Method for evaluating colorectal cancer metastasis based on nf-kb pathway activity

Abstract

The application discloses a colorectal cancer metastasis evaluation method based on NF-kappa B pathway activity, relates to the technical field of medical treatment, and comprises the following steps: obtaining a colorectal cancer tissue sample and a paired peripheral blood sample, separating exosomes, and dividing the tissue sample into a spatial resolution sub-sample and a function verification sub-sample; performing multiple immunofluorescence imaging on the spatial resolution sub-sample, identifying a phosphorylated p65 positive cancer cell group and a phosphorylated STAT3 positive immune cell group, and extracting cell phenotype data; calculating the spatial interaction weight value between high-activity cells based on the cell phenotype data to obtain a tumor microenvironment paracrine activation index; detecting the abundance of an upstream phosphorylated protein of the NF-kappa B pathway and the expression amount of a downstream metastasis effect gene in the function verification sub-sample, and obtaining a pathway function coupling score in combination with the matching degree of the two; and determining the content of a phosphorylated IKK protein and the content of a specific metastasis-promoting microRNA in the exosomes, and obtaining an exosome double-lock activity score by using the product relationship between the two.

Description

Technical Field

[0001] This invention relates to the field of medical technology, and in particular to a method for assessing colorectal cancer metastasis based on NF-κB pathway activity. Background Technology

[0002] Colorectal cancer, a highly prevalent malignant tumor of the digestive tract worldwide, is primarily caused not by the primary tumor itself, but by postoperative recurrence and distant metastasis. The nuclear factor κB signaling pathway, as a key link between the inflammatory microenvironment and tumor development, plays a central role in regulating epithelial-mesenchymal transition, invasion and migration, and anti-apoptosis of cancer cells. In current technologies, the assessment of NF-κB pathway activity mainly relies on immunohistochemical detection of the nuclear localization of the p65 subunit, Western blotting to determine phosphorylated protein levels, or qPCR analysis of downstream target gene expression.

[0003] In actual clinical applications, existing assessment models often separate local tissue information from systemic blood information, failing to establish a dynamic correlation between the two. Clinically, there is often a separation phenomenon where tissue tests are negative after primary lesion resection, but blood indicators are abnormally elevated, indicating the existence of occult micrometastases. However, existing technologies lack mathematical modeling and risk penalty mechanisms for the inconsistency between tissue and blood activity, and cannot use this difference itself as a diagnostic basis. Summary of the Invention

[0004] In view of the aforementioned existing problems, the present invention is proposed.

[0005] Therefore, this invention provides a method for assessing colorectal cancer metastasis based on NF-κB pathway activity to address the problem of failing to establish a dynamic correlation between the two. Clinically, there is often a separation phenomenon where tissue tests are negative after primary lesion resection but blood indicators are abnormally elevated, indicating the existence of occult micrometastases. However, existing technologies lack mathematical modeling and risk penalty mechanisms for the inconsistency between tissue and blood activity, and cannot use this difference itself as a diagnostic basis.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0007] In a first aspect, the present invention provides a method for assessing colorectal cancer metastasis based on NF-κB pathway activity, which includes obtaining colorectal cancer tissue samples and paired peripheral blood samples, isolating exosomes and dividing the tissue samples into spatial analysis sub-samples and functional verification sub-samples.

[0008] Multiple immunofluorescence imaging was performed on the spatial resolution subsamples to identify phosphorylated p65-positive cancer cell populations and phosphorylated STAT3-positive immune cell populations and extract cell phenotypic data.

[0009] The spatial interaction weight values ​​between highly active cells were calculated based on cell phenotype data to obtain the paracrine activation index of the tumor microenvironment.

[0010] The abundance of phosphorylated proteins upstream of the NF-κB pathway and the expression levels of downstream transfer effect genes in the functional verification subsample were detected, and the pathway functional coupling score was obtained by combining the degree of matching between the two.

[0011] The levels of phosphorylated IKK protein and specific pro-transfer microRNA in exosomes were measured, and the product of the two was used to obtain the exosome dual-lock activity score.

[0012] The fitted value of the tumor microenvironment paracrine activation index and the pathway function coupling score is defined as the local tissue activity characteristic, and the exosome dual-lock activity score is defined as the systemic circulating activity characteristic. The difference between the systemic circulating activity characteristic and the local tissue activity characteristic is calculated and multiplied by a preset penalty coefficient to obtain the occult metastasis penalty item. The local tissue activity characteristic, the systemic circulating activity characteristic, and the occult metastasis penalty item are added together to obtain the total risk score. The colorectal cancer metastasis risk rating is determined based on the preset threshold range in which the total risk score falls. A comprehensive assessment report is generated based on the colorectal cancer metastasis risk rating.

