Application of CST6 as early diagnosis marker for brain metastasis of lung cancer

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to application of CST6 as a lung cancer brain metastasis early diagnosis marker. The core of the invention is to confirm that CST6 is used as a marker for early diagnosis and prediction of brain metastasis of lung cancer for the first time. In experimental verification, the CST6 protein expression quantity of the lung cancer cell strain H1975-BrM with high brain metastasis potential is remarkably higher than that of a common cell strain, an animal model further shows that overexpression of CST6 can promote formation of brain metastasis, and knock-down expression can inhibit metastasis capacity; clinical sample analysis shows that the serum CST6 concentration of brain metastasis patients is far higher than that of non-metastasis patients and healthy people, and the diagnostic value of CST6 is supported. In the aspect of diagnosis efficiency, the area under the ROC curve of the serum CST6 reaches 0.848, and high accuracy is shown. On the whole, the clinical blank is filled, and a reliable tool is provided for non-invasive lung cancer brain metastasis risk management.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically involving the application of CST6 as an early diagnostic marker for brain metastases in lung cancer. Background Technology

[0002] Lung cancer is one of the most common malignant tumors worldwide, with the highest incidence and mortality rates, seriously threatening human life and health. In the course of lung cancer, distant organ metastasis is a major cause of treatment failure and patient death. The brain is one of the most common organs for lung cancer metastasis, with approximately 20-40% of lung cancer patients developing brain metastasis (BM) during the course of the disease. Once brain metastasis occurs, the patient's prognosis deteriorates dramatically, with a median survival of less than six months, accompanied by severe neurological symptoms such as severe headaches, seizures, cognitive impairment, and limited limb movement, significantly reducing the patient's quality of life.

[0003] Currently, the main clinical methods for diagnosing brain metastases from lung cancer are imaging examinations, among which contrast-enhanced MRI has the highest sensitivity and is considered the "gold standard" for diagnosis, followed by computed tomography (CT). These imaging methods primarily rely on morphological observation of existing intracranial space-occupying lesions to make a judgment.

[0004] Early detection and timely intervention are crucial for the treatment of lung cancer brain metastases. If treatment is initiated in the "early" stage—when the metastatic lesions are small, few in number, or have not yet caused significant neurological symptoms—using methods such as stereotactic radiosurgery (SRS), whole-brain radiotherapy (WBRT), or targeted drug therapy, disease progression can be effectively controlled, significantly prolonging patient survival and maintaining a better quality of life. Therefore, achieving early diagnosis of lung cancer brain metastases is a key step in improving patient prognosis.

[0005] While existing imaging techniques play an irreplaceable role in diagnosing established brain metastases, they have significant limitations and drawbacks in "early diagnosis" and "screening of high-risk groups": Early diagnosis lacks sensitivity: Imaging examinations such as MRI can only identify solid metastases that have already reached a certain size (usually larger than millimeters). For micrometastases or earlier stages where cancer cells have just invaded the brain parenchyma, imaging is not effective. By the time lesions can be clearly identified on images, the metastasis process has often already occurred for a considerable time, missing the optimal window for intervention.

[0006] The practicality and cost-effectiveness of screening are poor: Due to the high cost and long time required for MRI examinations, as well as the need for specialized equipment and personnel, it cannot be used as a routine and frequent screening method for brain metastases for all lung cancer patients, especially high-risk groups. Clinically, examinations are usually only performed after patients develop related neurological symptoms, which is essentially a "passive" late-stage diagnosis rather than an "active" early screening.

[0007] Lack of early warning and predictive capabilities: Current technologies cannot effectively predict which lung cancer patients are at high risk of brain metastasis before it occurs. Clinically, there is a severe lack of biomarkers that can prospectively assess the risk of brain metastasis, thereby enabling focused monitoring of high-risk patients.

[0008] Invasiveness of diagnostic methods: In rare cases where imaging is insufficient to differentiate the diagnosis, a brain biopsy is required for confirmation. This is a highly invasive and risky procedure that patients have poor tolerance for.

[0009] Therefore, there is an urgent need in the existing technology for a non-invasive (or minimally invasive), highly sensitive, highly specific and cost-effective detection method to achieve early diagnosis, risk warning and screening of high-risk groups for lung cancer brain metastases. Summary of the Invention

[0010] The present invention aims to address the shortcomings of the prior art by providing a novel serum or cerebrospinal fluid biomarker, CST6, for the early diagnosis of lung cancer brain metastases, thereby solving the technical problem of the lack of effective early diagnostic tools in clinical practice.

[0011] The first objective of this invention is to provide the application of CST6 as a biomarker in the diagnosis and prediction of brain metastases in lung cancer.

[0012] A second aspect of the present invention aims to provide the application of substances that detect CST6 in the preparation of products for diagnosing and predicting brain metastases in lung cancer.

[0013] The third aspect of this invention aims to provide a method for assisting in predicting the risk of brain metastasis in lung cancer.

[0014] To achieve the above-mentioned objectives of this invention, the technical solution adopted by this invention is as follows: In a first aspect, the present invention provides the application of CST6 as a biomarker in the diagnosis and prediction of brain metastases in lung cancer.

[0015] CST6 refers to Cystatin E / M, a member of the Cystatin Superfamily, whose main function is to inhibit the activity of papain-like cysteine ​​proteases. The sequence information of the human CST6 gene (Gene ID: 1474) is publicly available, and the accession number of a representative mRNA transcript is NCBI Reference Sequence: NM_001323.3. The protein it encodes (Protein ID: NP_001314.2) consists of 149 amino acids.

[0016] In some embodiments of this invention, lung cancer brain metastasis refers to the spread of primary lung cancer cells to the brain parenchyma, meninges, or ventricles via the bloodstream or other pathways, forming one or more metastatic tumors. The clinical diagnostic criteria are mainly based on the following points: 1. Clinical symptoms: The patient presents with new-onset neurological symptoms that cannot be explained by other causes, such as persistent headache, nausea and vomiting, seizures, visual impairment, speech impairment, limb weakness or sensory abnormalities, and cognitive decline. 2. Imaging examinations: This is the most important basis for diagnosing lung cancer brain metastasis. Contrast-enhanced MRI of the head is the "gold standard" for diagnosis, clearly showing the number, size, location, and relationship of metastatic lesions to surrounding tissues. Enhanced CT scans can also be used for diagnosis, but their sensitivity is lower than MRI. 3. Pathological diagnosis: In a few cases where imaging findings are atypical or need to be differentiated from other intracranial lesions (such as primary brain tumors or abscesses), surgical resection or stereotactic biopsy may be necessary to obtain brain lesion tissue. Pathological examination confirms lung cancer metastasis, which is the most definitive diagnostic basis. The lung cancer brain metastasis referred to in this invention refers to cases that meet any of the above-mentioned imaging or pathological diagnostic criteria.

[0017] A second aspect of the invention provides the use of substances that detect CST6 in the preparation of products for diagnosing and predicting brain metastases in lung cancer.

[0018] In some embodiments of the present invention, the substance for detecting CST6 includes reagents for detecting CST6 at the protein level or the gene level.

[0019] In some embodiments of the present invention, the reagent for detecting CST6 at the protein level is selected from reagents of one or more detection methods from the group consisting of: chemiluminescence, immunofluorescence, protein chip, proteometry, immunohistochemistry, patch tracing based on labeling technology, Western blotting, and enzyme-linked immunosorbent assay (ELISA).

[0020] In some embodiments of the present invention, the reagent for detecting CST6 at the gene level is selected from reagents of one or more detection methods from the group consisting of: high-throughput sequencing, digital PCR, and quantitative real-time PCR.

[0021] In some embodiments of the present invention, the product includes a test kit, a test chip, or a test strip.

[0022] In some embodiments of the present invention, the test sample for the product is serum, plasma, cerebrospinal fluid or tissue sample.

[0023] In some embodiments of the present invention, the test subjects of the product include humans.

[0024] A third aspect of this invention aims to provide a method for constructing a model for the diagnosis and prediction of brain metastases in lung cancer, comprising the following steps: Model building was performed using the expression levels of CST6.

[0025] In some embodiments of the present invention, the model construction algorithm includes at least one of logistic regression, linear discriminant analysis, support vector machine, random forest, and recursive partitioning tree.

[0026] A fourth aspect of the present invention provides a system for diagnosing and predicting brain metastases in lung cancer, the system comprising a computing device for diagnosing and predicting brain metastases in lung cancer based on the expression level detection results of CST6.

[0027] In some embodiments of the present invention, the detection results include protein level results or RNA level results.

[0028] In some embodiments of the present invention, the system further includes any one or more of the following: 1) Detection result collection device, also known as detection result input device, can specifically be one or more of the following: mouse, keyboard, touch screen display, one or more buttons, one or more switches, one or more triggers, etc. 2) Diagnostic result output device, also known as diagnostic result display device, can specifically be one or more of the following: liquid crystal display (LCD), light-emitting diode (LED) display, plasma display, projection display, touch screen display, etc. 3) Diagnostic result sending device, which can send the results of distinguishing whether the subject is in a strong or weak risk group to an information communication terminal device that can be viewed by the patient or medical staff.

[0029] A third aspect of the present invention provides a method for assisting in predicting the risk of brain metastasis in lung cancer, comprising the following steps: a) Obtain biological samples from the individual to be tested; b) The concentration of CST6 protein in the biological sample was determined using an immunological detection method; c) Compare the concentration measured in step b) with the preset diagnostic threshold; d) Based on the comparison results, assist in predicting whether the individual has lung cancer brain metastases or the level of risk of developing brain metastases.

[0030] When the measured CST6 protein concentration is significantly higher than the diagnostic threshold, it indicates that the individual has lung cancer brain metastases or is at high risk.

[0031] The detection methods include, but are not limited to, immunological detection methods such as enzyme-linked immunosorbent assay (ELISA) or Western blotting that can be used to detect the CST6 content in patient serum or plasma samples.

[0032] This method is not intended for direct diagnosis of diseases, but is only intended to provide reference for medical professionals.

[0033] The beneficial effects of this invention are: Compared with existing technologies, this invention provides a novel application for the early diagnosis of lung cancer brain metastases based on the biomarker CST6, which has the following significant beneficial effects: 1. Achieves early diagnosis and warning, significantly advancing the timing of intervention: This invention can detect brain metastases before the onset of clinical neurological symptoms and before solid lesions can be detected by imaging methods. This molecular-level early diagnosis can detect the disease weeks or even months earlier than existing technologies, gaining a valuable time window for clinical treatment and greatly improving treatment outcomes and patients' chances of survival.

[0034] 2. Minimally invasive and highly safe detection method: This invention mainly detects patients' blood (serum / plasma) samples. Compared with radioactive CT scans or MRI scans that require the injection of contrast agents, as well as high-risk brain tissue biopsies, this method greatly reduces the pain and risks for patients, has high safety, and good patient compliance.

[0035] 3. Applicable to dynamic monitoring and screening of high-risk groups: Due to its simple operation and relatively low cost, the method of this invention is suitable as a routine and periodic screening tool for lung cancer patients, especially those at high risk of brain metastasis. By dynamically monitoring changes in CST6 levels, continuous assessment and early warning of brain metastasis risk can be achieved, filling a gap in existing technologies in the field of screening.

[0036] 4. Improved diagnostic sensitivity and specificity: By setting the optimal diagnostic threshold, the CST6 detection method provided by this invention exhibits excellent sensitivity and specificity in diagnosing brain metastases in lung cancer. It can effectively distinguish between patients with brain metastases, lung cancer patients without brain metastases, and healthy individuals, providing a reliable diagnostic basis for clinical practice. Attached Figure Description

[0037] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 This indicates increased expression of CST6 in lung cancer brain metastasis cell lines.

[0038] Figure 2 The results show the effect of CST6 expression on the brain metastasis ability of lung cancer cells in mice.

[0039] Figure 3 The results show the measurement of CST6 protein expression levels in clinical samples.

[0040] Figure 4 The results of the evaluation of CST6's efficacy in diagnosing and predicting brain metastases in lung cancer. Detailed Implementation

[0041] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0042] Example 1: Determination of CST6 protein expression level in lung cancer brain metastases 1. Experimental materials Parental lung cancer cell line: human lung adenocarcinoma cell line H1975, which has a low spontaneous brain metastasis ability in vivo.

[0043] High brain metastasis potential lung cancer cell line: H1975-BrM cell line.

[0044] This cell line was constructed as follows: Log-growing parental H1975 cells were resuspended in PBS and injected into BALB / c nude mice via left ventricular injection. After the mice developed neurological symptoms, brain metastases were confirmed using in vivo imaging. The mice were euthanized, and brain tissue was aseptically harvested, digested with collagenase, and the brain metastases were isolated and cultured. Once the cells reached stable growth, the next round of in vivo screening was performed. This process was repeated four times to ultimately establish the H1975-BrM cell line with stable and high brain metastatic potential.

[0045] 2. Experimental Methods The expression level of CST6 protein in the two groups of cells was detected by enzyme-linked immunosorbent assay (ELISA).

[0046] Sample preparation: H1975 cells and H1975-BrM cells were cultured to the logarithmic growth phase, and the cell supernatant was collected, concentrated, and set aside for use. A commercially available human CST6 ELISA kit (Sinochem, KIT10438) was used. Cell supernatant samples and standards were added to pre-coated antibody-impregnated ELISA plates for incubation. Subsequent steps were strictly performed according to the kit instructions, including the addition of detection antibodies, enzyme conjugates, substrate, color development, and reaction termination. Finally, the absorbance (OD) value was measured at 450 nm using an ELISA reader, and the concentration of CST6 protein in the sample was calculated based on the standard curve. Results are expressed as nanograms of CST6 (ng / ml) per milliliter of cell supernatant.

[0047] 3. Experimental Results The results are as follows Figure 1 As shown in the ELISA results, the concentration of CST6 protein in the lysate of H1975-BrM cells with high brain metastasis potential (average value of approximately 2.28 ng / ml) was significantly higher than that in their parent H1975 cells (average value of approximately 0.75 ng / ml), and the difference between the two was statistically significant (P<0.01).

[0048] This embodiment demonstrates at the cellular level that the brain metastatic potential of lung cancer cells is positively correlated with the expression level of CST6 protein. Cell lines with high brain metastatic potential express higher levels of CST6 protein. This result provides cellular biological evidence that elevated serum CST6 levels can indicate the clinical phenomenon of lung cancer brain metastasis, further confirming the crucial role of CST6 in the process of lung cancer brain metastasis.

[0049] Example 2: Determination of the effect of CST6 expression on the brain metastasis ability of lung cancer cells in mice. 1. Experimental Methods Cell line construction: The full-length cDNA of the human CST6 gene was cloned into the pSin-Puro lentiviral vector to construct a stable CST6 overexpression vector (H1975-CST6), with an empty vector (H1975-Vector) as a control. The shRNA sequence targeting the CST6 gene was cloned into the pLKO.1-puro vector to construct an interference vector (H1975-BrM-shCST6), with a negative control shRNA sequence (H1975-BrM-shVector) as a control. The effective shRNA sequence targeting CST6 is: 5'-GCAGGTTCTACGGGAAGAAAT (SEQ ID NO: 1)-3'. The human lung adenocarcinoma cell line H1975 was selected. Using lentiviral transfection technology, a stable CST6 overexpression cell line (H1975-CST6) and a control cell line (H1975-Vector) were constructed. Simultaneously, a cell line stably interfering with CST6 expression (H1975-BrM-shCST6) and a negative control cell line (H1975-BrM-shVector) were constructed based on the highly metastatic brain cell line H1975-BrM. All constructed cell lines stably expressed luciferase simultaneously for subsequent in vivo imaging.

[0050] Animal Model: BALB / c nude mice aged 4-6 weeks were randomly divided into 4 groups of 5 mice each. An experimental brain metastasis model was established using the left ventricular injection method. Cells in the logarithmic growth phase of each group (H1975-Vector, H1975-CST6, H1975-BrM-shVector, H1975-BrM-shCST6) were resuspended in PBS, and the cell density was adjusted to 5 × 10⁻⁶ cells / mL. 5 cells / 100μL. Under anesthesia, 100μL of cell suspension was slowly injected into the left ventricle of each nude mouse.

[0051] In vivo imaging monitoring: Starting from day 7 post-injection, mice were subjected to in vivo biofluorescence imaging once a week. Mice were anesthetized by intraperitoneal injection of D-fluorescein sodium solution (150 mg / kg), and the intensity of fluorescence signals in the brain was observed and quantified using an imaging system.

[0052] 2. Experimental Results The results are as follows Figure 2As shown, in vivo imaging results revealed that at week 4 post-injection, the fluorescence signal intensity in the brains of H1975-CST6 mice was significantly higher than that in their control group, H1975-Vector. Conversely, the fluorescence signal in the brains of H1975-BRM-CST6 sh1 mice was significantly lower than that in their control group, H1975-BRM-Vector. At the experimental endpoint (week 4), the mice were euthanized and perfused for fixation, and their brain tissue was collected for paraffin sectioning. H&E staining results showed that the number and volume of brain metastases in the H1975-CST6 group were significantly greater than those in the control group; while the number and size of brain metastases in the H1975-BRM-CST6 sh1 group were significantly fewer and smaller than those in the control group.

[0053] The animal experimental results of this embodiment strongly demonstrate that the expression level of CST6 can directly affect the brain metastasis ability of lung cancer cells in vivo. Upregulation of CST6 expression promotes the formation of brain metastases, while inhibition of its expression slows down the process of brain metastasis. This provides strong functional evidence for CST6 as a diagnostic biomarker and potential therapeutic target for lung cancer brain metastases.

[0054] Example 3: Determination of CST6 protein expression level in clinical samples 1. Experimental Samples Serum samples were collected from 75 clinically diagnosed lung cancer patients. Based on imaging (enhanced MRI) and clinical diagnosis, they were divided into three groups: early-stage lung cancer (non-met group, n=14), lung cancer with brain metastasis (brain group, n=25), and lung cancer without brain metastasis (no-brain group, n=20). Sixteen healthy individuals undergoing routine physical examinations during the same period were selected as a healthy control group (normal group). This study protocol has been reviewed and approved by the Ethics Committee of Sun Yat-sen University.

[0055] 2. Detection Method A commercially available human CST6 ELISA kit (Sino Bioscience, KIT10438) was used, strictly following the instructions. The simplified procedure was as follows: Diluted serum samples and standards were added to an ELISA plate coated with anti-human CST6 antibody and incubated at 37°C for 60 minutes. After washing, biotin-labeled detection antibody was added and incubated at 37°C for 60 minutes. The plate was washed again, and horseradish peroxidase-labeled streptavidin was added and incubated at 37°C for 30 minutes. Finally, the plate was washed, and TMB substrate was added. The reaction was stopped after 15 minutes at room temperature in the dark, followed by the addition of stop solution. The absorbance (OD) of each well was measured at 450 nm using an ELISA reader, and the concentration of CST6 protein in each sample (in ng / mL) was calculated based on the standard curve.

[0056] 3. Experimental Results The results are as follows Figure 3As shown in the figure, statistical analysis revealed that the serum CST6 protein concentration in the lung cancer brain metastasis group (mean approximately 14.76 ng / ml) was significantly higher than that in the lung cancer without brain metastasis group (no-brain group, mean approximately 3.51 ng / ml) and the healthy control group (normal group, mean approximately 1.35 ng / ml), with statistically significant differences (p<0.001). This result indicates that serum CST6 levels are specifically elevated in lung cancer patients with brain metastases.

[0057] Example 4: Evaluation of the efficacy of CST6 in diagnosing lung cancer brain metastases 1. Experimental Methods Using the data obtained in Example 3, the diagnostic efficacy of serum CST6 for lung cancer brain metastases was evaluated using receiver operating characteristic (ROC) curves. ROC curves were plotted with the brain metastasis group as the positive sample and the non-brain metastasis group as the negative sample.

[0058] 2. Experimental Results like Figure 4 As shown in the ROC curve analysis, the area under the curve (AUC) for serum CST6 in diagnosing lung cancer brain metastases was 0.848 (95% confidence interval: 0.738–0.958), indicating high diagnostic accuracy. Based on the Youden index maximization principle, the optimal diagnostic cut-off value was determined to be 9.71 ng / ml. At this threshold, the sensitivity of CST6 in diagnosing lung cancer brain metastases was 100%, and the specificity was 56%.

[0059] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. Application of CST6 as a biomarker in the diagnosis and prediction of brain metastases in lung cancer.

2. Application of substances that detect CST6 in the preparation of products for diagnosing and predicting brain metastases in lung cancer.

3. The application according to claim 2, characterized in that: The substances used to detect CST6 include reagents for detecting CST6 at the protein or gene level.

4. The application according to claim 3, characterized in that: The reagents for detecting CST6 at the protein level are selected from one or more of the following detection methods: chemiluminescence, immunofluorescence, protein chip, proteometry, immunohistochemistry, labeling-based plaque tracing, Western blotting, and enzyme-linked immunosorbent assay (ELISA). The reagents for detecting CST6 at the gene level are selected from reagents of one or more detection methods from the group consisting of: high-throughput sequencing, digital PCR, and quantitative real-time PCR.

5. The application according to claim 2, characterized in that: The products include test kits, test chips, or test strips.

6. The application according to claim 2, characterized in that: The test samples for the product are serum, plasma, cerebrospinal fluid, or tissue samples.

7. A method for constructing a model for the diagnosis and prediction of lung cancer brain metastases, comprising the following steps: Model building was performed using the expression levels of CST6.

8. The construction method according to claim 7, characterized in that: The model construction algorithm includes at least one of logistic regression, linear discriminant analysis, support vector machine, random forest, and recursive partitioning tree.

9. A system for diagnosing and predicting brain metastases in lung cancer, the system comprising a computational device for diagnosing and predicting brain metastases in lung cancer based on the detection results of CST6 expression levels.

10. The system according to claim 9, characterized in that: The system further includes one or more of b1) to b3): b1) Device for collecting test results; b2) Diagnostic result output device; b3) Diagnostic result transmission device.

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