Detection kit for detecting interferon-related activating genes in neutrophil in tumor microenvironment

Abstract

The invention provides a detection kit for detecting interferon-related activating genes in neutrophil in a tumor microenvironment. The detection kit comprises an RNA extraction reagent, a reverse transcription reaction reagent, a quantitative PCR reagent, a probe, a positive reference substance and a negative reference substance. And the quantitative PCR reagent comprises upstream and downstream primers of nine interferon related genes, namely, ISG15 (Interferon Sorting Gene 15), RSAD2 (Registered Sequence Administration 2), IFIT1 (Interferon Institute of Transcription 1), IFI44L (Interferon Institute 44L), MX1, OAS1, STAT1, IRF7 and CXCL10. The detection kit can be used for detecting the expression quantity of nine genes including ISG15, RSAD2, IFIT1, IFI44L, MX1, OAS1, STAT1, IRF7 and CXCL10, accurate curative effect grouping is realized through dynamic monitoring of ISG15 + neutrophile granulocyte subpopulation, and meanwhile, improvement of curative effect prediction capability can be realized through combination with mrTRG.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a detection kit for detecting interferon-related activation genes in neutrophils in the tumor microenvironment. Background Technology

[0002] With the advent of immunotherapy in cancer treatment, approximately 5% of patients with microsatellite unstable (MSI-H) rectal cancer can benefit from immunotherapy, while the remaining 95% of MSS-type colorectal cancer patients have "cold tumors" and are resistant to immunotherapy. Therefore, finding ways to improve the efficacy of immunotherapy for patients with microsatellite stable (MSS) colorectal cancer is an urgent problem to be solved.

[0003] A nationwide multicenter phase III clinical trial conducted by our research group showed that a novel adjuvant therapy modality centered on short-course radiotherapy combined with immunotherapy and chemotherapy can increase the postoperative pathological response rate of MSS-type locally advanced rectal cancer to 39.8%. This suggests that a deeper analysis of radiotherapy-induced changes in the tumor microenvironment may provide clues for identifying key targets to enhance immunotherapy for MSS-type rectal cancer. Furthermore, although this novel adjuvant therapy modality can significantly increase the postoperative pathological response rate and improve patients' quality of life and sphincter preservation rates in locally advanced rectal cancer, there is currently a lack of robust indicators in clinical practice to effectively predict this treatment approach and help stratify patients for personalized treatment plans.

[0004] ISG15, short for Interferon-stimulated gene 15 protein, consists of two ubiquitin-like domains linked by a flexible linker. This structure shares similarities with ubiquitin, allowing it to participate in post-translational modifications like ubiquitin. In antiviral processes, ISG15 can covalently bind to viral proteins, affecting their function and thus interfering with the viral life cycle. In immune regulation, ISG15 can regulate the expression and release of cytokines such as interleukins, thereby influencing the recruitment and activation of immune cells. Furthermore, ISG15 can modify cyclin proteins, affecting cell cycle progression, and modify antioxidant enzymes or regulate redox pathways, reducing reactive oxygen species production. However, the role of ISG15 in neutrophils remains unexplored.

[0005] Neutrophils, comprising 50-70% of all white blood cells, are the most abundant type of white blood cell in the blood. They also form the first line of defense against bacterial and fungal infections. Recent studies have shown that they survive much longer than previously thought, with neutrophils surviving for more than five days in certain specific microenvironments. This implies that neutrophils possess exceptional plasticity, capable of being modulated and differentiated into different subsets by varying microenvironments, each performing distinct biological functions. The role of neutrophils in tumorigenesis and development remains controversial. Some studies have found that neutrophils promote tumor progression by releasing MMP9. However, other studies suggest that neutrophils may exert anti-tumor effects by activating anti-tumor immune responses and enhancing tumor cell clearance. Existing research has depicted functional subsets of neutrophils in the tumor microenvironment through single-cell sequencing; however, their dynamic changes during treatment remain largely unexplored.

[0006] Therefore, it is necessary to design a detection kit for detecting interferon-related activation genes in neutrophils in the tumor microenvironment. Summary of the Invention

[0007] The purpose of this invention is to provide a detection kit for detecting interferon-related activation genes in neutrophils in the tumor microenvironment. By detecting the dynamic changes of ISG15+ neutrophils, patient efficacy grouping can be achieved. At the same time, the combination with mrTRG can improve the predictive ability of efficacy.