[0013] As a preferred embodiment of the colorectal cancer metastasis assessment method based on NF-κB pathway activity described in this invention, the steps of obtaining colorectal cancer tissue samples and paired peripheral blood samples, isolating exosomes, and dividing the tissue samples into spatial resolution subsamples and functional verification subsamples are as follows:

[0014] Fresh colorectal cancer tissue samples were obtained through surgical resection or puncture biopsy, and paired peripheral blood samples were collected simultaneously at the same time point.

[0015] Some fresh colorectal cancer tissue samples were fixed by soaking in fixative, dehydrated, cleared and paraffin-impregnated, then embedded in paraffin and cut into tissue sections, which were used as sub-samples for spatial analysis.

[0016] Fresh colorectal cancer tissue samples were flash-frozen using cryogenic freezing medium and then stored in an ultra-low temperature environment as functional validation sub-samples.

[0017] Paired peripheral blood samples were centrifuged stepwise using differential centrifugation to remove cell debris and platelets. The supernatant was collected and then subjected to ultracentrifugation. The precipitate was resuspended in buffer to obtain a high-purity exosome suspension.

[0018] As a preferred embodiment of the colorectal cancer metastasis assessment method based on NF-κB pathway activity described in this invention, the specific steps of performing multiplex immunofluorescence imaging on spatially analyzed subsamples to identify phosphorylated p65-positive cancer cell populations and phosphorylated STAT3-positive immune cell populations and extracting cell phenotypic data are as follows:

[0019] Spatial analysis subsamples were cyclically stained using multiple immunofluorescence staining techniques. The antibody combination included cancer cell-specific marker antibodies, nuclear factor κBp65 subunit phosphorylation site-specific antibodies, macrophage-specific marker antibodies, and signal transduction and transcription activator factor 3 phosphorylation site-specific antibodies.

[0020] The stained spatial resolution subsamples were scanned in their entirety using a multispectral microscopy system to obtain high-resolution digital images;

[0021] High-resolution digital images are used to identify cell nuclei and cytoplasm regions using image segmentation algorithms. Based on fluorescence intensity thresholds, cancer cells with intranuclear phosphorylated p65 signals stronger than a preset cancer cell threshold are selected to form a phosphorylated p65 positive cancer cell population. At the same time, immune cells with intracytoplasmic phosphorylated STAT3 signals stronger than a preset immune cell threshold are selected to form a phosphorylated STAT3 positive immune cell population.

[0022] The two-dimensional coordinates of the center point of each cell in the phosphorylated p65-positive cancer cell population and the phosphorylated STAT3-positive immune cell population, along with the corresponding fluorescence intensity value, were extracted using a coordinate extraction program to form cell phenotypic data.

[0023] As a preferred embodiment of the colorectal cancer metastasis assessment method based on NF-κB pathway activity described in this invention, the specific steps for calculating the spatial interaction weight values ​​between highly active cells based on cell phenotype data to obtain the paracrine activation index of the tumor microenvironment are as follows:

[0024] The distance relationship between each phosphorylated p65-positive cancer cell and phosphorylated STAT3-positive immune cells within a predetermined radius in the cell phenotype data is modeled using an exponential decay function model to simulate the physical law that cytokine diffusion weakens with increasing distance.

[0025] The paracrine activation index of the tumor microenvironment is calculated by multiplying the nucleus-to-cytoplasm ratio of each cancer cell as the baseline activity coefficient by the logarithm of the sum of the weighted fluorescence intensities of neighboring immune cells, and then averaging the results over all cancer cells. The expression is as follows:

[0026] ;

[0027] in, It represents the paracrine activation index of the tumor microenvironment. The total number of cells in the phosphorylated p65-positive cancer cell population. and The first The average fluorescence intensity of the nucleus and cytoplasm regions of each cancer cell. For the first The number of phosphorylated STAT3-positive immune cells within a predetermined radius of action centered on a cancer cell. For the first The average fluorescence intensity of each immune cell This represents the maximum fluorescence intensity of all immune cells within the current field of view. For the first The first cancer cell and the first The Euclidean distance between the centroids of immune cells This is the spatial decay constant characterizing the diffusion capacity of cytokines.

[0028] As a preferred embodiment of the colorectal cancer metastasis assessment method based on NF-κB pathway activity described in this invention, the method involves detecting the abundance of phosphorylated proteins upstream of the NF-κB pathway and the expression levels of downstream metastasis effect genes in the functional verification subsample, and combining the degree of matching between the two to obtain a pathway functional coupling score. The specific steps are as follows:

[0029] The functional validation subsamples were lysed with protein extraction reagent and total protein was extracted. The concentrations of phosphorylated IKK protein and total IKK protein were detected by enzyme-linked immunosorbent assay.

