Application of IV-type collagen and coding gene thereof in prognosis evaluation and treatment of recurrent nasopharynx cancer

Abstract

The invention provides an application of type IV collagen and a coding gene thereof in prognosis evaluation and treatment of recurrent nasopharynx cancer. Researches find that high expression of the IV-type collagen, the coding gene COL4A1 of the IV-type collagen and the protein is remarkably related to lower T cell infiltration in patients with recurrent nasopharyngeal carcinoma; compared with recurrent nasopharyngeal carcinoma patients with low expression of IV collagen, recurrent nasopharyngeal carcinoma patients with high expression of IV collagen have poorer LRRFS and OS; the expression level of the IV type collagen coding gene COL4A1 is obviously related to poorer PFS of a patient with recurrent nasopharynx cancer. Therefore, the IV-type collagen and the coding gene COL4A1 thereof can be used as a specific prognosis evaluation biomarker for the recurrent nasopharyngeal carcinoma patient, and can be used for evaluating the prognosis condition of the recurrent nasopharyngeal carcinoma patient. Furthermore, the growth of the nasopharyngeal carcinoma can be effectively inhibited by inhibiting the expression of the IV type collagen, so that the reagent for inhibiting the expression of the IV type collagen can be used as a medicine for inhibiting the immune escape of the nasopharyngeal carcinoma and is used for treating the nasopharyngeal carcinoma.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular diagnosis, and in particular relates to the application of type IV collagen and its encoding gene in the prognosis evaluation and treatment of recurrent nasopharyngeal carcinoma. Background Art

[0002] Nasopharyngeal carcinoma (NPC) is highly prevalent in my country, and a large number of infiltrating immune cells have been found around tumor foci, suggesting the presence of a specific tumor microenvironment (TME) in NPC. The TME is a complex microecological system characterized by spatiotemporal interactions between heterogeneous cell types, including malignant cells, immune cells, and stromal cells. The TME and its heterogeneity are closely associated with 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 radical radiotherapy. