A kit for predicting the risk of recurrence and metastasis of a cancer patient after surgery

Abstract

The application discloses a kit for predicting the risk of recurrence and metastasis of a cancer patient after operation, and discloses that the lactylation modification of Vimentin at the 373th lysine (VIM K373la) is significantly up-regulated in metastatic renal cancer and is significantly negatively correlated with the recurrence-free survival of a patient. The kit provided by the application comprises rabbit-derived antibodies specifically recognizing VIM K373la and immunohistochemical detection reagents, and the expression level of VIM K373la in tumor tissue is quantitatively evaluated by an H-score scoring system. Experiments show that the recurrence and metastasis risk of the high-expression group of VIM K373la is 12.15 times that of the low-expression group, the area under the ROC curve reaches 0.8654, and is significantly better than that of a traditional marker total Vimentin. The kit of the application is simple to operate, has superior prediction performance, can guide individualized treatment, and also has prediction value for breast cancer metastasis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical detection technology, specifically relating to a detection kit that uses the lactylation modification of lysine at position 373 of Vimentin protein (VIM K373la) as a biomarker to predict the risk of postoperative recurrence and metastasis in cancer patients (especially kidney cancer), and belongs to the field of tumor prognostic diagnosis technology. Background Technology

[0002] Renal cell carcinoma (RCC) is one of the most common malignant tumors of the urinary system. Although surgical resection is the preferred treatment for localized renal cell carcinoma, some patients still experience local recurrence or distant metastasis after surgery. Currently, clinical prognosis is mainly assessed using indicators such as TNM staging and Fuhrman classification, but their predictive accuracy is limited, making it difficult to achieve individualized early warning of recurrence and metastasis. Therefore, the development of highly sensitive and specific biomarkers is of significant clinical value in guiding postoperative adjuvant therapy and follow-up strategies.

[0003] Post-translational modifications play a crucial role in tumor progression. Lactylation, a newly discovered acylation modification mediated by lactate, is involved in regulating gene expression and cellular metabolism. Studies have shown that lactate accumulation can induce protein lactylation, promoting epithelial-mesenchymal transition (EMT) and migration in tumor cells. Vimentin, as an intermediate filament protein, is a core marker of EMT, and its post-translational modifications are essential for cytoskeleton remodeling and migration.

[0004] Although current research has confirmed that Vimentin can promote the proliferation, migration, adhesion, invasion and apoptosis of various tumor cells, its expression is upregulated in tumor tissues such as gastrointestinal tumors, lung cancer, breast cancer, prostate cancer and kidney cancer, and is closely related to the clinical characteristics of patients, affecting their prognosis. It is a clinical biological marker of various tumors (Zhang Yong, Yin Duo. Research progress of vimentin in gynecological malignant tumors [J]. Tumor Research and Clinic, 2018, 30(3):5.).

[0005] The detection of Vimentin primarily employs antibodies capable of recognizing total Vimentin protein, i.e., the TotalVimentin assay. This type of assay reflects the overall expression level of Vimentin in tissues and has been widely used in pathological studies and related detection of tumor tissues. Patent application CN117430700A discloses an antibody or its antigen-binding fragment against vimentin, a kit, and its applications. The antibody or its antigen-binding fragment provided by this invention can specifically bind to vimentin and has good binding activity and affinity; therefore, it can be used for the detection, prognosis, and recurrence monitoring of Vimentin-related diseases.

[0006] However, Vimentin protein undergoes various post-translational modifications within cells, and different modification states, such as lactation, affect its protein structure and cytoskeleton assembly. Measuring only the total Vimentin protein level is insufficient to further distinguish different post-translational modification states of Vimentin, nor can it fully reflect its specific functional state associated with tumor cell invasion and metastasis. Therefore, it is necessary to develop antibodies that can specifically recognize lactation modifications and further establish clinical diagnostic kits based on these antibodies. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a biomarker and its detection kit that can effectively predict the risk of recurrence and metastasis after surgery in patients with cancer, especially renal cell carcinoma. The lactylation modification of lysine at position 373 of Vimentin (VIM K373la) can serve as a molecular marker for postoperative recurrence and metastasis in renal cell carcinoma patients. Detection of this molecular marker can accurately determine the risk of postoperative recurrence and metastasis in renal cell carcinoma patients, thereby enabling physicians to develop effective treatment plans.

[0008] This invention is the first to discover that VIM K373la is significantly upregulated in metastatic renal cell carcinoma and is significantly negatively correlated with recurrence-free survival (AUC up to 0.8654). Based on this, this invention provides a diagnostic kit and its application for predicting renal cell carcinoma recurrence and metastasis, filling a gap in the existing technology.

[0009] To achieve the above objectives, the technical solution adopted by the present invention includes: In a first aspect, the present invention provides the application of a reagent for detecting VIM K373la levels in the preparation of a kit for predicting the risk of postoperative recurrence and metastasis in cancer patients, wherein VIM K373la is a lysine lactylated modification at position 373 of Vimentin protein (UniProt ID: P08670).

[0010] Preferably, the cancer is kidney cancer or breast cancer.