[0008] To achieve the above objectives, the present invention provides a detection kit for detecting interferon-related activation genes in neutrophils within the tumor microenvironment, comprising the following components:

[0009] RNA extraction reagent: Contains Trizol reagent for extracting total RNA from neutrophils;

[0010] Reverse transcription reaction reagents: contain reverse transcriptase, reverse transcription reaction buffer, reverse transcription storage solution, dNTP mixture, random primers, and RNase inhibitors; the reverse transcription reaction reagents reverse transcribe the extracted RNA into cDNA, providing a template for subsequent quantitative PCR detection;

[0011] Quantitative PCR reagent: Used for quantitative PCR amplification and detection of target genes; the quantitative PCR reagent contains Taq DNA polymerase, dNTPs, SYBR Green fluorescent dye, reaction buffer, and upstream and downstream primers for interferon-related genes ISG15, RSAD2, IFIT1, IFI44L, MX1, OAS1, STAT1, IRF7, and CXCL10.

[0012] ISG15

[0013] Pre-primer 5'-CGCAGATCACCCAGAAGATCG-3'

[0014] Back primer 5'-TTCGTCGCATTTGTCCACCA-3'

[0015] RSAD2

[0016] Pre-primer 5'-TGGGTGCTTACACCTGCTG-3'

[0017] Back primer 5'-GAAGTGATAGTTGACGCTGGTT-3'

[0018] IFIT1

[0019] Pre-primer 5'-TTGATGACGATGAAATGCCTGA-3'

[0020] Back primer 5'-CAGGTCACCAGACTCCTCAC-3'

[0021] IFI44L

[0022] Pre-primer 5'-AGCCGTCAGGGATGTACTATAAC-3'

[0023] Back primer 5'-AGGGAATCATTTGGCTCTGTAGA-3'

[0024] MX1

[0025] Pre-primer 5'-GTTTCCGAAGTGGACATCGCA-3'

[0026] Back primer 5'-CTGCACAGGTTGTTCTCAGC-3'

[0027] OAS1

[0028] Pre-primer 5'-TGTCCAAGGTGGTAAAGGGTG-3'

[0029] Back primer 5'-CCGGCGATTTAACTGATCCTG-3'

[0030] STAT1

[0031] Pre-primer 5'-CAGCTTGACTCAAAATTCCTGGA-3'

[0032] Back primer 5'-TGAAGATTACGCTTGCTTTTCCT-3'

[0033] IRF7

[0034] Pre-primer 5'-GCTGGACGTGACCATCATGTA-3'

[0035] Back primer 5'-GGGCCGTATAGGAACGTGC-3'

[0036] CXCL10

[0037] Pre-primer 5'-GTGGCATTCAAGGAGTACCTC-3'

[0038] Back primer 5'-TGATGGCCTTCGATTCTGGATT-3'

[0039] The sequence of the fluorescently labeled probe is as follows:

[0040] 5'-FAM-TGCTGCACTTCTTCATATGCCAACA-TAMRA-3';

[0041] In addition to positive and negative controls.

[0042] As a further improvement of the present invention, the concentrations of both upstream and downstream primers are 10 μM.

[0043] As a further improvement of the present invention, the quantitative PCR reagent comprises: 18.5 mmol Tris-HCl, 2.78 mmol / L MgCl2, 92.6 mmol / L KCl, 10 μmol / L upstream and downstream primers, 0.1 U / μl Taq enzyme, 400 nmol / L dNTP mixture, and H2O.

[0044] As a further improvement of the present invention, the reverse transcription reaction reagent comprises: 200 U / μl transcriptase and its matching 5× reaction buffer, 2.5 mM dATP, 2.5 mM dCTP, 2.5 mM dGTP, 2.5 mM dTTP, 50 μM random primers, and 40 U / μL LRNase inhibitor.

[0045] As a further improvement of the present invention, the RNA extraction reagent includes cell lysis buffer I, cell lysis buffer II, RNA washing buffer I, RNA washing buffer II, RNase-free water, and RNA separation column.

[0046] As a further improvement of the present invention, the cell lysis buffer I comprises 0.1 mol / L Tris-HCl, 0.05 mol / L magnesium chloride, H2O, and 0.1 mol / L NaCl; the cell lysis buffer II comprises: 3 mol / L guanidine isothiocyanate, 2 mol / L guanidine hydrochloride, 0.3 mol / L sodium acetate, 0.2% sodium dodecyl sulfate, and H2O;

[0047] RNA Washing Buffer I: 0.2 mol / L sodium acetate, 0.1 mol / L sodium chlorate, 0.1 mol / L Tris-HCl, H2O;

[0048] RNA Wash Buffer II: 1.2 mol / L sodium citrate, 0.5 mol / L Tris-HCl, H2O.

[0049] As a further improvement of the present invention, the reverse transcription reaction buffer contains: 9.1 μmol / L Oligo(dT) 12-18 3.6 U / ml RNase Inhibitor, 72.7 mmol / L dithiothreitol, 182 mmol / L Tris-HCl, 273 mmol / L KCl, 11 mmol / L MgCl2.