[0030] The functional validation subsamples were lysed with RNA extraction reagent and total RNA was extracted. After reverse transcription, the relative expression levels of matrix metalloproteinase 9 gene, CXC chemokine receptor 4 gene and vascular endothelial growth factor gene relative to the internal reference gene were detected by conventional quantitative polymerase chain reaction.

[0031] The expression for the pathway functional coupling score is:

[0032] ;

[0033] in, For pathway functional coupling scoring, and These represent the concentrations of phosphorylated IKK protein and total IKK protein, respectively. For the first The relative expression levels of downstream transfer effect genes, For the first Preset weighting coefficients for each gene, The Gaussian error function is... This is a commonly used logarithmic function.

[0034] As a preferred embodiment of the colorectal cancer metastasis assessment method based on NF-κB pathway activity described in this invention, the steps of measuring the phosphorylated IKK protein content and the content of specific pro-metastatic microRNAs in exosomes, and obtaining the exosome dual-lock activity score using the product of the two, are as follows:

[0035] The exosome suspension was subjected to lysis buffer to break down the membrane, releasing the internal and membrane surface contents;

[0036] The concentration of phosphorylated IKK protein in the lysate was detected by electrochemiluminescence immunoassay, and the concentrations of microRNA-21 and microRNA-221 in the lysate were detected by nucleic acid hybridization capture assay.

[0037] The expression for the exosome double-lock activity score is:

[0038] ;

[0039] in, The exosome dual-lock activity score, This refers to the concentration of phosphorylated IKK protein in exosomes. and These represent the concentrations of microRNA-21 and microRNA-221 in exosomes, respectively. The preset half-saturation constant, Here, delta is the preset microRNA activation threshold, and delta is the preset coefficient of variation. It is the arctangent function. Pi is the mathematical constant of a circle.

[0040] As a preferred embodiment of the colorectal cancer metastasis assessment method based on NF-κB pathway activity described in this invention, the following steps are taken: The fitted value of the tumor microenvironment paracrine activation index and the pathway function coupling score is defined as the local tissue activity characteristic; the exosome dual-lock activity score is defined as the systemic circulating activity characteristic; the difference between the systemic circulating activity characteristic and the local tissue activity characteristic is calculated and multiplied by a preset penalty coefficient to obtain a occult metastasis penalty item; the local tissue activity characteristic, the systemic circulating activity characteristic, and the occult metastasis penalty item are summed to obtain a total risk score.

[0041] The tumor microenvironment paracrine activation index and pathway function coupling score were multiplied to obtain the tissue local activity characteristic value, and the exosome double-lock activity score was directly assigned as the systemic circulation activity characteristic value.

[0042] The expression for the total risk score of colorectal cancer metastasis is:

[0043] ;

[0044] in, The total risk score for colorectal cancer metastasis. This represents the characteristic value of local tissue activity. This represents the characteristic value of systemic circulatory activity. and These are the preset local weighting coefficients and the whole-body weighting coefficients, respectively. This is the activity difference value, when hour ,no , The preset penalty intensity coefficient, The preset sensitivity constant, It is an exponential function.

[0045] As a preferred embodiment of the colorectal cancer metastasis assessment method based on NF-κB pathway activity described in this invention, the steps of determining the colorectal cancer metastasis risk rating based on the preset threshold range into which the total risk score falls, and generating a comprehensive assessment report based on the colorectal cancer metastasis risk rating, are as follows:

[0046] The total risk score is compared with the preset upper limit of low risk threshold and lower limit of high risk threshold using the interval comparison method;

[0047] When the total risk score is less than the upper limit of the low risk threshold, the risk rating of colorectal cancer metastasis is determined to be low risk.

[0048] When the total risk score is greater than or equal to the upper limit of the low-risk threshold and less than the lower limit of the high-risk threshold, it is judged as a medium-risk level;

[0049] When the total risk score is greater than or equal to the lower limit of the high-risk threshold, it is judged as a high-risk level;

[0050] The risk rating of colorectal cancer metastasis, the paracrine activation index of the tumor microenvironment, the pathway functional coupling score, and the exosome dual-lock activity score are integrated into a comprehensive assessment report through the report generation template.

[0051] In a second aspect, the present invention provides a computer device including a memory and a processor, wherein the memory stores a computer program, wherein: when the computer program is executed by the processor, it implements any step of the method for assessing colorectal cancer metastasis based on NF-κB pathway activity as described in the first aspect of the present invention.

[0052] Thirdly, the present invention provides a computer-readable storage medium having a computer program stored thereon, wherein: when the computer program is executed by a processor, it implements any step of the method for assessing colorectal cancer metastasis based on NF-κB pathway activity as described in the first aspect of the present invention.

[0053] The beneficial effects of this invention are as follows: by measuring the content of phosphorylated IKK protein and specific pro-metastatic microRNA in exosomes and constructing a dual-lock activity score using the Michaelis equation and logic gating function, the synergistic quantification and nonlinear switching control of exosomal proteins and nucleic acid markers are realized, thereby greatly reducing the misjudgment caused by the increase of a single indicator due to nonspecific inflammation or benign lesions, improving the specificity of identifying occult micrometastases and overcoming the interference of background noise in liquid biopsy. Attached Figure Description

[0054] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0055] Figure 1 This is a flowchart of a method for assessing colorectal cancer metastasis based on NF-κB pathway activity. Detailed Implementation

[0056] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0057] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0058] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0059] Reference Figure 1 This is one embodiment of the present invention, which provides a method for assessing colorectal cancer metastasis based on NF-κB pathway activity, comprising the following steps:

[0060] S1. Obtain colorectal cancer tissue samples and paired peripheral blood samples, isolate exosomes, and divide the tissue samples into spatial resolution subsamples and functional verification subsamples.

[0061] Furthermore, fresh colorectal cancer tissue samples were obtained through surgical resection or puncture biopsy, and paired peripheral blood samples were collected simultaneously at the same time point.

[0062] Some fresh colorectal cancer tissue samples were fixed by soaking in fixative, dehydrated, cleared and paraffin-impregnated, then embedded in paraffin and cut into tissue sections, which were used as sub-samples for spatial analysis.

[0063] Fresh colorectal cancer tissue samples were flash-frozen using cryogenic freezing medium and then stored in an ultra-low temperature environment as functional validation sub-samples.

[0064] Paired peripheral blood samples were centrifuged stepwise using differential centrifugation to remove cell debris and platelets. The supernatant was collected and then subjected to ultracentrifugation. The precipitate was resuspended in buffer to obtain a high-purity exosome suspension.

[0065] It should be noted that by employing physical segmentation and differentiated processing strategies, an optimal balance between preserving the spatial structure and maintaining the molecular integrity of the same sample source was achieved. This not only avoids the spatial information loss or protein / RNA degradation problems caused by a single fixation method, but also ensures strict consistency between the local tissue state and the whole blood state in the time dimension through synchronous acquisition, laying a reliable material foundation for the accurate fusion of subsequent multimodal data.

[0066] S2. Perform multiplex immunofluorescence imaging on the spatial resolution subsamples to identify phosphorylated p65-positive cancer cell populations and phosphorylated STAT3-positive immune cell populations and extract cell phenotypic data.

[0067] Furthermore, the spatial resolution subsamples were cyclically stained using multiple immunofluorescence staining techniques. The antibody combination included cancer cell-specific marker antibodies, nuclear factor κBp65 subunit phosphorylation site-specific antibodies, macrophage-specific marker antibodies, and signal transduction and transcription activator factor 3 phosphorylation site-specific antibodies.

[0068] The stained spatial resolution subsamples were scanned in their entirety using a multispectral microscopy system to obtain high-resolution digital images;

[0069] High-resolution digital images are used to identify cell nuclei and cytoplasm regions using image segmentation algorithms. Based on fluorescence intensity thresholds, cancer cells with intranuclear phosphorylated p65 signals stronger than a preset cancer cell threshold are selected to form a phosphorylated p65 positive cancer cell population. At the same time, immune cells with intracytoplasmic phosphorylated STAT3 signals stronger than a preset immune cell threshold are selected to form a phosphorylated STAT3 positive immune cell population.

[0070] The two-dimensional coordinates of the center point of each cell in the phosphorylated p65-positive cancer cell population and the phosphorylated STAT3-positive immune cell population, along with the corresponding fluorescence intensity value, were extracted using a coordinate extraction program to form cell phenotypic data.

[0071] It should be noted that by simultaneously capturing the dual activation states of effector cells and regulatory cells through multiple labeling, and by using single-cell resolution spatial coordinate extraction technology, abstract biochemical signals are transformed into quantifiable geometric location data. This not only clarifies the activation sites of the NF-κB pathway, but also identifies potential paracrine interaction entities in the microenvironment, providing high-precision data input for the subsequent construction of spatial interaction models.

[0072] S3. Calculate the spatial interaction weight values ​​between highly active cells based on cell phenotype data to obtain the paracrine activation index of the tumor microenvironment.

[0073] Furthermore, the distance relationship between each phosphorylated p65-positive cancer cell and the phosphorylated STAT3-positive immune cells within a predetermined radius in the cell phenotype data is modeled using an exponential decay function model to simulate the physical law that cytokine diffusion weakens with increasing distance.