[0011] Preferably, the reagent is an antibody that specifically recognizes VIM K373la; The antibody is a rabbit-derived anti-human VIM K373la antibody. The antibody is prepared by the following method: rabbits are immunized with KLH conjugated with VIM K373la peptide A and / or peptide B; serum is collected and subjected to Protein A affinity chromatography, VIM K373la peptide affinity chromatography, and negative selection with unmodified VIM peptides; wherein, the sequence of VIM K373la peptide A is: CLQDEIQNM-(lactyl)K-EEMA (Vimentin 365-377); The sequence of VIM K373la polypeptide B is as follows: CDEIQNM-(lactyl)K-EEMA (Vimentin 367-377); The sequence of VIM unmodified polypeptide C is as follows: CLQDEIQNMKEEMA (Vimentin 365-377).

[0012] Vimentin, as an intermediate filament protein, is a core biomarker for EMT and is closely related to tumor metastasis. However, the application of VIM expression alone in predicting tumor metastasis risk has significant limitations and weak predictive ability. In our cohort, VIM expression was negatively correlated with recurrence-free survival, but the AUC was 0.6283. Further differentiation of the post-translational modification status of VIM can significantly improve the clinical application of VIM and fill the gap in clinical prediction of renal cell carcinoma metastasis potential.

[0013] This invention is the first to discover that lactylation modification of lysine 373 of Vimentin (VIM K373la) is significantly upregulated in metastatic renal cell carcinoma and is significantly negatively correlated with recurrence-free survival (AUC up to 0.8654). Therefore, by detecting the expression level of VIM K373la in renal cell carcinoma tissue, its expression level can be conveniently and quickly used to help predict the risk of postoperative recurrence and metastasis in renal cell carcinoma patients and provide a reference for disease treatment selection.

[0014] Preferably, the prediction refers to comparing the detection data of VIM K373la in tumor tissue to assess the expression level of VIM K373la.

[0015] Furthermore, the kit is an immunohistochemical detection kit, and the method for detecting the VIM K373la level is immunohistochemistry, with quantitative evaluation performed using the H-score scoring system.

[0016] Specifically, the evaluation steps of the H-score scoring system include: The staining intensity and percentage of positive cells in tumor cells were assessed under a microscope. The grading and scoring criteria for staining intensity are as follows: No staining, score 0; The staining intensity is light yellow, indicating a weak positive result, worth 1 point; The staining intensity is brownish-yellow, which is considered a moderate positive result, and is scored as 2 points. A staining intensity of brownish-red indicates a strong positive result, scored as 3 points. Calculate the H-score using the following formula: H-score = (1 × percentage of weakly positive cells) + (2 × percentage of moderately positive cells) + (3 × percentage of strongly positive cells); The percentage values ​​of weakly positive cells, moderately positive cells, and strongly positive cells are the percentages of cells that are weakly positive, moderately positive, and strongly positive, respectively, counted under a microscope after removing the percentage sign. Each percentage ranges from 0 to 100%, and the corresponding percentage value ranges from 0 to 100. The sum of the three is 100%. The total H-score ranges from 0 to 300. The criteria used to predict the risk of recurrence and metastasis are as follows: When the H-score is ≥140, it is considered as high expression of VIM K373la, indicating a high risk of postoperative recurrence and metastasis in patients; When the H-score is <140, it is considered as low expression of VIM K373la, indicating a low risk of postoperative recurrence and metastasis. The cutoff value of 140 points was determined by receiver operating characteristic (ROC) curves, with an area under the ROC curve ≥ 0.8654.

[0017] The above criteria are examples in specific embodiments of the present invention, but are not limited thereto.

[0018] Secondly, the present invention provides an immunohistochemical detection kit for predicting the risk of postoperative recurrence and metastasis in cancer patients, comprising an anti-human VIM K373la rabbit antibody for detecting VIM K373la levels and an immunohistochemical detection reagent. The VIM K373la is a lactylated modification of the lysine residue at position 373 of the Vimentin protein.

[0019] Preferably, the kit further includes a secondary antibody, a chromogenic substrate, a positive control, a negative control, and instructions for use.

[0020] Preferably, the positive control is renal cell carcinoma tissue with known high expression of VIM K373la, and the negative control is subcutaneous tumorigenic tissue of mouse human renal cell carcinoma cell line 786-O VIM K373R.

[0021] Specifically, the kit also contains one or more components selected from the following: Dewaxing and rehydration reagent (xylene, ethanol), endogenous catalase blocker, goat serum blocking working solution, antibody diluent, reaction enhancement solution, secondary antibody working solution, DAB chromogenic reagent, hematoxylin, EDTA antigen retrieval solution, positive control and negative control.

[0022] To accurately assist clinicians in the diagnosis and treatment of renal cell carcinoma, this invention provides a novel predictive technology for renal cell carcinoma progression. Specifically, it relates to a kit for predicting renal cell carcinoma recurrence and metastasis. This kit can be used to detect the expression level of VIM K373la in tumor tissue to diagnose the possibility of postoperative recurrence and metastasis in renal cell carcinoma patients. Furthermore, the kit is simple and rapid to operate, with high detection sensitivity and specificity. It can perform qualitative and semi-quantitative detection on selected clinical samples without the need for other special instruments. Clinical samples can be stored for a long time, and the process can be completed in general hospital laboratories or pathology departments. Therefore, this kit is expected to become a new technology for the diagnosis of renal cell carcinoma after surgery.

[0023] Preferably, the endogenous peroxidase inhibitor is a 3% hydrogen peroxide solution; and the goat serum has a 5% concentration.

[0024] Preferably, the primary antibody is diluted 200 times.