[0050] As a further improvement of the present invention, the reverse transcription storage solution comprises: 20 mmol / L Tris-HCl, 100 mmol / L NaCl, 0.1 mmol / L EDTA, 1.0 mmol / L DTT, 50% (V / V) glycerol, 0.01% (V / V) Nonidet p-40, 200 U / L reverse transcriptase, and H2O.

[0051] The beneficial effects of this invention are:

[0052] The aggregation of ISG15+ neutrophils can disrupt the immunosuppressive microenvironment in tumor diseases such as rectal cancer, while suppression of this subset of neutrophils exacerbates the immunosuppressive microenvironment within tumor tissue. This invention uses the dynamic changes in ISG15+ neutrophils to group patients based on their treatment efficacy, and the combination with mrTRG can enhance the predictive ability of treatment efficacy. Attached Figure Description

[0053] Figure 1 This study examines the differences in ISG15+ neutrophil levels in patient tumor tissue across different treatment response groups.

[0054] Figure 2 To detect the expression of ISG15+ neutrophils and CD8+ T lymphocytes in rectal cancer patients with different treatment effects after treatment using immunofluorescence.

[0055] Figure 3 To target and inhibit the immunosuppressive microenvironment in tumor tissues of tumor-bearing mice that is aggravated by ISG15+ neutrophils.

[0056] Figure 4 The dynamic changes of ISG15+ neutrophils in tumor tissue of rectal cancer patients were combined with mrTRG as a predictive factor to predict treatment efficacy. Detailed Implementation

[0057] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0058] It should also be noted that, in order to avoid obscuring the present invention with unnecessary details, only the structures and / or processing steps closely related to the present invention are shown in the accompanying drawings, while other details that are not closely related to the present invention are omitted.

[0059] Additionally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0060] Single-cell sequencing revealed that ISG15+ neutrophil subsets were significantly enriched in the tumor microenvironment of rectal cancer patients after short-course radiotherapy and with good treatment outcomes, and were associated with better prognosis in rectal cancer patients. Further research showed that type I interferon released from tumors by short-course radiotherapy could significantly induce the formation of ISG15+ neutrophil subsets, suggesting that ISG15+ neutrophil subsets are key to improving the efficacy of immunotherapy for MSS-type rectal cancer after short-course radiotherapy.

[0061] In this application, we further discovered that radiotherapy-induced type I interferon promotes the expression of MHC class I molecules on the cell surface by activating the NOD1 / NF-κB signaling pathway, thereby improving the antigen-presenting function of neutrophils and ultimately activating the anti-tumor function of T cells in the tumor microenvironment. Furthermore, we explored the optimal induction conditions for ISG15+ neutrophil subsets in vitro, and the induced ISG15+ neutrophil subsets, when reinfused into vivo, demonstrated a synergistic anti-tumor effect with immune checkpoint inhibitors. These findings suggest that ISG15+ neutrophil subsets may be an important biomarker for predicting the prognosis of rectal cancer, and that the in vitro-induced ISG15+ neutrophil subsets have the function of an immune adjuvant in improving the cold tumor immune microenvironment.

[0062] In Experiment 1, ISG15+ neutrophils were significantly enriched in patients with locally advanced rectal cancer who achieved pathological complete remission after neoadjuvant therapy.

[0063] Differences in the dynamic changes of ISG15+ neutrophil content in tumor tissue among different treatment response groups.

[0064] After obtaining approval from the Ethics Committee of Union Hospital affiliated to Tongji Medical College of Huazhong University of Science and Technology and signing informed consent forms from patients, tumor tissues were collected from patients with locally advanced rectal cancer who underwent short-course radiotherapy followed by chemotherapy and immunotherapy before and after neoadjuvant therapy.

[0065] Based on their response to neoadjuvant therapy, patients were divided into a pathologically complete response (pCR) group and a non-pathologically complete response (npCR) group. A total of 10 pairs of tumor tissue samples were collected before and after treatment, including 5 pCR patients and 5 npCR patients.

[0066] Multiplex immunofluorescence assay was used to detect the composition and distribution of immune cells in rectal tumor tissues before and after treatment: Fresh tissue specimens were serially sectioned, fixed in 40 g / L paraformaldehyde for 12 h, dehydrated in 200 mL / L sucrose solution for 12 h, embedded in OCT, frozen, and serially sectioned for routine immunofluorescence staining: primary antibody against CD66b and ISG15 (CD8 dilution 1:200, secondary antibody dilution 1:100), washed three times with PBS for 5 min each time in the dark, and 70 μl of DAPI dye was added and incubated at room temperature for 5 min in the dark. The tissues were washed three times with PBS for 5 min each time in the dark, and then an appropriate amount of anti-fluorescence quenching mounting medium was added. The tissues were observed under a laser confocal microscope, and images were photographed and saved.

