[0034] Exclusion criteria: (1) melanoma occurring in non-skin sites; (2) patients with other tumors or unknown primary lesions; (3) patients with insufficient or missing paraffin specimens.

[0035] II. Main instruments and experimental reagents Table 1 Major Instruments and Manufacturers

[0036] Table 2 Main Reagents and Manufacturers

[0037] The working concentration of GLUL antibody was diluted 1:100, the working concentration of C3 antibody was diluted 1:500, and the CD163 antibody was a ready-to-use reagent.

[0038] III. Experimental Procedure Immunohistochemical staining uses antibodies to detect antigens in cells or tissues and uses chromogenic agents to label the antibodies to reveal the sites of antigen-antibody binding, thereby enabling qualitative, local, or quantitative studies of the expression of the protein under study.

[0039] Experimental steps: (1) Paraffin-embedded tissue sections: After the tissue has been embedded in paraffin, it is placed on a tissue section freezer for 15 minutes in advance, cut into 2μm tissue sections, spread in a constant temperature slide spreader (46℃), and then picked up with a glass slide to prevent detachment and placed in a slide rack.

[0040] (2) Baking the slices at a constant temperature: Place the paraffin slices in a constant temperature oven and bake for 1.5 hours (85℃).

[0041] (3) Dewaxing treatment: After the paraffin slices are taken out of the oven, they are placed in xylene tanks 1, 2 and 3 in sequence for dewaxing, 10 minutes in each tank.

[0042] (4) Hydration treatment: Place the completely dewaxed slices in a gradient of 100%, 95%, 90%, 85%, 80%, and 75% alcohol in a tank for 2 minutes in each tank, and then rinse with running water for 3 minutes.

[0043] (5) Place the hydrated slices into a gel basket, and then put them into PBS solution in tanks 1, 2 and 3 for 5 minutes each.

[0044] (6) Prepare fresh antigen retrieval solution: Dilute the pH 9.0 EDTA retrieval solution 50 times with distilled water for later use. Prepare 1L of pH 6.0 citrate retrieval solution (3.2g / bag) with distilled water for later use.

[0045] (7) Antigen retrieval: Place the prepared pH 9.0 EDTA antigen retrieval solution (for C3 and CD163) / pH 6.0 citrate retrieval solution (for GLUL) in a pressure cooker, completely immerse the slides in the retrieval solution, tighten the pressure cooker lid, turn on the induction cooker switch, adjust to 2000W, and when the pressure cooker valve starts to rotate and release steam, reduce the 2000W to 1600W and start timing for 2 minutes and 30 seconds. After timing is complete, turn off the induction cooker, wait for the antigen retrieval solution to cool to room temperature, turn on the induction cooker, take out the slides and place them in PBS solution tank 1 and tank 2 for washing, 3 minutes in each tank.

[0046] (8) Prepare a 3% hydrogen peroxide solution: Dilute the 30% hydrogen peroxide solution with distilled water by 10 times to prepare a 3% hydrogen peroxide solution for later use.

[0047] (9) Inactivation of endogenous peroxidase: Place the cleaned slices in a 3% hydrogen peroxide solution and incubate at room temperature for 10 minutes. Then rinse with distilled water for 30 seconds and wash with PBS 3 times for 2 minutes each time.

[0048] (10) Staining

[0049] ① Primary antibody incubation: Use an immunohistochemical pen to draw circles at a distance of 2-3 mm from the tissue, then wash the slides 3 times with PBS solution for 2 minutes each time. After shaking off the liquid on the slides, place them in a humidified chamber. Place an appropriate amount of PBS solution at the bottom of the chamber in advance, and add the prepared primary antibody to the tissue slides to completely cover the tissue. Place the humidified chamber in a refrigerator at 4°C for overnight incubation.

[0050] ② Secondary antibody incubation: The next day, remove the humidified chamber from the 4℃ refrigerator and allow it to warm to room temperature for 30 minutes. Discard the primary antibody and wash the slides three times with PBS for 2 minutes each time. Add an appropriate amount of 1gG polymer of goat anti-mouse / rabbit enzyme-labeled polymer and incubate in a 37℃ water bath for 30 minutes. After incubation, wash the slides three times with PBS for 2 minutes each time, and then spin dry after thorough washing.

[0051] ③Staining: 5 minutes before staining, prepare the AEC staining working solution (add 50 μl of 1% AEC dimethyl sulfoxide solution (Solution A), 50 μl of 0.1 mol / L acetate-sodium acetate buffer solution (pH 5.0), and 50 μl of 0.3% H2O2 solution (Solution B), and mix well to prepare 1 ml of AEC staining agent) to 800 μl of distilled water, drop the solution onto the tissue section, place the section under a microscope to observe the staining intensity, and stop the staining with tap water when the staining is optimal (about 5 minutes), and rinse the section with running water for 3 minutes.

[0052] ④ Counterstaining: Place the cleaned sections in hematoxylin for 30 seconds, then rinse with running water for 3 minutes.

[0053] ⑤ Blueing: Place the slice in the blueing solution for 1 minute and 30 seconds, then rinse with running water for 3 minutes.

[0054] (11) Mounting: Place the slide in a constant temperature oven to dry for 5 minutes, add an appropriate amount of GVA glyceryl vinyl alcohol water-soluble mounting medium, and cover with a coverslip.

[0055] IV. Criteria for Interpreting Immunohistochemical Detection Results Immunohistochemical results were interpreted by two experienced pathologists. Because SKCM tissue contains a high amount of pigment, it is difficult to distinguish positive staining from pigment when using DAB chromogenic agent. Therefore, this experiment used AEC red chromogenic agent, with positive cell cytoplasmic staining ranging from light red to red and reddish-brown.

[0056] (1) GLUL interpretation criteria Immunohistochemical staining was assessed using the IRS (Immunoreactive Score), as follows: Staining intensity was determined by the presence of pale red, red, or reddish-brown granules in the cytoplasm, and was graded into four levels: 0 points: no staining; 1 point: pale red; 2 points: red; 3 points: reddish-brown. Figure 1(AD). In each slide, positive cell counts were performed on the stromal cells within the tumor and at the tumor infiltration front. The percentage was divided into four intervals: <10% for positive cells per field of view (1 point); 11%-50% (2 points); 51%-80% (3 points); >80% (4 points). The scoring formula was: IRS = staining intensity × percentage of positive cells, with a score range of 0-12. <4 points indicated low expression, and ≥4 points indicated high expression. Each field of view was scored in three high-power fields (×400), and the average score was the final score.

[0057] GLUL cytoplasmic staining score in SKCM as follows Figure 1 As shown, A: No positive cells observed (0 points) (10×40). B: Positive cells are light red (1 point) (10×40). C: Positive cells are red (2 points) (10×40). D: Positive cells are reddish-brown (3 points) (10×40). The arrows indicate positive cells.

[0058] (2) C3 interpretation criteria Immunohistochemical staining was assessed using the H-score (Histochemistry score), as follows: Staining intensity was determined by the presence of pale red, red, or reddish-brown granules in the cytoplasm, and was graded into four levels: 0 points - negative (no staining); 1 point - weakly positive (pale red); 2 points - moderately positive (red); 3 points - strongly positive (reddish-brown). Figure 2 (AD). In each slide, positive cells were counted within the tumor and at the tumor infiltration front, and the percentage of positive cells in each staining intensity region was calculated. The scoring formula was: H-score = 0 × (percentage of negative cells) + 1 × (percentage of weakly positive cells) + 2 × (percentage of moderately positive cells) + 3 × (percentage of strongly positive cells), with a score range of 0-300. ≤100 points indicated low expression, and >100 points indicated high expression. Each field of view (×400) was scored separately in three high-power fields, and the average score was the final score.

[0059] C3 cytoplasmic staining score in SKCM as follows: Figure 2 As shown, A: No positive cells observed (0 points) (10x40). B: Positive cells are light red (1 point) (10x40). C: Positive cells are red (2 points) (10x40). D: Positive cells are reddish-brown (3 points) (10x40). The arrows indicate positive cells.

[0060] (3) Criteria for interpreting CD163 Immunohistochemical scoring was performed using the positive cell counting method, as follows: Five fields of view were randomly selected under a 400x light microscope, and the number of positively stained cells in each field of view was counted, and the average number of positive cells was calculated.

[0061] All experimental data in this study were statistically analyzed using SPSS 26.0 software. Spearman's rank correlation was used for correlation analysis. Chi-square test was used for ordinal data. If the chi-square test conditions were not met, the continuity correction test or Fisher's exact test was used. P < 0.05 was considered statistically significant.

[0062] V. Immunohistochemical Detection Results (1) Clinical data of SKCM patients A retrospective study screened and collected 62 SKCM patients diagnosed at the Second Affiliated Hospital of Dalian Medical University from January 2018 to December 2023. Three patients had both primary lesions and metastatic lesions. Among them, primary lesion specimens were collected from 43 cases and recurrent metastatic lesion specimens were collected from 22 cases (see Tables 3 and 4).

[0063] Table 3. General information of 43 patients with primary SKCM

[0064] SSM, superficial diffuse melanoma; ALM, acral lentiginesoid melanoma; NM, nodular melanoma.

[0065] Table 4 General information of 22 patients with recurrent and metastatic SKCM

[0066] (2) The relationship between GLUL, C3 and CD163 expression in SKCM tissues Since this invention studies the expression of GLUL and C3 in macrophages within the stroma of SKCM tumors, macrophages were labeled using CD163 immunohistochemical staining. Simultaneously, GLUL and C3 immunohistochemical staining was performed, and GLUL and C3 positive cells were observed microscopically within the tumor and in the stroma at the tumor infiltration front. It was found that in the same field of view of the same SKCM tissue, CD163-labeled macrophages expressed GLUL and C3 to varying degrees. Figure 3 and Figure 4 ), Figure 3 In Figure A: SKCM and CD163 expression in the tissue; the arrow indicates CD163-positive cells (10×40). In Figure B: GLUL expression in the same field of view as Figure A; the arrow indicates GLUL-positive cells (10×40). Figure 4 In Figure A: CD163 expression in SKCM tissue, with arrows indicating CD163-positive cells (10×40). In Figure B: C3 expression in the same field of view as Figure A, with arrows indicating C3-positive cells (10×40).

[0067] Spearman correlation analysis was used to analyze the relationship between the expression of GLUL, C3 and CD163 in the primary lesions and recurrent metastatic lesions of SKCM patients. The results showed that GLUL expression was positively correlated with CD163 expression (RS=0.448, P<0.001), as shown in Table 5; C3 expression was positively correlated with CD163 expression (RS=0.325, P=0.008), as shown in Table 6.

[0068] Table 5. Relationship between GLUL expression and CD163 expression in SKCM tissues.

[0069] * This means P < 0.05.

[0070] Table 6. Relationship between C3 expression and CD163 expression in SKCM tissues.

[0071] * This means P < 0.05.

[0072] (3) Expression of GLUL and C3 in macrophages of primary and recurrent metastatic SKCM lesions

[0073] Immunohistochemical staining was used to detect the expression of GLUL in macrophages from the primary lesions of 43 SKCM patients and the recurrent metastatic lesions of 22 SKCM patients. The results showed that the expression level of GLUL in macrophages from the recurrent metastatic lesions was significantly higher than that from the primary lesions (P=0.025), as shown in Table 7. Figure 5 . Figure 5 In the diagram, A: GLUL is expressed at low levels (10×40) in the primary lesion of SKCM patients. B: GLUL is expressed at high levels (10×40) in recurrent metastatic lesions of SKCM patients.

[0074] Table 7. Expression of GLUL in macrophages of primary and recurrent metastatic SKCM lesions

[0075] * This means P < 0.05.

[0076] Immunohistochemical staining was used to detect the expression of C3 in macrophages from the primary and recurrent metastatic lesions of SKCM patients. The results showed that the expression level of C3 in macrophages from recurrent metastatic lesions was significantly higher than that from the primary lesion (P=0.022), as shown in Table 8. Figure 6 . Figure 6 In the diagram, A: C3 was expressed at low levels (10×40) in the primary lesion of SKCM patients. B: C3 was expressed at high levels (10×40) in the recurrent metastatic lesions of SKCM patients.

[0077] Table 8. C3 expression in macrophages of primary and recurrent metastatic SKCM lesions.

[0078] * This means P < 0.05.

[0079] (4) Correlation between GLUL and C3 expression in macrophages of primary and recurrent metastatic SKCM lesions

[0080] The relationship between GLUL and C3 expression in macrophages of primary and recurrent metastatic lesions of SKCM patients was analyzed using Spearman correlation analysis. The results showed that GLUL expression was positively correlated with C3 expression (RS=0.407, P=0.001), as shown in Table 9.

[0081] Table 9. Relationship between GLUL and C3 expression in macrophages of primary and recurrent metastatic SKCM lesions. RS stands for Spearman correlation coefficient; * This means P < 0.05.

[0082] (5) Relationship between GLUL and C3 expression and clinicopathological parameters in SKCM patients The chi-square test was used to analyze the relationship between GLUL expression in macrophages of the primary lesion of 43 SKCM patients and various clinicopathological parameters. The results showed that GLUL expression was not correlated with gender, age, tumor location, histopathological type, presence or absence of ulceration, Breslow thickness, Clark grade, degree of lymphocyte infiltration, nuclear mitotic count, presence or absence of metastasis, or T stage of SKCM patients (P>0.05), as shown in Table 10.

[0083] Table 10 Relationship between GLUL expression in macrophages and clinicopathological features in 43 cases of primary SKCM lesions

[0084] The chi-square test was used to analyze the relationship between C3 expression in macrophages of primary lesions in 43 SKCM patients and various clinicopathological parameters. Comparison of primary lesion specimens from patients with and without metastasis revealed that C3 expression was significantly higher in macrophages from metastatic primary lesions (P=0.043). C3 expression was not correlated with gender, age, tumor location, histological type, presence or absence of ulceration, Breslow thickness, Clark grade, T stage, degree of lymphocyte infiltration, or mitotic count in SKCM patients (P>0.05) (Table 11).

[0085] Table 11 Relationship between C3 expression in macrophages and clinicopathological features in 43 cases of primary SKCM lesions

[0086] * This means P < 0.05.

[0087] (6) Relationship between GLUL and C3 expression in SKCM macrophages and disease-free survival (DFS) in patients Forty-three patients with primary SKCM lesions were followed up by telephone. By the final follow-up date, 3 patients experienced relapse, 7 metastases, and 10 deaths. Survival analysis was performed using the Karl von Meckel (KM) method to obtain survival curves. Log-rank comparisons of survival rates showed that patients with high expression of GLUL and C3 on macrophages had significantly lower disease-free survival (DFS) than those with low expression (P=0.034, P=0.032). Figure 7 and Figure 8 As shown.

[0088] The above research shows that: (1) GLUL and C3 are highly expressed in macrophages of SKCM. (2) High expression of GLUL and C3 in macrophages is positively correlated with SKCM metastasis. (3) High expression of GLUL and C3 in macrophages is associated with shorter DFS (disease-free survival) in SKCM patients.

[0089] GLUL is expressed in the cytoplasm of SKCM tumor stromal cells, but is not expressed or is weakly expressed in melanoma cells. This invention adds immunohistochemical staining for CD163, a marker of M2 macrophages, to the case studies. Spearman's rank correlation method was used to analyze the correlation between GLUL and CD163 in the same field of view of the same tissue. The results showed a strong positive correlation between GLUL and CD163 (RS=0.448, P<0.001), suggesting that immunohistochemical scoring of GLUL-positive cells in the tumor stromal can represent the expression status of GLUL in M2 macrophages. The expression level of GLUL in macrophages from SKCM relapse and metastasis lesions was significantly higher than that in the primary lesion, with a statistically significant difference (P=0.025). The proportion of macrophages with high GLUL expression in patients with metastatic SKCM was greater than that in patients without metastasis (40.0% vs 22.9%), although the P-value was 0.076, which did not reach a statistically significant difference, but the difference showed a certain trend. The above findings suggest that GLUL, a protein associated with mitochondrial dysfunction in macrophages, may be involved in the regulation of SKCM metastasis, and high expression of GLUL in macrophages may promote SKCM metastasis.

[0090] Furthermore, the Kaplan-Meier (KM) survival analysis of DFS in SKCM patients showed that patients with high GLUL expression had lower DFS than those with low expression, and the difference was statistically significant (P=0.034), suggesting that GLUL can serve as a potential biomarker for SKCM prognosis monitoring.

[0091] Furthermore, C3 is expressed in the cytoplasm of SKCM tumor stromal cells, but not expressed or weakly expressed in melanoma cells. In the same field of view within the same tissue, Spearman's rank correlation method was used to analyze the correlation between C3 and CD163, showing a strong positive correlation (RS=0.325, P=0.008). Therefore, immunohistochemical scoring of C3-positive cells in the tumor stromal can represent the expression status of C3 in M2 macrophages. Chi-square test revealed that the expression level of C3 in macrophages from SKCM recurrence and metastasis lesions was higher than that in primary lesions, and the expression level in macrophages of metastatic SKCM patients was higher than that in patients without metastasis, with statistically significant differences (P=0.043). These results suggest that C3, a mitochondrial dysfunction-related protein in macrophages, may be involved in the regulation of SKCM metastasis, and high expression of C3 in macrophages may promote SKCM metastasis. KM survival analysis of disease-free survival (DFS) in SKCM patients showed that patients with high C3 expression had lower DFS than those with low C3 expression, and the difference was statistically significant (P=0.032).

[0092] C3 has high prognostic value in SKCM, and targeting C3, a protein associated with mitochondrial dysfunction in macrophages, may provide a potentially effective treatment for patients with metastatic SKCM. Furthermore, this invention demonstrates a positive correlation between GLUL expression and C3 expression (RS=0.407, P=0.001), suggesting that these two proteins may play a synergistic role in the SKCM metastasis pathway.

[0093] The above experimental results provide solid data support for GLUL and C3 as biomarkers for predicting melanoma metastasis, confirming the scientific validity and feasibility of the application scheme of this invention.

[0094] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A biomarker for predicting the metastasis of cutaneous melanoma, characterized in that: The biomarkers are mitochondrial dysfunction-related proteins, including GLUL and C3.

2. The use of the biomarker as described in claim 1 in the preparation of a biological product for predicting the metastasis of cutaneous melanoma.

3. The application as described in claim 2, characterized in that: The biological products include reagents, reagent kits, or chips.

4. The application as described in claim 2, characterized in that: The prediction process includes using GLUL and C3 as biomarkers to predict the metastasis of cutaneous melanoma by detecting the expression levels of GLUL and C3 in macrophages.

5. The application as described in claim 2, characterized in that: The metastasis prediction refers to the upregulation of GLUL and C3 expression, indicating a risk of metastasis in cutaneous melanoma.

6. The application as described in claim 2, characterized in that: The biopharmaceutical for predicting melanoma metastasis was analyzed by immunohistochemical staining to detect the expression levels of GLUL and C3.

7. The application as described in claim 6, characterized in that: In the immunohistochemical staining, macrophages are first marked by CD163 immunohistochemical staining, and then GLUL and C3 are specifically immunohistochemically stained. The specific antibodies are labeled with a chromogenic agent to show the antigen-antibody binding site. The expression levels of GLUL and C3 in the sample are then analyzed to target the expression of GLUL and C3 in macrophages in skin melanoma tissue.

8. The application as described in claim 7, characterized in that: In the immunohistochemical staining, the objects of detection and analysis are paraffin sections of primary and / or metastatic lesions of cutaneous melanoma.

9. The application as described in claim 7, characterized in that: The colorimetric reagent is AEC red colorimetric reagent.

10. The use of the biomarker as described in claim 1 in the preparation of a therapeutic agent for combating melanoma metastasis.

Images