[0025] In immunohistochemical assay kits, parameters such as antibody dilution factor and component concentration have a significant impact on the quality and accuracy of the test results. When the above-mentioned optimized parameter conditions are used for sample testing, the sensitivity and specificity of the test can be effectively improved, thereby ensuring the repeatability and reliability of the experimental results.

[0026] Specifically, the kit also includes instructions that specify the following criteria: Immunohistochemistry was used to detect the H-score of VIM K373la in tumor tissue. An H-score ≥ 140 was considered high expression, indicating a high risk of recurrence and metastasis in patients; an H-score < 140 was considered low expression, indicating a low risk of recurrence and metastasis in patients.

[0027] Thirdly, the present invention provides an apparatus for predicting the risk of postoperative recurrence and metastasis in cancer patients. The apparatus includes: an acquisition unit for acquiring the expression level of VIM K373la in a sample to be tested, wherein the sample to be tested is a paraffin section of tumor tissue, and VIM K373la is a lysine lactylated modification of Vimentin protein at position 373; the expression level of VIM K373la is quantitatively assessed using an H-score scoring system. The evaluation steps of the H-score scoring system include: The staining intensity and percentage of positive cells in tumor cells were assessed under a microscope. The grading and scoring criteria for staining intensity are as follows: No staining, score 0; The staining intensity is light yellow, indicating a weak positive result, worth 1 point; The staining intensity is brownish-yellow, which is considered a moderate positive result, and is scored as 2 points. A staining intensity of brownish-red indicates a strong positive result, scored as 3 points. Calculate the H-score using the following formula: H-score = (1 × percentage of weakly positive cells) + (2 × percentage of moderately positive cells) + (3 × percentage of strongly positive cells); The percentage values ​​of weakly positive cells, moderately positive cells, and strongly positive cells are the percentages of cells that are weakly positive, moderately positive, and strongly positive, respectively, after removing the percentage sign when counting positive cells under a microscope. Each percentage ranges from 0 to 100%, and the corresponding percentage value ranges from 0 to 100. The sum of the three is 100%. The total H-score ranges from 0 to 300. When the H-score is ≥140, the expression level of VIM K373la is high expression of VIM K373la. When the H-score is <140, the expression level of VIM K373la is low expression of VIM K373la. The processing unit is used to input the expression level of VIM K373la into the model to obtain the prediction results of the risk of postoperative recurrence and metastasis in cancer patients. Specifically, when the expression level of VIM K373la is high, the predicted result is a high risk of postoperative recurrence and metastasis; when the expression level of VIM K373la is low, the predicted result is a low risk of postoperative recurrence and metastasis.

[0028] Fourthly, the present invention provides a method for assessing the risk of postoperative recurrence and metastasis in cancer patients. This method is not intended for disease diagnosis or treatment, and includes the following steps: (1) Obtain the sample to be tested, wherein the sample is a paraffin section of tumor tissue; (2) The expression level of VIM K373la in the sample to be tested was detected using the above-mentioned immunohistochemical detection kit. VIM K373la is a lysine lactylated modification of Vimentin protein at position 373. (3) Compare the detected value with the preset threshold. If it is higher than the threshold, it is judged as high risk of recurrence and metastasis. If it is lower than the threshold, it is judged as low risk of recurrence and metastasis.

[0029] Furthermore, the threshold is determined by the receiver operating characteristic curve, with an area under the curve ≥ 0.8654; The level of VIM K373la was detected by immunohistochemistry and quantitatively evaluated using the H-score scoring system. The evaluation steps of the H-score scoring system include: The staining intensity and percentage of positive cells in tumor cells were assessed under a microscope. The grading and scoring criteria for staining intensity are as follows: No staining, score 0; The staining intensity is light yellow, indicating a weak positive result, worth 1 point; The staining intensity is brownish-yellow, which is considered a moderate positive result, and is scored as 2 points. A staining intensity of brownish-red indicates a strong positive result, scored as 3 points. Calculate the H-score using the following formula: H-score = (1 × percentage of weakly positive cells) + (2 × percentage of moderately positive cells) + (3 × percentage of strongly positive cells); The percentage values ​​of weakly positive cells, moderately positive cells, and strongly positive cells are the percentages of cells that are weakly positive, moderately positive, and strongly positive, respectively, after removing the percentage sign when counting positive cells under a microscope. Each percentage ranges from 0 to 100%, and the corresponding percentage value ranges from 0 to 100. The sum of the three is 100%. The total H-score ranges from 0 to 300. The criteria used to assess the risk of recurrence and metastasis are as follows: When the H-score is ≥140, it is considered as high expression of VIM K373la, indicating a high risk of postoperative recurrence and metastasis in patients; When the H-score is <140, it is considered as low expression of VIM K373la, indicating a low risk of postoperative recurrence and metastasis.

[0030] Compared with the prior art, the present invention has the following beneficial effects: High specificity: The prepared antibody specifically recognizes VIM K373la, and VIM K373R (K373 site arginine mutation, unable to undergo lactylation modification) tissues show no positive staining.

[0031] High sensitivity: It can detect modification levels down to the nanogram level, making it suitable for clinical micro-samples.

[0032] Superior predictive performance: In renal cell carcinoma tissue microarrays, the risk of recurrence and metastasis in the high-expression group of VIM K373la was 12.15 times that of the low-expression group, with an AUC of 0.8654, which is superior to traditional prognostic indicators such as total VIM (AUC=0.6283).

[0033] Easy to use: The kit adopts the standard IHC procedure and is suitable for widespread use in clinical pathology departments.

[0034] Cross-cancer potential: Preliminary experiments showed that VIM K373la also has predictive value in breast cancer metastasis (AUC=0.8504), suggesting that its application may not be limited to renal cell carcinoma. Attached Figure Description

[0035] Figure 1 Construction of VIM K373la specific antibody. Where A: Immunogen peptide information; BD: Immunogen mass spectrometry identification.

[0036] Figure 2 Validation of VIM K373la specific antibodies. Specifically: A: Antibody enzyme-linked immunosorbent assay (ELISA); B: Antibody dot blot hybridization; C: Antibody Western blot (WB) detection; D: Western blot validation of antibodies in renal cell carcinoma lines; E: Antibody immunohistochemical specificity detection, scale bar 100 μm. After subcutaneous tumor formation in nude mice using 786-O cells containing VIM WT and VIMK373R (which cannot be lactylated), tumors were embedded for HE and immunohistochemical staining.

[0037] Figure 3 VIM K373la was significantly upregulated in renal cell carcinoma metastases. A: Representative images of renal cell carcinoma tissue immunohistochemical staining, primary lesion and corresponding metastases, scale bar at 200 μm; B: Statistical analysis, where scatter plots represent different samples, **** indicates p < 0.0001.

[0038] Figure 4Kaplan-Meier survival curve analysis results for renal cell carcinoma and breast cancer cohorts. A: Disease-free survival rate in the renal cell carcinoma cohort between the high VIM K373la expression group (n=88) and the low expression group (n=81); B: Disease-free survival rate in the breast cancer cohort between the high VIM K373la expression group (n=53) and the low expression group (n=50).

[0039] Figure 5 : Detection of the predictive ability of VIM K373la for renal cell carcinoma and breast cancer recurrence and metastasis. Among them, A: ROC curve of VIM K373la for predicting renal cell carcinoma recurrence and metastasis (AUC=0.8654, significantly better than total VIM AUC=0.6283); B: ROC curve of VIM K373la for predicting breast cancer recurrence and metastasis (AUC=0.8504, significantly better than total VIM AUC=0.6722).

[0040] Figure 6 Examples of clinical predictions of renal cell carcinoma and breast cancer recurrence and metastasis using VIM K373la. A: Staining results of two representative renal cell carcinoma tissues (same clinical stage, recurrence and metastasis vs. no recurrence and metastasis), scale bar 200 μm; B: Staining results of two representative breast cancer tissues (same clinical stage, recurrence and metastasis vs. no recurrence and metastasis), scale bar 100 μm. Detailed Implementation

[0041] Example 1: Preparation and validation of rabbit-derived antibody against human VIM K373la 1. Immunogen design and synthesis Based on the human Vimentin protein sequence (UniProt ID: P08670), a peptide containing lysine at position 373 (K373) was selected as the immunogen. Three peptides were synthesized using a solid-phase synthesis method: two were lactylated peptides at the K373 site, and the other was an unmodified control peptide (with an unmodified lysine at K373). Figure 1 (A in the middle).

[0042] The sequence of VIM K373la polypeptide A is as follows: CLQDEIQNM-(lactyl)K-EEMA (Vimentin 365-377); The sequence of VIM K373la polypeptide B is as follows: CDEIQNM-(lactyl)K-EEMA (Vimentin 367-377); The sequence of VIM unmodified polypeptide C is as follows: CLQDEIQNMKEEMA (Vimentin 365-377).

[0043] The three peptides were identified by liquid chromatography-tandem mass spectrometry (LC-MS / MS) for both mass and sequence determination. The results showed that the measured mass of each peptide differed from the theoretical mass by less than 10 ppm, and the sequence identification results were consistent with the design, indicating that the synthesized peptides met the immunogen design requirements. Figure 1 B to Figure 1 (D in the middle).

[0044] 2. Animal immunization and antiserum screening Four healthy SPF-grade New Zealand white rabbits were selected as experimental animals. VIM K373la peptide A and peptide B were used to immunize the animals. Rabbits R1 and R2 were immunized with VIM K373la peptide A, while rabbits R3 and R4 were immunized with VIM K373la peptide B.

[0045] Prior to animal immunization, the modified peptides were conjugated with KLH to prepare immunogens. The immunogens were appropriately diluted with physiological saline and then mixed with the corresponding adjuvants at a 1:1 volume ratio to form a stable emulsion. Immunization was administered via subcutaneous and intramuscular injections at multiple sites, with each rabbit receiving a total of six immunizations on days 1, 7, 14, 21, 28, and 40.

[0046] Blood samples were collected on days 28, 35, 47 and 52, and serum was obtained. Antiserum was screened by ELISA and dot blot hybridization to evaluate antibody titers and specific recognition ability of VIM K373la modified peptides.

[0047] Serum ELISA results showed that the antisera from rabbits R1, R2, R3, and R4 could all recognize the VIM K373la modified peptide, with antibody titers reaching 1:64,000 (OD). 450 >0.5) and above. Further Western blotting was used to detect the recognition ability of antisera on endogenous VIMK373la. The results showed that antisera R1, R2, R3, and R4 could all detect specific protein bands with a molecular weight of approximately 55 kDa.

[0048] Based on the combined results of serum ELISA and WB, antisera from R1, R2, R3, and R4 were selected for subsequent antibody purification.