[0067] The results are as follows Figure 1 As shown: Figure 1 Images a, b, c, and d in the figure show representative images of multicolor immunofluorescence. Figure 1 Figures e and f show that ISG15+ neutrophils infiltrated significantly more tissues in the pCR group than in the npCR group, and that ISG15+ neutrophil infiltration increased significantly after patients received neoadjuvant therapy.

[0068] Figure 1 The results in g and h show that in patients who achieved pCR after treatment, the infiltration of ISG15+ neutrophils was significantly increased compared to before treatment, while this change was not observed in patients in the npCR group. This indicates that the ISG15 interferon-related gene is significantly correlated with patient prognosis.

[0069] Experiment Example 2: Immunosuppressive Microenvironment in Tumor Tissue of Tumor-Bearing Mice by Targeted Inhibition of ISG15+ Neutrophils

[0070] Six- to eight-week-old C57BL / 6J mice, regardless of sex, were anesthetized and subcutaneously injected with 3 × 10⁵ MC38 colorectal cancer cells to establish a subcutaneous tumor model. After tumor formation, mice were randomly assigned to groups according to tumor size: control group, ISG15 inhibition (SARS-CoV-PLo) group, neutrophil clearance group, and ISG15 inhibition + neutrophil clearance (SARS-CoV-PLo) group. Tumors were targeted until they reached a size of 100-150 mm. 3 At that time, each group was given the appropriate treatment. The changes in tumor tissue size and survival status of mice in each group were observed and recorded. After 14 days of tumor cell injection, the tumor cells were weighed and analyzed by multicolor flow cytometry.

[0071] The results are as follows Figure 2 As shown: Targeted inhibition of ISG15+ neutrophils promotes tumor growth in C57 tumor-bearing mice. Figure 2 (a, b, and c) Combined inhibition of ISG15+ neutrophils and clearance of neutrophils eliminates this pro-tumor effect. Flow cytometry shows that targeted inhibition of ISG15+ neutrophils reduces the infiltration of CD8+ T cells in the tumor, while the reduction in CD8+ T cells disappears after combined inhibition of ISG15+ neutrophils and clearance of neutrophils. Figure 2 (d, e).

[0072] The results show that ISG15 plays an important role in tumor tissue, and this role depends on neutrophils.

[0073] Experiment 3: Infusion of ISG15+ neutrophils inhibited tumor growth in tumor-bearing mice and improved the immunosuppressive microenvironment.

[0074] Six- to eight-week-old C57BL / 6J mice, regardless of sex, were anesthetized and subcutaneously injected with 3 × 10⁵ MC38 colorectal cancer cells to establish a subcutaneous tumor model. After tumor formation, mice were randomly assigned to groups according to tumor size: control group, ISG15+ neutrophil group induced by radiotherapy culture medium, neutrophil group induced by IFN-α infusion, and IFN-α intraperitoneal injection group. Tumors were targeted until they reached a size of 100-150 mm. 3 At that time, each group was given the appropriate treatment. The changes in tumor tissue size and survival status of mice in each group were observed and recorded. After 14 days of tumor cell injection, the tumor cells were weighed and analyzed by multicolor flow cytometry.

[0075] The results are as follows Figure 3 As shown: Reinfusion of in vitro induced ISG15+ neutrophils can inhibit tumor growth in tumor-bearing mice. Figure 3(a, b, c) but its effect is slightly less than that of direct use of IFN-α recombinant cytokines. Flow cytometry showed that reinfusion of in vitro induced ISG15+ neutrophils promoted the infiltration of CD8+ T cells into the tumor. Figure 3 (d, e).

[0076] The results show that the reinfusion of in vitro induced ISG15+ neutrophils has an anti-tumor effect, but its anti-tumor effect is less than that of IFN-α alone.

[0077] Experiment Example 4

[0078] Dynamic changes in blood ISG15 score before and after treatment in cancer patients, combined with mrTRG index, can enhance the accuracy of predicting treatment efficacy. Treatment response information in tumor tissues, expression of interferon-related genes characterized by ISG15+ in peripheral blood neutrophils, and mrTRG index results were collected. Receptor operating characteristic (ROC) curves were used to further evaluate the prognostic value of dynamic changes in peripheral blood ISG15 score in predicting the efficacy of neoadjuvant therapy.

[0079] The results are as follows Figure 4 As shown, the AUC value of the ISG15 score in the tumor microenvironment before and after treatment in tumor patients was 0.530 when used alone, the AUC value of the mrTRG index was 0.527 when used alone, and the AUC value of the combined use of the two indices was 0.820. This suggests that the combined use of peripheral blood ISG15 score and mrTRG index in tumor patients can serve as a molecular marker for predicting the efficacy of short-course chemoradiotherapy combined with immunotherapy.

[0080] This invention provides a detection kit for detecting interferon-related activation genes in neutrophils in the tumor microenvironment, comprising the following components: RNA extraction reagent, reverse transcription reaction reagent, quantitative PCR reagent, probe, positive control, and negative control.

[0081] RNA extraction reagent for extracting total RNA from neutrophils.

[0082] RNA extraction reagents include Trizol reagent.

[0083] Trizol reagent effectively lyses cells, separating RNA from proteins and DNA, ensuring high purity and integrity of the extracted RNA to meet the needs of subsequent gene expression detection. In addition, it is equipped with reagents such as chloroform, isopropanol, and 75% ethanol for phase separation, precipitation, and washing steps during RNA extraction.

[0084] Specifically, RNA extraction reagents include cell lysis buffer I, cell lysis buffer II, RNA washing buffer I, RNA washing buffer II, RNase-free water, and RNA separation column.

[0085] Cell lysis buffer I contains: 0.1 mol / L Tris-HCl (pH 7.6), 0.05 mol / L magnesium chloride, H2O, and 0.1 mol / L NaCl.

[0086] Cell lysis buffer II contains: 3 mol / L guanidine isothiocyanate, 2 mol / L guanidine hydrochloride, 0.3 mol / L sodium acetate, 0.2% sodium dodecyl sulfate, and H2O.

[0087] RNA Wash Buffer I contains: 0.2 mol / L sodium acetate, 0.1 mol / L sodium chlorate, 0.1 mol / L Tris-HCl, and H2O.

[0088] RNA Wash Buffer II contains: 1.2 mol / L sodium citrate, 0.5 mol / L Tris-HCl (pH 7.5), and H2O.

[0089] RNase-free water: Add diethyl pyrocarbonate to deionized water at a concentration of 0.05%, let stand at room temperature for 10 hours, then autoclave at 121°C for 20 minutes, and store at room temperature.

[0090] RNA separation column: purchased from OMAGE Biotechnology, USA. It can specifically bind to RNA and can be washed away with water to achieve the purpose of separating and purifying RNA.

[0091] The reverse transcription reagent reverse transcribes the extracted RNA into cDNA, providing a template for subsequent quantitative PCR detection.

[0092] Reverse transcription reaction reagents: including reverse transcriptase, reverse transcription reaction buffer, reverse transcription storage solution, dNTP mixture, random primers, and RNase inhibitors.

[0093] Reverse transcriptase uses RNA as a template to synthesize cDNA according to the base pairing principle. The reverse transcription reaction buffer provides a suitable reaction environment, the dNTP mixture provides raw materials for cDNA synthesis, random primers are used to initiate the reverse transcription reaction, and RNase inhibitors are used to prevent RNA from being degraded during the reaction.

[0094] Specifically, the reverse transcription reaction reagents include: 200 U / μl transcriptase and its matching 5× reverse transcription reaction buffer, 2.5 mM dATP, 2.5 mM dCTP, 2.5 mM dGTP, 2.5 mM dTTP, 50 μM random primers, and 40 U / μL RNase inhibitor.

[0095] The reverse transcription reaction buffer contains: 9.1 μmol / L Oligo(dT) 12-18 3.6 U / ml RNase Inhibitor, 72.7 mmol / L dithiothreitol, 182 mmol / L Tris-HCl (pH 8.3), 273 mmol / L KCl, 11 mmol / L MgCl2.

[0096] The reverse transcription storage solution contains: 20 mmol / L Tris-HCl (pH 7.5), 100 mmol / L NaCl, 0.1 mmol / L EDTA, 1.0 mmol / L DTT, 50% (V / V) glycerol, 0.01% (V / V) Nonidet p-40, 200 U / L reverse transcriptase, and H2O.

[0097] Quantitative PCR reagents are used for quantitative PCR amplification and detection of target genes.

[0098] In this application, the commercially available reagent SYBR Green PCR Master Mix was used.

[0099] The quantitative PCR reagent contains Taq DNA polymerase, dNTPs, SYBR Green fluorescent dye, reaction buffer, and upstream and downstream primers for interferon-related genes ISG15, RSAD2, IFIT1, IFI44L, MX1, OAS1, STAT1, IRF7, and CXCL10.

[0100] ISG15

[0101] Pre-primer 5'-CGCAGATCACCCAGAAGATCG-3'

[0102] Back primer 5'-TTCGTCGCATTTGTCCACCA-3'

[0103] RSAD2

[0104] Pre-primer 5'-TGGGTGCTTACACCTGCTG-3'

[0105] Back primer 5'-GAAGTGATAGTTGACGCTGGTT-3'

[0106] IFIT1

[0107] Pre-primer 5'-TTGATGACGATGAAATGCCTGA-3'

[0108] Back primer 5'-CAGGTCACCAGACTCCTCAC-3'

[0109] IFI44L

[0110] Pre-primer 5'-AGCCGTCAGGGATGTACTATAAC-3'

[0111] Back primer 5'-AGGGAATCATTTGGCTCTGTAGA-3'

[0112] MX1

[0113] Pre-primer 5'-GTTTCCGAAGTGGACATCGCA-3'

[0114] Back primer 5'-CTGCACAGGTTGTTCTCAGC-3'

[0115] OAS1

[0116] Pre-primer 5'-TGTCCAAGGTGGTAAAGGGTG-3'

[0117] Back primer 5'-CCGGCGATTTAACTGATCCTG-3'

[0118] STAT1

[0119] Pre-primer 5'-CAGCTTGACTCAAAATTCCTGGA-3'

[0120] Back primer 5'-TGAAGATTACGCTTGCTTTTCCT-3'

[0121] IRF7

[0122] Pre-primer 5'-GCTGGACGTGACCATCATGTA-3'

[0123] Back primer 5'-GGGCCGTATAGGAACGTGC-3'

[0124] CXCL10

[0125] Pre-primer 5'-GTGGCATTCAAGGAGTACCTC-3'

[0126] Back primer 5'-TGATGGCCTTCGATTCTGGATT-3'.

[0127] In this application, Taq DNA polymerase catalyzes DNA strand elongation in the PCR reaction, dNTPs serve as the raw materials for synthesizing new DNA strands, and SYBR Green fluorescent dye specifically binds to double-stranded DNA, generating a fluorescence signal during PCR amplification. The PCR reaction progress can be monitored in real time by detecting the fluorescence intensity, thereby enabling quantitative analysis of the target gene. Furthermore, it is equipped with specific primers for nine genes: ISG15, RSAD2, IFIT1, IFI44L, MX1, OAS1, STAT1, IRF7, and CXCL10. The primer sequences have been optimized to ensure primer specificity and amplification efficiency, avoiding non-specific amplification.

[0128] The primer concentration is 10 μM, and can be appropriately diluted according to actual experimental needs.

[0129] Specifically, the quantitative PCR reagent contains: 18.5 mmol Tris-HCl, 2.78 mmol / L MgCl2, 92.6 mmol / L KCl, 0.37 μmol / L upstream and downstream primers, 0.1 U / μl Taq enzyme, 400 nmol / L dNTP mixture, and H2O.

[0130] The sequence of the fluorescently labeled probe is as follows:

[0131] 5'-FAM-TGCTGCACTTCTTCATATGCCAACA-TAMRA-3'.

[0132] The reagent kit is prepared as follows:

[0133] 1) Reagent Specifications and Packaging: Cell Lysis Buffer I, 1 bottle (30ml / bottle); Cell Lysis Buffer II, 1 bottle (10ml / bottle); RNA Wash Buffer I, 1 bottle (10ml / bottle); RNA Wash Solution II, 1 bottle (3ml / bottle); RNase-free, 1 bottle (2ml / bottle); RNA Separation Column (20 columns); Reverse Transcription Reaction Buffer, 1 tube (27.5u / tube); dNTP Mixture, 1 tube (5μl / tube); Reverse Transcription Enzyme, 1 tube (2.5μl / tube); PCR Reaction Solution, 1 tube (216μl / tube)

[0134] Accurately weigh or measure the corresponding chemical reagents according to their instructions and experimental requirements, and prepare them in a sterile, nuclease-free environment. For example, dilute Trizol reagent to prepare 5× reverse transcription buffer, dNTP mixture, etc. After preparation, use sterile, nuclease-free pipettes to aliquot each reagent into different centrifuge tubes or microplates, ensuring that the reagent content in each tube or well is accurate and consistent. During aliquoting, take care to avoid cross-contamination of reagents and clearly label them with the reagent name, concentration, aliquoting date, and other information.

[0135] 2) Primer Design and Synthesis: Primers were designed based on the sequences of nine genes: ISG15, RSAD2, IFIT1, IFI44L, MX1, OAS1, STAT1, IRF7, and CXCL10, using professional primer design software (such as Primer Premier 5.0). Basic primer design principles were followed, such as primer length generally being 18-25 bp, GC content between 40% and 60%, and avoiding primer dimers and hairpin structures. After the designed primer sequences were evaluated and confirmed to be correct, they were synthesized by a professional biotechnology company. The synthesized primers were purified by PAGE or HPLC to remove impurities, ensuring primer quality and purity. After dissolving the primers, a 10 μM stock solution was prepared, aliquoted, and stored at -20°C for later use.

[0136] 3) Kit Assembly: The pre-packaged RNA extraction reagent, reverse transcription reagent, quantitative PCR reagent, positive control, negative control, and sample processing aids are loaded into the kit according to a specific order and specifications. A detailed instruction manual is also provided, including the kit's purpose, operating principles, procedures, result interpretation methods, and precautions, ensuring users can use the kit correctly and appropriately. The kit's outer packaging should clearly indicate the kit name, manufacturer, production date, expiration date, and storage conditions for easy management and use.

[0137] Reagent kit application examples

[0138] 1. Sample Collection and Processing: Tumor tissue samples were collected from patients with locally advanced rectal cancer before and after neoadjuvant therapy, with approval from the ethics committee and informed consent from the patients. The tumor tissue samples were cut into small pieces, and an appropriate amount of PBS buffer was added. The samples were homogenized using a tissue homogenizer to ensure thorough dispersion of the cells. Then, proteinase K solution was added, and the mixture was incubated at 56°C for 1-2 hours to digest proteins and promote cell lysis. After incubation, RNA was extracted according to the Trizol reagent instructions. The extracted RNA was dissolved in an appropriate amount of nuclease-free water, and the concentration and purity of the RNA were determined to ensure that the RNA quality met the requirements for subsequent experiments.

[0139] 2. Reverse transcription reaction: Take an appropriate amount of RNA sample, and add 5× reverse transcription reaction buffer, dNTP mixture, random primers, RNase inhibitor, reverse transcriptase and other reagents to the reaction system in sequence. After gently mixing, place it in a PCR instrument to carry out the reverse transcription reaction.

[0140] The reaction conditions were: incubation at 25°C for 10 minutes, at 42°C for 60 minutes, and at 70°C for 10 minutes. After the reaction, the obtained cDNA could be used directly for subsequent quantitative PCR detection or stored at -20°C for later use.

[0141] 3. Quantitative PCR Detection: Using cDNA obtained from reverse transcription as a template, quantitative PCR was performed using SYBR Green PCR Master Mix and specific primers. Add appropriate amounts of SYBR Green PCR Master Mix, upstream and downstream primers for nine genes (ISG15, RSAD2, IFIT1, IFI44L, MX1, OAS1, STAT1, IRF7, and CXCL10), cDNA template, and nuclease-free water to the reaction system, bringing the total volume to 20 μL. Gently mix the reaction system and then place it in a quantitative PCR instrument for amplification.

[0142] The reaction conditions were: pre-denaturation at 95℃ for 3 minutes; followed by 40 cycles, each cycle consisting of denaturation at 95℃ for 15 seconds, annealing at 60℃ for 30 seconds, and extension at 72℃ for 30 seconds. During amplification, changes in fluorescence signals were monitored in real time using a quantitative PCR instrument, and the expression levels of nine genes—ISG15, RSAD2, IFIT1, IFI44L, MX1, OAS1, STAT1, IRF7, and CXCL10—were calculated based on a standard curve.

[0143] 4. Results analysis and efficacy group prediction: ISG score is calculated based on the calculated expression levels of each gene.

[0144] The specific method is as follows: First, the expression levels of each gene are standardized and their Z values ​​are calculated. Then, the Z values ​​of the nine genes are added together to obtain the ISG score.

[0145] ISGscore = Σ (Z-value of gene expression level)

[0146] Simultaneously, immunohistochemistry was used to detect the density of ISG15+ neutrophils (cells / mm²) in tumor tissue. 2 ).

[0147] Input ISG15+ neutrophil density and ISG score into the prediction model:

[0148] Response probability = 1 / (1+e^-(0.87×a+1.24×ISGscore-2.56)).

[0149] In the formula, 'a' refers to the density of ISG15+ neutrophils.

[0150] A probability value ≥ 0.65 indicates a treatment-sensitive group; a probability value < 0.65 indicates a treatment-resistant group. The accuracy and reliability of this kit in predicting treatment efficacy grouping were verified through sample testing and analysis of multiple patients with locally advanced rectal cancer.

[0151] The results showed that using this kit in conjunction with a predictive model can accurately predict patients' responses to short-course chemoradiotherapy combined with immunotherapy, providing an important reference for clinical development of personalized treatment plans.

[0152] 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.