[0074] The paracrine activation index of the tumor microenvironment is calculated by multiplying the nucleus-to-cytoplasm ratio of each cancer cell as the baseline activity coefficient by the logarithm of the sum of the weighted fluorescence intensities of neighboring immune cells, and then averaging the results over all cancer cells. The expression is as follows:

[0075] ;

[0076] in, It represents the paracrine activation index of the tumor microenvironment. The total number of cells in the phosphorylated p65-positive cancer cell population. and The first The average fluorescence intensity of the nucleus and cytoplasm regions of each cancer cell. For the first The number of phosphorylated STAT3-positive immune cells within a predetermined radius of action centered on a cancer cell. For the first The average fluorescence intensity of each immune cell This represents the maximum fluorescence intensity of all immune cells within the current field of view. For the first The first cancer cell and the first The Euclidean distance between the centroids of immune cells This is the spatial decay constant characterizing the diffusion capacity of cytokines.

[0077] It should be noted that by introducing the exponential decay mechanism from biophysics, the physical distance between cells is creatively transformed into a functional interaction weight, which effectively solves the shortcomings of traditional methods that only count the number of positive cells and ignore spatial distribution characteristics. This allows for the accurate identification of high-risk cell clusters at the center of the inflammatory storm, improving the sensitivity and specificity of the assessment method for microenvironment-driven metastasis.

[0078] S4. Detect the abundance of phosphorylated proteins upstream of the NF-κB pathway and the expression level of downstream transfer effector genes in the functional verification subsample, and obtain a pathway functional coupling score by combining the degree of matching between the two.

[0079] Furthermore, the functional validation subsamples were lysed with protein extraction reagents and total protein was extracted. The concentrations of phosphorylated IKK protein and total IKK protein were detected by enzyme-linked immunosorbent assay (ELISA).

[0080] The functional validation subsamples were lysed with RNA extraction reagent and total RNA was extracted. After reverse transcription, the relative expression levels of matrix metalloproteinase 9 gene, CXC chemokine receptor 4 gene and vascular endothelial growth factor gene relative to the internal reference gene were detected by conventional quantitative polymerase chain reaction.

[0081] The expression for the pathway functional coupling score is:

[0082] ;

[0083] in, For pathway functional coupling scoring, and These represent the concentrations of phosphorylated IKK protein and total IKK protein, respectively. For the first The relative expression levels of downstream transfer effect genes, For the first Preset weighting coefficients for each gene, The Gaussian error function is... This is a commonly used logarithmic function.

[0084] It should be noted that a signal transduction efficiency verification mechanism was constructed, using a Gaussian error function as a consistency penalty term to force a high degree of matching between upstream kinase activation and downstream gene transcription. This effectively filters out false activation signals caused by cellular stress, feedback blockade, or detection noise, ensuring that the evaluation results truly reflect the biological ability of tumor cells to execute metastasis procedures.

[0085] S5. The content of phosphorylated IKK protein and specific pro-transfer microRNA in exosomes were measured, and the exosome double-lock activity score was obtained by using the product relationship between the two.

[0086] Furthermore, the exosome suspension is subjected to lysis buffer to break down the membrane, releasing the internal and membrane surface contents;

[0087] The concentration of phosphorylated IKK protein in the lysate was detected by electrochemiluminescence immunoassay, and the concentrations of microRNA-21 and microRNA-221 in the lysate were detected by nucleic acid hybridization capture assay.

[0088] The expression for the exosome double-lock activity score is:

[0089] ;

[0090] in, The exosome dual-lock activity score, This refers to the concentration of phosphorylated IKK protein in exosomes. and These represent the concentrations of microRNA-21 and microRNA-221 in exosomes, respectively. The preset half-saturation constant, Here, delta is the preset microRNA activation threshold, and delta is the preset coefficient of variation. It is the arctangent function. Pi is the mathematical constant of a circle.

[0091] It should be noted that a protein-miRNA dual-lock logic gating mechanism is adopted, using the Michaelis equation to simulate saturation dynamics and combining it with the arctangent function to construct a nonlinear switch. High scores are only triggered when two biomarkers of different properties are simultaneously highly expressed, which reduces the misjudgment rate caused by the elevation of a single indicator due to nonspecific inflammation or benign lesions and improves the specificity of identifying occult micrometastases.

[0092] S6. Define the fitted value of the paracrine activation index of the tumor microenvironment and the functional coupling score of the pathway as the local tissue activity feature, define the exosome dual-lock activity score as the systemic circulating activity feature, calculate the difference between the systemic circulating activity feature and the local tissue activity feature and multiply it by a preset penalty coefficient to obtain the occult metastasis penalty item, sum the local tissue activity feature, the systemic circulating activity feature and the occult metastasis penalty item to obtain the total risk score, determine the colorectal cancer metastasis risk rating based on the preset threshold range in which the total risk score falls, and generate a comprehensive assessment report based on the colorectal cancer metastasis risk rating.

[0093] Furthermore, the tumor microenvironment paracrine activation index and pathway function coupling score are multiplied to obtain the tissue local activity characteristic value, and the exosome double-lock activity score is directly assigned as the systemic circulation activity characteristic value.

[0094] The expression for the total risk score of colorectal cancer metastasis is:

[0095] ;

[0096] in, The total risk score for colorectal cancer metastasis. This represents the characteristic value of local tissue activity. This represents the characteristic value of systemic circulatory activity. and These are the preset local weighting coefficients and the whole-body weighting coefficients, respectively. This is the activity difference value, when hour ,no , The preset penalty intensity coefficient, The preset sensitivity constant, It is an exponential function;

[0097] The total risk score is compared with the preset upper limit of low risk threshold and lower limit of high risk threshold using the interval comparison method;

[0098] When the total risk score is less than the upper limit of the low risk threshold, the risk rating of colorectal cancer metastasis is determined to be low risk.

[0099] When the total risk score is greater than or equal to the upper limit of the low-risk threshold and less than the lower limit of the high-risk threshold, it is judged as a medium-risk level;

[0100] When the total risk score is greater than or equal to the lower limit of the high-risk threshold, it is judged as a high-risk level;

[0101] The risk rating of colorectal cancer metastasis, the paracrine activation index of the tumor microenvironment, the pathway functional coupling score, and the exosome dual-lock activity score are integrated into a comprehensive assessment report through the report generation template.

[0102] It should be noted that by using the inconsistency of tissue blood activity as a risk amplification factor, the exponential integral penalty model can keenly capture the abnormal pattern of high blood activity and low blood activity. This pattern often indicates the presence of distant micrometastases outside the primary lesion that have not been biopsied. This overcomes the limitation of traditional single-point biopsy in reflecting the systemic disease burden and achieves dynamic, panoramic, and accurate early warning of colorectal cancer metastasis risk.

[0103] This embodiment also provides a computer device applicable to the assessment method of colorectal cancer metastasis based on NF-κB pathway activity, comprising: a memory and a processor; the memory is used to store computer-executable instructions, and the processor is used to execute the computer-executable instructions to implement the assessment method of colorectal cancer metastasis based on NF-κB pathway activity as proposed in the above embodiment.

[0104] The computer device can be a terminal, comprising a processor, memory, communication interface, display screen, and input devices connected via a system bus. The processor provides computing and control capabilities. The memory includes non-volatile storage media and internal memory. The non-volatile storage media stores the operating system and computer programs. The internal memory provides an environment for the operation of the operating system and computer programs stored in the non-volatile storage media. The communication interface is used for wired or wireless communication with external terminals; wireless communication can be achieved through Wi-Fi, carrier networks, NFC (Near Field Communication), or other technologies. The display screen can be an LCD screen or an e-ink screen. The input devices can be a touch layer covering the display screen, buttons, a trackball, or a touchpad on the computer device's casing, or an external keyboard, touchpad, or mouse.

[0105] This embodiment also provides a storage medium storing a computer program that, when executed by a processor, implements the method for assessing colorectal cancer metastasis based on NF-κB pathway activity as proposed in the above embodiments. The storage medium can be implemented by any type of volatile or non-volatile storage device or a combination thereof, such as Static Random Access Memory (SRAM), Electrically Erasable Programmable Read-Only Memory (EEPROM), Erasable Programmable Read Only Memory (EPROM), Programmable Red-Only Memory (PROM), Read-Only Memory (ROM), magnetic storage, flash memory, magnetic disk, or optical disk.

[0106] In summary, this invention achieves synergistic quantification and nonlinear switching control of exosomal proteins and nucleic acid markers by measuring the content of phosphorylated IKK protein and specific pro-metastatic microRNA in exosomes and constructing a dual-lock activity score using the Michaelis equation and logic gating functions. This greatly reduces misjudgments caused by single-indicator elevations due to nonspecific inflammation or benign lesions, improves the specificity of identifying occult micrometastases, and overcomes background noise interference from liquid biopsy.

[0107] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for assessing colorectal cancer metastasis based on NF-κB pathway activity, characterized in that: include: Colorectal cancer tissue samples and paired peripheral blood samples were obtained, exosomes were isolated, and the tissue samples were divided into spatial resolution subsamples and functional verification subsamples. Multiple immunofluorescence imaging was performed on the spatial resolution subsamples to identify phosphorylated p65-positive cancer cell populations and phosphorylated STAT3-positive immune cell populations and extract cell phenotypic data. The spatial interaction weight values ​​between highly active cells were calculated based on cell phenotype data to obtain the paracrine activation index of the tumor microenvironment. The abundance of phosphorylated proteins upstream of the NF-κB pathway and the expression levels of downstream transfer effect genes in the functional verification subsample were detected, and the pathway functional coupling score was obtained by combining the degree of matching between the two. The levels of phosphorylated IKK protein and specific pro-transfer microRNA in exosomes were measured, and the product of the two was used to obtain the exosome dual-lock activity score. The fitted value of the tumor microenvironment paracrine activation index and the pathway function coupling score is defined as the local tissue activity characteristic, and the exosome dual-lock activity score is defined as the systemic circulating activity characteristic. The difference between the systemic circulating activity characteristic and the local tissue activity characteristic is calculated and multiplied by a preset penalty coefficient to obtain the occult metastasis penalty item. The local tissue activity characteristic, the systemic circulating activity characteristic, and the occult metastasis penalty item are added together to obtain the total risk score. The colorectal cancer metastasis risk rating is determined based on the preset threshold range in which the total risk score falls. A comprehensive assessment report is generated based on the colorectal cancer metastasis risk rating.