[0049] 3. Antibody purification Sufficient rabbit serum from sources R1, R2, R3 and R4 was collected, and the IgG in the serum was first purified by Protein A affinity chromatography.

[0050] After packing the Protein A column, the column was equilibrated with PBS buffer. Serum was filtered appropriately and loaded onto the column, and the eluent was collected as a process control sample. The column was then washed with PBS buffer, and bound IgG was eluted with 150 mM glycine buffer. The eluent was collected and the pH was immediately adjusted to approximately 7.0 with neutralization buffer.

[0051] The crude pure IgG obtained by Protein A purification was further loaded into a VIM K373la immunogenic peptide affinity chromatography column to enrich antibodies that can specifically recognize VIM K373la modified epitopes.

[0052] To further reduce the background of nonspecific antibodies, the enriched products were loaded onto a VIM unmodified peptide affinity column, and the efflux fractions that did not bind to the unmodified peptides were collected, thereby removing antibody components that could recognize the unmodified epitopes of Vimentin.

[0053] Purified antibodies derived from R1, R2, R3, and R4 were obtained and numbered Ab1, Ab2, Ab3, and Ab4, respectively. After determining the protein concentration of the purified antibodies, glycerol was added for stable storage, and the antibodies were aliquoted and stored at -20°C.

[0054] 4. Antibody specificity verification ELISA: VIM K373la-modified peptides and VIM-unmodified peptides were coated onto 96-well microplates and incubated overnight at 4°C. After washing, the plates were blocked with 1% BSA. Purified Ab1, Ab2, Ab3, and Ab4 were serially diluted 3-fold at a starting concentration of 1:1,000 and added to the microplates, then incubated at 37°C for 1.5 h. After washing, HRP-labeled anti-rabbit IgG secondary antibody was added, and TMB was used for color development. The absorbance at 450 nm was measured using a microplate reader.

[0055] The results showed that Ab1 and Ab3 had high binding activity to the VIM K373la modified peptide, and positive detection signals could still be obtained at an antibody dilution of 1:54,000. Compared with the unmodified Vimentin peptide, the positive signal differed by at least two gradients, indicating that Ab1 and Ab3 had good modification specificity for VIM K373la. Figure 2 (A in the middle).

[0056] Dotted hybridization: VIM K373la-modified peptide and VIM-unmodified peptide were spotted onto PVDF membranes at gradients of 1 ng, 4 ng, 16 ng, and 64 ng. After blocking the membranes, the target antibody was added and incubated. Subsequently, HRP-labeled anti-rabbit IgG secondary antibody was added, and detection was performed using chemiluminescence immunoassay.

[0057] The results showed that Ab1, Ab3, and Ab4 produced significant positive signals for VIM K373la-modified peptides, while exhibiting virtually no binding to unmodified VIM peptides. Notably, Ab1 and Ab4 showed detectable signals at a level of 4 ng of the modified peptide, indicating high detection sensitivity and modification specificity. Figure 2 (B in the middle).

[0058] WB: HeLa and 293T cells were used, and sodium lactate (NaLac) treated and untreated groups were set up. Total protein was extracted from cells after treatment with 25 mM NaLac for 24 h, and Western blotting was performed using Ab1, Ab2, Ab3, and Ab4, respectively. The results showed that Ab1, a specific protein band of approximately 55 kDa, was detectable in both the NaLac treated and untreated groups of HeLa and 293T cells, and lactate treatment upregulated its expression. Figure 2 (C in the text). The molecular weight of the bands mentioned above is consistent with the expected molecular weight of Vimentin protein.

[0059] Western blot analysis was performed on renal cell carcinoma cells 786-O and ACHN to evaluate the recognition ability of the candidate antibody Ab1 for endogenous VImentin 373la. Cells were treated with different concentrations of NaLac to induce changes in intracellular lactylation levels. The results showed that Ab1 could stably detect a specific band of approximately 55 kDa in both 786-O and ACHN cells, and the VImentin 373la signal detected by Ab1 gradually increased with increasing sodium lactate treatment concentration, while the total vimentin protein level remained consistent. Figure 2 (D in the middle).

[0060] Based on the combined results of ELISA, dot blot hybridization, and WB detection, Ab1 exhibits good VIM K373la recognition specificity and is therefore used as the VIM K373la detection antibody in this invention.

[0061] 5. Immunohistochemical specificity verification To verify the specificity of VIM-K373la-mAb (Ab1) in tissue sections, 786-O cell lines containing VIM WT and VIM K373R (an arginine mutation at the K373 site, preventing lactylation modification) were constructed. 1×10 6 Cells were subcutaneously inoculated into the right axilla of nude mice. Mice were sacrificed when the tumor diameter reached 1.5 cm, and tumor tissue was collected and embedded in paraffin. The immunohistochemical detection method described in this invention (see Example 2) was used to stain both tissue sections, using VIM-K373la-mAb (1:200) as the primary antibody.

[0062] The results showed that VIM WT tumor tissue exhibited strong positive brownish-yellow staining (mainly located in the cytoplasm), while VIMK373R tumor tissue showed no positive staining. Figure 2 E in the text). HE staining confirmed that the tumor cells in the two tissues had the same morphology ( Figure 2 (E in the original text). This result confirms that the antibody prepared in this invention specifically recognizes VIM K373la modification at the tissue level and has no cross-reactivity with unmodified or mutated tissues.

[0063] Example 2: Detection and prognostic assessment of VIM K373la in renal cell carcinoma tissue based on immunohistochemistry (IHC) 1. Clinical Sample Collection Surgically resected tissue specimens were collected from patients with renal cell carcinoma. All patients had signed informed consent forms and were pathologically diagnosed. Inclusion criteria: (1) initial diagnosis of primary renal cell carcinoma; (2) radical nephrectomy; (3) complete clinicopathological data and follow-up data. Exclusion criteria: (1) prior radiotherapy, chemotherapy or targeted therapy; (2) other malignant tumors; (3) a total of 172 patients with renal cell carcinoma were included. All samples were fixed in 10% neutral formalin and embedded in paraffin.

[0064] 2. Immunohistochemical detection steps The immunohistochemical staining kit for assessing the risk of recurrence and metastasis of renal cell carcinoma provided by this invention was used for staining, and its components are shown in Table 1.

[0065] Table 1

[0066] All components should be stored at 2-8°C. Shelf life is 12 months from the date of manufacture. Repeated freeze-thaw cycles are not recommended after primary antibody dilution; it should be aliquoted and stored at -20°C.

[0067] The specific steps for immunohistochemical detection are as follows: a) The prepared paraffin tissue sections were baked and placed in a 60°C incubator overnight; b) Dewaxing and hydration were performed sequentially: the sections were soaked in xylene three times at 37°C and 80 rpm in a shaker for 15 minutes each time, and then hydrated in anhydrous ethanol, graded alcohols (95%, 80%, 75%), and double-distilled water for 3 minutes each. c) Soak the slides in 10% neutral formalin for 10 min, then wash the slides three times with double-distilled water, 1 min each time; d) Antigen retrieval: Place the slide in a microwave oven containing sodium citrate antigen retrieval solution, heat on high until boiling, then reduce to low heat and maintain for 15 minutes. After heating, allow it to cool naturally. e) Wash the sections three times with PBS buffer, 3 min each time (horizontal shaker, 80 rpm). f) Block endogenous peroxidase: After outlining the tissue with a histochemical pen, add catalase blocking agent and incubate at room temperature for 10 minutes. Then wash the sections three times with PBS buffer, 3 minutes each time (horizontal shaker, 80 rpm). g) Blocking: After discarding the PBS, add goat serum and block at room temperature for 30 minutes; h) Primary antibody incubation: Discard the serum, add the primary antibody that specifically recognizes VIM K373la diluted 1:200, and incubate overnight at 4°C in a humidified chamber; i) Rewarming: Remove the humidified chamber and allow it to warm to room temperature. Then wash the slides three times with PBS buffer, 3 min each time (horizontal shaker, 80 rpm). j) Add reaction enhancement solution and incubate in a humidified chamber at 37°C for 20 min; wash the sections three times with PBS buffer, 3 min each time (horizontal shaker, 80 rpm). k) Secondary antibody incubation: Add enzyme-labeled goat anti-rabbit secondary antibody and incubate in a humidified chamber at 37°C for 30 min; l) Wash the slides three times with PBS buffer, 3 min each time (horizontal shaker, 80 rpm). m) DAB chromogenic reaction: Prepare 1x DAB chromogenic reaction solution in the dark and set aside. Wipe away the moisture around the tissue, add the DAB chromogenic reaction solution, and develop the color at room temperature in the dark for 1-3 minutes. Observe the color development results under a microscope. After the positive control has fully developed, rinse with running water to stop the color development. n) Counterstain: Counterstain with hematoxylin for 5 minutes, then rinse with clean water.

[0068] o) Differentiation and blueing: Differentiation takes about 5 seconds, then rinse with clean water to stop; Blueing takes 30 seconds, then rinse thoroughly with clean water. p) Dehydration, clearing and mounting: Dehydrated sequentially with 70%, 95% and 100% alcohol gradients, cleared twice with xylene for 10 minutes each time, and finally mounted with neutral resin. q) Scan the histochemical sections and use the H-score method to assess the expression level of VIM K373la (intensity score × percentage of positive cells).

[0069] 3. Result Judgment and Scoring Criteria Two pathologists independently interpreted the data without knowledge of the clinicopathological information, and the results were averaged. The standard H-score scoring method was used, as follows: (1) First, assess the staining intensity and percentage of positive cells of tumor cells under a microscope (200x field of view).

[0070] (2) Staining intensity grading: no staining is 0 points; light yellow is 1 point (weak positive); brownish yellow is 2 points (moderate positive); brownish brown is 3 points (strong positive).

[0071] (3) H-score calculation formula: H-score = (1 × percentage of weakly positive cells) + (2 × percentage of moderately positive cells) + (3 × percentage of strongly positive cells); The percentage of positive cells at each intensity ranges from 0 to 100%, and the corresponding percentage values ​​range from 0 to 100. The sum of the three is 100%, and the total score is 0 to 300.

[0072] For example: If 20% of cells in a certain field of view are weakly positive, 50% are moderately positive, and 30% are strongly positive, then the H-score = (1×20) + (2×50) + (3×30) = 20 + 100 + 90 = 210 points.

[0073] According to the ROC curve ( Figure 5 In section B), the optimal cutoff value is determined: an H-score ≥ 140 is defined as high expression of VIM K373la, and an H-score < 140 is defined as low expression. This cutoff value is used in this invention as a threshold for judging a high risk of recurrence and metastasis.

[0074] 4. Detection Results and Prognostic Analysis The detection method described in this invention was used to detect renal cell carcinoma tissue. The results showed that the H-score of VIM K373la in metastatic lesions (202±18) was significantly higher than that in primary lesions (131±26), and the difference was statistically significant (P<0.001). Figure 3 Using VIM K373la expression levels, 172 renal cell carcinoma tissues were divided into a high-expression group (H-score ≥ 140, n=88) and a low-expression group (H-score < 140, n=81). Survival analysis: Kaplan-Meier curves showed that the median disease-free survival in the high-expression group was significantly shorter than that in the low-expression group, with a hazard ratio (HR) of 12.15 (95% CI: 4.77–20.97), and a Log-rank test showed P < 0.001. Figure 4 (A) Using VIM K373la expression levels, 103 breast cancer tissue samples were divided into a high-expression group (H-score ≥ 140, n=53) and a low-expression group (H-score < 140, n=50). Survival analysis: Kaplan-Meier curves showed that the median disease-free survival in the high-expression group was significantly shorter than that in the low-expression group, with a hazard ratio (HR) of 5.79 (95% CI: 3.13–10.73), and a Log-rank test result of P < 0.001. Figure 4 (B in the middle).

[0075] 5. ROC curve analysis The predictive efficacy of VIM K373la for renal cell carcinoma and breast cancer recurrence and metastasis was evaluated using receiver operating characteristic (ROC) curves, and the expression of total Vimentin protein was compared (using a commercially available anti-Vimentin antibody, CST: D21H3).

[0076] The area under the ROC curve (AUC) of VIM K373la in the renal cell carcinoma cohort was 0.8654, and the AUC of total Vimentin was 0.6283. Figure 5 In the A group of breast cancer cohorts, the area under the ROC curve (AUC) for VIM K373la was 0.8504, and the AUC for total Vimentin was 0.6722. Figure 5 (B in the text). The results show that the AUC of the present invention for detecting VIM K373la is significantly better than that for total Vimentin.

[0077] Figure 6 Figure A shows the staining results of representative renal cell carcinoma tissues from two patients with the same clinical stage (both T2aN0M0) but different prognoses: the left patient had no recurrence or metastasis 58 months after surgery, and VIM K373la was expressed at low levels (H-score=105); the right patient developed lung metastasis 24 months after surgery, and VIM K373la was expressed at high levels (H-score=205). Figure 6 Figure B shows the staining results of representative triple-negative breast cancer tissues from two patients with the same clinical stage (both T2aN0M0) but different prognoses: the left patient was relapse-free 131 months post-surgery, with low expression of VIM K373la (H-score=95); the right patient developed lung metastasis 76 months post-surgery, with high expression of VIM K373la (H-score=180). These results demonstrate that the detection method described in this invention can effectively identify individuals with a high risk of recurrence and metastasis among patients with the same clinical stage. In contrast, conventional total Vimentin antibody therapy under the same conditions could not effectively distinguish the clinical outcomes of these two patients.

[0078] Example 3: Clinical Application Case ( Figure 6 A) Case 1: Patients with low recurrence risk Patient A, a 56-year-old male, was clinically diagnosed with clear cell renal cell carcinoma (T2aN0M0). Postoperatively, paraffin sections of tumor tissue were obtained, and VIM K373la was detected using the kit and detection method described in Example 2 of this invention. The staining result showed an H-score of 105, indicating low expression of VIM K373la. The patient was followed up for 58 months postoperatively, and no local recurrence or distant metastasis occurred, consistent with the predicted result of this invention (low risk of recurrence and metastasis).

[0079] Case 2: Patients at high risk of relapse Patient B, a 62-year-old female, was clinically diagnosed with clear cell renal cell carcinoma (T2aN0M0, same clinical stage as Case 1). Postoperatively, paraffin sections of tumor tissue were obtained, and VIM K373la was detected using the same method. The staining result showed an H-score of 205, indicating high expression of VIM K373la. This patient developed lung metastases 24 months postoperatively. The predicted outcome (high risk of recurrence and metastasis) of this invention is consistent with the actual clinical outcome.

[0080] This comparative case demonstrates that, among patients with renal cell carcinoma at the same clinical stage, the present invention can effectively distinguish individuals with different risks of recurrence and metastasis, providing an important basis for developing individualized follow-up and treatment plans in clinical practice.

[0081] Example 4: Clinical Application Case ( Figure 6 (B) Case 1: Patients with low recurrence risk Patient A, a 51-year-old female, was clinically diagnosed with invasive ductal carcinoma of the breast (T2aN0M0, triple negative). Postoperatively, paraffin sections of tumor tissue were obtained, and VIM K373la was detected using the kit and detection method described in Example 2 of this invention. The staining result showed H-score = 95, indicating low expression of VIM K373la. The patient was followed up for 131 months postoperatively, and no local recurrence or distant metastasis occurred, consistent with the predicted result of this invention (low risk of recurrence and metastasis).

[0082] Case 2: Patients at high risk of relapse Patient B, a 56-year-old female, was clinically diagnosed with invasive ductal carcinoma of the breast (T2aN0M0, same clinical stage as Case 1, also triple-negative breast cancer). Postoperatively, paraffin sections of tumor tissue were obtained, and VIM K373la was detected using the same method. The staining result showed an H-score of 180, indicating high expression of VIM K373la. This patient developed systemic metastasis 58 months postoperatively. The predicted outcome (high risk of recurrence and metastasis) of this invention is consistent with the actual clinical outcome.

[0083] This comparative case demonstrates that, among breast cancer patients at the same clinical stage, the present invention can also effectively distinguish individuals with different risks of recurrence and metastasis, providing an important basis for the development of individualized follow-up and treatment plans in clinical practice.

Claims

1. Application of reagents for detecting VIM K373la levels in the preparation of kits for predicting the risk of postoperative recurrence and metastasis in cancer patients, wherein VIM K373la is a lysine lactylated modification of Vimentin protein at position 373.

2. The application according to claim 1, characterized in that, The cancer in question is either kidney cancer or breast cancer.

3. The application according to claim 1 or 2, characterized in that, The reagent is an antibody that specifically recognizes VIM K373la; The antibody is a rabbit-derived antibody against human VIM K373la.

4. The application according to claim 1, characterized in that, The kit is an immunohistochemical detection kit. The method for detecting VIM K373la level is immunohistochemistry, and quantitative evaluation is performed using the H-score scoring system.

5. The application according to claim 4, characterized in that, The evaluation steps of the H-score scoring system include: The staining intensity and percentage of positive cells in tumor cells were assessed under a microscope. The grading and scoring criteria for staining intensity are as follows: No staining, score 0; The staining intensity is light yellow, indicating a weak positive result, worth 1 point; The staining intensity is brownish-yellow, which is considered a moderate positive result, and is scored as 2 points. A staining intensity of brownish-red indicates a strong positive result, scored as 3 points. Calculate the H-score using the following formula: H-score = (1 × percentage of weakly positive cells) + (2 × percentage of moderately positive cells) + (3 × percentage of strongly positive cells); The percentage values ​​of weakly positive cells, moderately positive cells, and strongly positive cells are the percentages of cells that are weakly positive, moderately positive, and strongly positive, respectively, counted under a microscope after removing the percentage sign. Each percentage ranges from 0 to 100%, and the corresponding percentage value ranges from 0 to 100. The sum of the three is 100%. The total H-score ranges from 0 to 300. The criteria for predicting the risk of postoperative recurrence and metastasis in cancer patients are as follows: When the H-score is ≥140, it is considered as high expression of VIM K373la, indicating a high risk of postoperative recurrence and metastasis in patients; When the H-score is <140, it is considered as low expression of VIM K373la, indicating a low risk of postoperative recurrence and metastasis. The cutoff value of 140 points was determined using the receiver operating characteristic (ROC) curve, and the area under the ROC curve was ≥0.8654.

6. A kit for predicting the risk of postoperative recurrence and metastasis in cancer patients, characterized in that, This includes rabbit-derived anti-human VIM K373la antibody for detecting VIM K373la levels and immunohistochemical detection reagents; The VIM K373la is a lactylated modification of the lysine residue at position 373 of the Vimentin protein.

7. The reagent kit according to claim 6, characterized in that, The kit also contains one or more components selected from the following: Dewaxing and rehydration reagent, endogenous catalase inhibitor, goat serum blocking working solution, antibody diluent, reaction enhancement solution, secondary antibody working solution, DAB chromogenic reagent, hematoxylin, EDTA antigen retrieval solution, positive control and negative control.

8. The reagent kit according to claim 6, characterized in that, The kit also includes instructions that describe the following judgment criteria: Immunohistochemistry was used to detect the H-score of VIM K373la in tumor tissue. A score of ≥140 was considered high expression, indicating a high risk of recurrence and metastasis in patients. When the H-score is <140, it is considered low expression, indicating a low risk of recurrence and metastasis in the patient.

9. A device for predicting the risk of postoperative recurrence and metastasis in cancer patients, characterized in that, The device includes: an acquisition unit for acquiring the expression level of VIM K373la in a sample to be tested, wherein the sample to be tested is a paraffin section of tumor tissue, and VIM K373la is a lysine lactylated modification of Vimentin protein at position 373; the expression level of VIM K373la is quantitatively assessed using an H-score scoring system. The evaluation steps of the H-score scoring system include: The staining intensity and percentage of positive cells in tumor cells were assessed under a microscope. The grading and scoring criteria for staining intensity are as follows: No staining, score 0; The staining intensity is light yellow, indicating a weak positive result, worth 1 point; The staining intensity is brownish-yellow, which is considered a moderate positive result, and is scored as 2 points. A staining intensity of brownish-red indicates a strong positive result, scored as 3 points. Calculate the H-score using the following formula: H-score = (1 × percentage of weakly positive cells) + (2 × percentage of moderately positive cells) + (3 × percentage of strongly positive cells); The percentage values ​​of weakly positive cells, moderately positive cells, and strongly positive cells are the percentages of cells that are weakly positive, moderately positive, and strongly positive, respectively, counted under a microscope after removing the percentage sign. Each percentage ranges from 0 to 100%, and the corresponding percentage value ranges from 0 to 100. The sum of the three is 100%. The total H-score ranges from 0 to 300. When the H-score is ≥140, the expression level of VIM K373la is high expression of VIM K373la. When the H-score is <140, the expression level of VIM K373la is low expression of VIM K373la. The processing unit is used to input the expression level of VIM K373la into the model to obtain the prediction results of the risk of postoperative recurrence and metastasis in cancer patients. Specifically, when the expression level of VIM K373la is high, the predicted result is a high risk of postoperative recurrence and metastasis; when the expression level of VIM K373la is low, the predicted result is a low risk of postoperative recurrence and metastasis.

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