Application of collagen type iv and its encoding gene in prognosis evaluation and treatment of nasopharyngeal carcinoma

Abstract

This invention provides the application of type IV collagen and its encoding gene in the prognostic assessment and treatment of nasopharyngeal carcinoma (NPC). Research has shown that high expression of type IV collagen and its encoding gene COL4A1 is significantly associated with lower T-cell infiltration in patients with recurrent NPC; compared to NPC patients with low expression of type IV collagen, those with high expression have worse LRRFS and OS; and the expression level of the type IV collagen encoding gene COL4A1 is significantly associated with worse PFS in NPC patients. Therefore, type IV collagen and its encoding gene COL4A1 can serve as specific prognostic biomarkers for assessing the prognosis of NPC patients. Furthermore, inhibiting the expression of type IV collagen can effectively inhibit the growth of NPC; therefore, agents that inhibit type IV collagen expression can be used as drugs to inhibit immune escape in NPC and for the treatment of NPC.

Description

Technical Field

[0001] This invention belongs to the field of molecular diagnostic technology, specifically relating to the application of type IV collagen and its encoding gene in the prognostic assessment and treatment of nasopharyngeal carcinoma. Background Technology

[0002] Nasopharyngeal carcinoma (NPC) is highly prevalent in my country, and the presence of numerous infiltrating immune cells around tumor foci suggests the existence of a specific tumor microenvironment (TME) in NPC. The tumor microenvironment is a complex micro-ecosystem with spatiotemporal interactions among heterogeneous cell types (including malignant cells, immune cells, and stromal cells). The tumor microenvironment and its heterogeneity are closely related to tumor treatment resistance and recurrence. Radiotherapy is the most critical treatment for NPC patients. Approximately 10-20% of patients with endemic NPC experience local recurrence after previous radical radiotherapy, and the current standard treatment for locally recurrent NPC is two-course radiotherapy. However, this treatment often has significant toxic side effects, and the survival prognosis for recurrent NPC patients is poor, with a reported 5-year overall survival rate of only 44.9%. Immune checkpoint blockade (ICB) combats immune evasion by enhancing immune activation and has shown clinical efficacy, becoming an important rescue treatment for rNPC, although its response rate remains unsatisfactory.

[0003] TME and immune escape mechanisms can affect the treatment efficacy of ICB, but the mechanisms of immune escape in recurrent nasopharyngeal carcinoma remain unclear. Therefore, identifying biomarkers highly correlated with immune escape in patients with recurrent nasopharyngeal carcinoma could provide prognostic assessment and therapeutic targets for these patients. Summary of the Invention

[0004] Based on this, the purpose of this invention is to provide the application of type IV collagen and its encoding gene in the prognostic assessment and treatment of nasopharyngeal carcinoma.

[0005] To achieve the above objectives, the present invention adopts the following technical solution.

[0006] The first aspect of the present invention provides the use of reagents for detecting COL4A1 gene expression in the preparation of prognostic assessment products for nasopharyngeal carcinoma patients.

[0007] In some embodiments, the reagents include reagents for PCR detection, quantitative real-time PCR detection, and sequencing detection.

[0008] A second aspect of the invention provides the use of a reagent for detecting type IV collagen content in the preparation of prognostic assessment products for nasopharyngeal carcinoma patients.

[0009] In some embodiments, the reagents include reagents for immunohistochemical detection and Western blotting detection.

[0010] In some embodiments, the reagent is a specific antibody against type IV collagen.

[0011] In some embodiments, the specific antibody is a specific monoclonal antibody.

[0012] In some embodiments, the product is a reagent kit.

[0013] A third aspect of the invention provides the use of reagents that inhibit type IV collagen expression in the preparation of medicaments that inhibit immune escape from nasopharyngeal carcinoma.

[0014] A fourth aspect of the present invention provides the use of an agent for inhibiting COL4A1 gene expression in the preparation of a medicament for inhibiting immune escape from nasopharyngeal carcinoma.

[0015] This invention has found that high expression of type IV collagen and its encoding gene COL4A1 is significantly associated with lower T-cell infiltration in patients with recurrent nasopharyngeal carcinoma (NPC). Compared with NPC patients with low expression of type IV collagen, NPC patients with high expression of type IV collagen had worse LRRFS (p = 0.036) and OS (p = 0.031). Furthermore, the expression level of the type IV collagen encoding gene COL4A1 was significantly associated with worse PFS in NPC patients. Therefore, type IV collagen and its encoding gene COL4A1 can serve as specific prognostic biomarkers for assessing the prognosis of NPC patients.

[0016] Furthermore, inhibiting the expression of type IV collagen can promote T cell infiltration, suppress immune escape from nasopharyngeal carcinoma, and effectively inhibit its growth. Therefore, agents that inhibit type IV collagen expression can be used as drugs to suppress immune escape from nasopharyngeal carcinoma and for the treatment of nasopharyngeal carcinoma. Attached Figure Description

[0017] Figure 1 The results of the test for the type IV collagen and its encoding gene COL4A1 in Example 1 can be used for prognostic assessment of nasopharyngeal carcinoma patients.

[0018] Figure 2 ROC curves for type IV collagen used to assess the prognosis of nasopharyngeal carcinoma patients.

[0019] Figure 3 ROC curves for the COL4A1 gene used to assess the prognosis of nasopharyngeal carcinoma patients.

[0020] Figure 4 The results of a study on the therapeutic effect of COL4A1 gene knockout on nasopharyngeal carcinoma. Detailed Implementation

[0021] Experimental methods in the following embodiments of the present invention, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. All commonly used chemical reagents used in the embodiments are commercially available products.

[0022] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0023] The terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product, or device that includes a series of steps is not limited to the steps or modules listed, but may optionally include steps not listed, or may optionally include other steps inherent to such process, method, product, or device.

[0024] The following description is based on specific embodiments. Example 1

[0025] We collected fresh tissue samples from 11 newly diagnosed nasopharyngeal carcinoma (NPC) patients and 13 patients with recurrent NPC for single-cell sequencing. We also collected spatial transcriptome sequencing samples from 8 newly diagnosed NPC patients and 7 patients with recurrent NPC. Both single-cell sequencing and spatial transcriptome sequencing were performed by Guangzhou Yuanxin Biotechnology Co., Ltd.

[0026] Based on sequencing results, and by constructing a global tumor ecosystem map, we found that samples from patients with recurrent nasopharyngeal carcinoma exhibited higher levels of immune surveillance and immune escape characteristics. Figure 1 A), among which we found that genes significantly associated with immune escape, such as CD47, were significantly elevated in tumor cells in samples from patients with recurrent nasopharyngeal carcinoma, suggesting that they can inhibit dendritic cell infiltration and antigen presentation ( Figure 1 B).

[0027] We found significant changes in the immune microenvironment of recurrent nasopharyngeal carcinoma, which has a high ability to evade immune-mediated cell killing. Among these changes, we discovered that collagen can regulate lymphocyte infiltration in the tumor.

[0028] Compared with newly diagnosed nasopharyngeal carcinoma patients, patients with recurrent nasopharyngeal carcinoma showed significantly increased expression of type IV collagen.

[0029] Furthermore, multiplex immunohistochemistry of tissue samples from 40 paired, newly diagnosed-recurrent nasopharyngeal carcinoma patients in validation cohort 1 revealed that high expression of type IV collagen was associated with lower T cell infiltration. Type IV collagen expression was measured in all samples, with a median h-score of 30.4. Figure 1 C). Interestingly, we found that higher expression levels of type IV collagen were significantly negatively correlated with T cell infiltration (r = -0.545, p < 0.001) (Figure 1D).

[0030] Furthermore, because type IV collagen is primarily encoded by the genes COL4A1 and COL4A2, single-cell sequencing results also revealed significantly elevated expression levels of COL4A1 and COL4A2 in patients with recurrent nasopharyngeal carcinoma. Figure 1 E and F).

[0031] The above results indicate that the expression levels of type IV collagen and its encoding gene COL4A1 are related to the immune escape characteristics of patients with recurrent nasopharyngeal carcinoma, suggesting that the expression levels of type IV collagen and COL4A1 gene can be used as prognostic indicators for nasopharyngeal carcinoma patients. Example 2

[0032] To verify the correlation between type IV collagen expression levels and prognosis in nasopharyngeal carcinoma patients, we enrolled 86 newly diagnosed nasopharyngeal carcinoma patients who had received radical radiotherapy (validation cohort 2) for validation.

[0033] The content of type IV collagen in tissue samples from all nasopharyngeal carcinoma patients in validation cohort 2 was detected using multiple immunohistochemical staining to determine the expression level of type IV collagen.

[0034] Based on the test results, the top 50% of patients with type IV collagen expression were defined as the type IV collagen high expression group, and the bottom 50% were defined as the type IV collagen low expression group. According to this standard, in validation cohort 2, there were 43 cases in the type IV collagen high expression group and 43 cases in the type IV collagen low expression group.

[0035] LRRFS and OS were analyzed in patients with high and low type IV collagen expression. Results showed that in validation cohort 2, the high type IV collagen expression group had significantly worse LRRFS (p = 0.036) and OS (p = 0.031) compared to the low type IV collagen expression group. Figure 1 (G and 1H). This indicates that the expression of type IV collagen is significantly associated with the prognosis of patients with recurrent nasopharyngeal carcinoma and can serve as a prognostic biomarker for these patients.

[0036] Based on patients' overall survival (OS) results, receiver operating characteristic (ROC) curve analysis was used to evaluate the predictive efficacy of type IV collagen for patient prognosis. Results ( Figure 2 The results showed that the area under the curve (AUC) was 0.731; at the optimal cutoff value of 1.5, the sensitivity was 82.61% and the specificity was 63.49%, indicating that type IV collagen has a good ability to assist in prognostic assessment and can be used to help determine the prognostic risk of nasopharyngeal carcinoma patients. Example 3

[0037] To verify the correlation between the expression level of the COL4A1 gene, which encodes type IV collagen, and the prognosis of nasopharyngeal carcinoma patients, we re-included 88 newly diagnosed nasopharyngeal carcinoma patients (validation cohort 4) for validation.

[0038] The expression level of COL4A1 in 88 newly diagnosed nasopharyngeal carcinoma patients was detected using bulk RNA sequencing (conducted by Guangzhou Yuanxin Biotechnology Co., Ltd.). Based on the COL4A1 expression level, patients were divided into COL4A1 high expression and COL4A1 low expression groups using the `surv_cutpoint` function in R. Progression-free survival (PFS) analysis was performed on patients in the COL4A1 high expression and low expression groups, revealing that the PFS in the COL4A1 high expression group was significantly worse than that in the COL4A1 low expression group. Figure 1 Middle I).

[0039] Based on patient overall survival (OS) results, ROC curve analysis was used to evaluate its predictive efficacy for patient prognosis. Results ( Figure 3 The results showed an AUC of 0.766 with a 95% confidence interval of 0.6433–0.8884. At the optimal cutoff value of 39.04, the sensitivity was 73.68% and the specificity was 73.91%, suggesting that the COL4A1 gene can be used to assist in assessing the prognostic risk of nasopharyngeal carcinoma patients.

[0040] Consistently, in validation cohort 4, we found that high levels of COL4A1 transcriptional expression were significantly associated with low expression of T cell-related genes (Figure 1J). We also found that, based on ST data, COL4A1 expression was negatively correlated with both the CD8+ T ratio (r = -0.554, p = 0.0323) and the CD4+ T ratio (r = -0.886, p < 0.0001). Figure 1 K).

[0041] The above results indicate that the expression level of COL4A1, the gene encoding type IV collagen, is significantly associated with the prognosis of nasopharyngeal carcinoma patients, and patients with recurrent nasopharyngeal carcinoma who have high COL4A1 gene expression have a worse prognosis. Example 4

[0042] Since type IV collagen and its encoding gene COL4A1 are significantly associated with immune escape and prognosis in recurrent nasopharyngeal carcinoma, we further investigated whether it could serve as a therapeutic target to inhibit immune escape in nasopharyngeal carcinoma.

[0043] I. Experimental Methods To better investigate the relationship between type IV collagen and immune escape formation in nasopharyngeal carcinoma, we constructed a humanized mouse model. MCAM+ tumor-associated fibroblasts with knockdown or non-knockdown of the COL4A1 gene were co-injected into mice with C666 tumor cells. The group co-injected with PBS and C666 served as the control group. The specific procedures were as follows: To construct a xenograft model of humanized nasopharyngeal carcinoma cells (CDX), NCG mice were irradiated with a dose of 80-100 cGy. Twenty-four hours after irradiation, 1×10⁻⁶ CCAM+ cells were injected into the mice. 5 A fresh human volunteer CD34 + Hematopoietic stem cells (HSCs) were intravenously injected into mice, producing various hematopoietic or immune cells. CD34 in peripheral blood... + Mice with HSCs content exceeding 15% were identified as peripherally humanized. Ten weeks after hematopoietic stem cell transplantation, before constructing a nasopharyngeal carcinoma xenograft model, mice were bled and humanized chimerism was confirmed by flow cytometry. Mice were randomly assigned to C666+COL4A1 groups. KO MCAM + mCAFs group, C666+MCAM + mCAFs group and C666+PBS group (6 mice in each group). C666 cells (1×10⁻⁶) were used to... 6 ) and COL4A1 KO MCAM + mCAFs or MCAM + mCAFs were mixed in a 3:1 ratio or injected subcutaneously into the buttocks of mice with PBS as a negative control. On day 10, when the tumors in the mice were measurable, the tumor volume (V) and weight of each mouse were measured every 2 days.

[0044] On day 28, the tumor was removed from the mouse for measurement.

[0045] During the experiment, the following indicators were measured: (1) Tumor volume: The volume of mice was measured every 2 days. After the mice were sacrificed on the 28th day, the tumors were dissected and measured. The calculation formula is V = (length × width 2) / 2.

[0046] (2) Tumor weight: After the mice were sacrificed on day 28, the tumors were dissected and weighed.

[0047] (3) CD8 +T-cell activation markers were detected using a tumor dissociation kit. Following the protocol of Miltenyi Biotec (China), tumors in hu-NCG mice were dissected, washed, minced, and digested at 37°C. The cell suspension was incubated with RBC lysis buffer three times, 10 minutes each time. Subsequently, according to the manufacturer's protocol, cells were first stained with surface antibodies, washed with a fixation / permeation kit (BD Pharmingen), blocked with Human BD Fc Block (Fc1.3216), and stained with the listed antibodies. Live CD3+ was detected. + T cells were sorted from the suspension using FACS (Beckman Coulter) and stained with the following antibodies: BB515 Mouse Anti-Human CD3 (UCHT1) (BDPharmingen), PerCP-Cy5.5 Mouse Anti-Human CD8 (RPA-T8), PE-Cy7 Mouse Anti-Human CD69 (FN50), BV421 Mouse Anti-Human CD45RA (HI100), BV605 Mouse Anti-Human CD45RO (UCHL1), PE Mouse Anti-Human CD44 (G44-26FLEX), and APC Mouse Anti-Human CD62L (DREG-56).

[0048] II. Experimental Results The results show that MCAM + Experimental group with COL4A1 knockdown in tumor-associated fibroblasts (COL4A1) KO The tumor-forming ability was weaker, and the tumor volume and weight were significantly lower than those of the COL4A1 non-knockout group (Vector). Figure 4 A and Figure 4 As shown in Figure B, COL4A1 expression promotes immune escape in nasopharyngeal carcinoma, thereby accelerating its growth. Knockdown of COL4A1 expression inhibited immune escape in nasopharyngeal carcinoma, restricted its growth, and significantly reduced tumor volume and weight.

[0049] Furthermore, in IF staining and flow cytometry analysis, CD4 in the COL4A1 non-knockdown group was significantly lower. + and CD8 + The proportion of T cells was significantly lower than that of COL4A1. KO Group and PBS group ( Figure 4C). This indicates that IV collagen significantly inhibits T cell infiltration and promotes immune escape.

[0050] In addition, we observed higher type IV collagen scores and lower CD4 scores. + T cell infiltration (r=-0.523, p=0.026) and CD8+ T cell infiltration (r=-0.752, p=0.0003) were significantly correlated. Figure 4 D). This illustrates the important role and therapeutic potential of type IV collagen in inhibiting T-cell infiltration in nasopharyngeal carcinoma.

[0051] The above results demonstrate the potential role of type IV collagen in inhibiting T cell infiltration in nasopharyngeal carcinoma. Inhibiting type IV collagen expression can suppress immune escape in nasopharyngeal carcinoma, thereby exerting a therapeutic effect. Therefore, type IV collagen and its encoding gene COL4A1 can serve as novel targets for inhibiting immune escape and treating nasopharyngeal carcinoma.

[0052] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0053] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. Application of reagents for detecting COL4A1 gene expression in the preparation of prognostic assessment products for nasopharyngeal carcinoma patients.

2. Use according to claim 1, wherein The reagents include those used for PCR detection, quantitative real-time PCR detection, and sequencing detection.

3. Application of reagents for detecting type IV collagen content in the preparation of prognostic assessment products for nasopharyngeal carcinoma patients.

4. The application as described in claim 3, characterized in that, The reagents include those used for immunohistochemical detection and Western blotting detection.

5. The application as described in claim 4, characterized in that, The reagent is a specific antibody against type IV collagen; preferably, the specific antibody is a specific monoclonal antibody.

6. The application as described in any one of claims 1 to 5, characterized in that, The product in question is a reagent kit.

7. Application of reagents that inhibit type IV collagen expression in the preparation of drugs that inhibit immune escape from nasopharyngeal carcinoma.

8. Application of reagents that inhibit COL4A1 gene expression in the preparation of drugs that inhibit immune escape from nasopharyngeal carcinoma.

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