2. The method for assessing colorectal cancer metastasis based on NF-κB pathway activity as described in claim 1, characterized in that: The specific steps for obtaining colorectal cancer tissue samples and paired peripheral blood samples, isolating exosomes, and dividing the tissue samples into spatial resolution sub-samples and functional verification sub-samples are as follows: Fresh colorectal cancer tissue samples were obtained through surgical resection or puncture biopsy, and paired peripheral blood samples were collected simultaneously at the same time point. Some fresh colorectal cancer tissue samples were fixed by soaking in fixative, dehydrated, cleared and paraffin-impregnated, then embedded in paraffin and cut into tissue sections, which were used as sub-samples for spatial analysis. Fresh colorectal cancer tissue samples were flash-frozen using cryogenic freezing medium and then stored in an ultra-low temperature environment as functional validation sub-samples. Paired peripheral blood samples were centrifuged stepwise using differential centrifugation to remove cell debris and platelets. The supernatant was collected and then subjected to ultracentrifugation. The precipitate was resuspended in buffer to obtain a high-purity exosome suspension.

3. The method for assessing colorectal cancer metastasis based on NF-κB pathway activity as described in claim 2, characterized in that: The specific steps for performing multiplex immunofluorescence imaging on the spatially analyzed subsamples to identify phosphorylated p65-positive cancer cell populations and phosphorylated STAT3-positive immune cell populations and extract cell phenotypic data are as follows: Spatial analysis subsamples were cyclically stained using multiple immunofluorescence staining techniques. The antibody combination included cancer cell-specific marker antibodies, nuclear factor κBp65 subunit phosphorylation site-specific antibodies, macrophage-specific marker antibodies, and signal transduction and transcription activator factor 3 phosphorylation site-specific antibodies. The stained spatial resolution subsamples were scanned in their entirety using a multispectral microscopy system to obtain high-resolution digital images; High-resolution digital images are used to identify cell nuclei and cytoplasm regions using image segmentation algorithms. Based on fluorescence intensity thresholds, cancer cells with intranuclear phosphorylated p65 signals stronger than a preset cancer cell threshold are selected to form a phosphorylated p65 positive cancer cell population. At the same time, immune cells with intracytoplasmic phosphorylated STAT3 signals stronger than a preset immune cell threshold are selected to form a phosphorylated STAT3 positive immune cell population. The two-dimensional coordinates of the center point of each cell in the phosphorylated p65-positive cancer cell population and the phosphorylated STAT3-positive immune cell population, along with the corresponding fluorescence intensity value, were extracted using a coordinate extraction program to form cell phenotypic data.

4. The method for assessing colorectal cancer metastasis based on NF-κB pathway activity as described in claim 3, characterized in that: The specific steps for calculating the spatial interaction weights between highly active cells based on cell phenotype data to obtain the paracrine activation index of the tumor microenvironment are as follows: The distance relationship between each phosphorylated p65-positive cancer cell and phosphorylated STAT3-positive immune cells within a predetermined radius in the cell phenotype data is modeled using an exponential decay function model to simulate the physical law that cytokine diffusion weakens with increasing distance. The paracrine activation index of the tumor microenvironment is calculated by multiplying the nucleus-to-cytoplasm ratio of each cancer cell as the baseline activity coefficient by the logarithm of the sum of the weighted fluorescence intensities of neighboring immune cells, and then averaging the results over all cancer cells. The expression is as follows: ; in, It represents the paracrine activation index of the tumor microenvironment. The total number of cells in the phosphorylated p65-positive cancer cell population. and The first The average fluorescence intensity of the nucleus and cytoplasm regions of each cancer cell. For the first The number of phosphorylated STAT3-positive immune cells within a predetermined radius of action centered on a cancer cell. For the first The average fluorescence intensity of each immune cell This represents the maximum fluorescence intensity of all immune cells within the current field of view. For the first The first cancer cell and the first The Euclidean distance between the centroids of immune cells This is the spatial decay constant characterizing the diffusion capacity of cytokines.

5. The method for assessing colorectal cancer metastasis based on NF-κB pathway activity as described in claim 4, characterized in that: The detection function verifies the abundance of phosphorylated proteins upstream of the NFκB pathway and the expression levels of downstream transfer effector genes in the subsample. A pathway functional coupling score is obtained by combining the degree of matching between the two. The specific steps are as follows: The functional validation subsamples were lysed with protein extraction reagent and total protein was extracted. The concentrations of phosphorylated IKK protein and total IKK protein were detected by enzyme-linked immunosorbent assay. The functional validation subsamples were lysed with RNA extraction reagent and total RNA was extracted. After reverse transcription, the relative expression levels of matrix metalloproteinase 9 gene, CXC chemokine receptor 4 gene and vascular endothelial growth factor gene relative to the internal reference gene were detected by conventional quantitative polymerase chain reaction. The expression for the pathway functional coupling score is: ; in, For pathway functional coupling scoring, and These represent the concentrations of phosphorylated IKK protein and total IKK protein, respectively. For the first The relative expression levels of downstream transfer effect genes, For the first Preset weighting coefficients for each gene, The Gaussian error function is... This is a commonly used logarithmic function.

6. The method for assessing colorectal cancer metastasis based on NF-κB pathway activity as described in claim 5, characterized in that: The determination of phosphorylated IKK protein content and specific pro-transfer microRNA content in exosomes, and the calculation of exosome dual-lock activity score using the product of the two, are as follows: The exosome suspension was subjected to lysis buffer to break down the membrane, releasing the internal and membrane surface contents; The concentration of phosphorylated IKK protein in the lysate was detected by electrochemiluminescence immunoassay, and the concentrations of microRNA-21 and microRNA-221 in the lysate were detected by nucleic acid hybridization capture assay. The expression for the exosome double-lock activity score is: ; in, The exosome dual-lock activity score, This refers to the concentration of phosphorylated IKK protein in exosomes. and These represent the concentrations of microRNA-21 and microRNA-221 in exosomes, respectively. The preset half-saturation constant, Here, delta is the preset microRNA activation threshold, and delta is the preset coefficient of variation. It is the arctangent function. Pi is the mathematical constant of a circle.

7. The method for assessing colorectal cancer metastasis based on NF-κB pathway activity as described in claim 6, characterized in that: The fitted value of the tumor microenvironment paracrine activation index and the pathway function coupling score is defined as the local tissue activity characteristic, and the exosome dual-lock activity score is defined as the systemic circulating activity characteristic. The difference between the systemic circulating activity characteristic and the local tissue activity characteristic is calculated and multiplied by a preset penalty coefficient to obtain the occult metastasis penalty item. The local tissue activity characteristic, the systemic circulating activity characteristic, and the occult metastasis penalty item are added together to obtain the total risk score. The specific steps are as follows: The tumor microenvironment paracrine activation index and pathway function coupling score were multiplied to obtain the tissue local activity characteristic value, and the exosome double-lock activity score was directly assigned as the systemic circulation activity characteristic value. The expression for the total risk score of colorectal cancer metastasis is: ; in, The total risk score for colorectal cancer metastasis. This represents the characteristic value of local tissue activity. This represents the characteristic value of systemic circulatory activity. and These are the preset local weighting coefficients and the whole-body weighting coefficients, respectively. This is the activity difference value, when hour ,no , The preset penalty intensity coefficient, The preset sensitivity constant, It is an exponential function.

8. The method for assessing colorectal cancer metastasis based on NF-κB pathway activity as described in claim 7, characterized in that: The process involves determining the colorectal cancer metastasis risk rating based on the preset threshold range within which the total risk score falls, and generating a comprehensive assessment report based on the colorectal cancer metastasis risk rating. The specific steps are as follows: The total risk score is compared with the preset upper limit of low risk threshold and lower limit of high risk threshold using the interval comparison method; When the total risk score is less than the upper limit of the low risk threshold, the risk rating of colorectal cancer metastasis is determined to be low risk. When the total risk score is greater than or equal to the upper limit of the low-risk threshold and less than the lower limit of the high-risk threshold, it is judged as a medium-risk level; When the total risk score is greater than or equal to the lower limit of the high-risk threshold, it is judged as a high-risk level; The risk rating of colorectal cancer metastasis, the paracrine activation index of the tumor microenvironment, the pathway functional coupling score, and the exosome dual-lock activity score are integrated into a comprehensive assessment report through the report generation template.

9. A computer device comprising a memory and a processor, wherein the memory stores a computer program, characterized in that: When the processor executes the computer program, it implements the steps of the method for assessing colorectal cancer metastasis based on NF-κB pathway activity as described in any one of claims 1 to 8.

10. A computer-readable storage medium having a computer program stored thereon, characterized in that: When the computer program is executed by the processor, it implements the steps of the method for assessing colorectal cancer metastasis based on NF-κB pathway activity as described in any one of claims 1 to 8.

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