Claims

1. A detection kit for detecting interferon-related activation genes in neutrophils within the tumor microenvironment, characterized in that, It contains the following ingredients: RNA extraction reagent: Contains Trizol reagent for extracting total RNA from neutrophils; Reverse transcription reaction reagents: including reverse transcriptase, reverse transcription reaction buffer, reverse transcription storage solution, dNTP mixture, random primers, and RNase inhibitors; The reverse transcription reagent reverse transcribes the extracted RNA into cDNA, providing a template for subsequent quantitative PCR detection; Quantitative PCR reagents: used for quantitative PCR amplification and detection of target genes; The quantitative PCR reagent contains Taq DNA polymerase, dNTPs, SYBR Green fluorescent dye, reaction buffer, and upstream and downstream primers for interferon-related genes ISG15, RSAD2, IFIT1, IFI44L, MX1, OAS1, STAT1, IRF7, and CXCL10. ISG15 Pre-primer 5'-CGCAGATCACCCAGAAGATCG-3' Back primer 5'-TTCGTCGCATTTGTCCACCA-3' RSAD2 Pre-primer 5'-TGGGTGCTTACACCTGCTG-3' Back primer 5'-GAAGTGATAGTTGACGCTGGTT-3' IFIT1 Pre-primer 5'-TTGATGACGATGAAATGCCTGA-3' Back primer 5'-CAGGTCACCAGACTCCTCAC-3' IFI44L Pre-primer 5'-AGCCGTCAGGGATGTACTATAAC-3' Back primer 5'-AGGGAATCATTTGGCTCTGTAGA-3' MX1 Pre-primer 5'-GTTTCCGAAGTGGACATCGCA-3' Back primer 5'-CTGCACAGGTTGTTCTCAGC-3' OAS1 Pre-primer 5'-TGTCCAAGGTGGTAAAGGGTG-3' Back primer 5'-CCGGCGATTTAACTGATCCTG-3' STAT1 Pre-primer 5'-CAGCTTGACTCAAAATTCCTGGA-3' Back primer 5'-TGAAGATTACGCTTGCTTTTCCT-3' IRF7 Pre-primer 5'-GCTGGACGTGACCATCATGTA-3' Back primer 5'-GGGCCGTATAGGAACGTGC-3' CXCL10 Pre-primer 5'-GTGGCATTCAAGGAGTACCTC-3' Back primer 5'-TGATGGCCTTCGATTCTGGATT-3' The sequence of the fluorescently labeled probe is as follows: 5'-FAM-TGCTGCACTTCTTCATATGCCAACA-TAMRA-3'; In addition to positive and negative controls.

2. The detection kit for detecting interferon-related activation genes in neutrophils in the tumor microenvironment according to claim 1, characterized in that: The concentrations of both upstream and downstream primers were 10 μM.

3. The detection kit for detecting interferon-related activation genes in neutrophils in the tumor microenvironment according to claim 1, characterized in that: The quantitative PCR reagent contains: 18.5 mmol Tris-HCl, 2.78 mmol / L MgCl2, 92.6 mmol / L KCl, 10 μmol / L upstream and downstream primers, 0.1 U / μl Taq enzyme, 400 nmol / L dNTP mixture, and H2O.

4. The detection kit for detecting interferon-related activation genes in neutrophils in the tumor microenvironment according to claim 1, characterized in that: The reverse transcription reaction reagent contains: 200 U / μl transcriptase and its matching 5× reaction buffer, 2.5 mM dATP, 2.5 mM dCTP, 2.5 mM dGTP, 2.5 mM dTTP, 50 μM random primers, and 40 U / μL RNase inhibitor.

5. The detection kit for detecting interferon-related activation genes in neutrophils in the tumor microenvironment according to claim 1, characterized in that: The RNA extraction reagents include cell lysis buffer I, cell lysis buffer II, RNA washing buffer I, RNA washing buffer II, RNase-free water, and an RNA separation column.

6. The detection kit for detecting interferon-related activation genes in neutrophils in the tumor microenvironment according to claim 5, characterized in that: The cell lysis buffer I contains 0.1 mol / L Tris-HCl, 0.05 mol / L magnesium chloride, H2O, and 0.1 mol / L NaCl; the cell lysis buffer II contains 3 mol / L guanidine isothiocyanate, 2 mol / L guanidine hydrochloride, 0.3 mol / L sodium acetate, 0.2% sodium dodecyl sulfate, and H2O. RNA Washing Buffer I: 0.2 mol / L sodium acetate, 0.1 mol / L sodium chlorate, 0.1 mol / L Tris-HCl, H2O; RNA Wash Buffer II: 1.2 mol / L sodium citrate, 0.5 mol / L Tris-HCl, H2O.

7. The detection kit for detecting interferon-related activation genes in neutrophils in the tumor microenvironment according to claim 1, characterized in that: The reverse transcription reaction buffer contains: 9.1 μmol / Loligo(dT) 12-18 3.6 U / ml RNase Inhibitor, 72.7 mmol / L dithiothreitol, 182 mmol / L Tris-HCl, 273 mmol / L KCl, 11 mmol / L MgCl2.

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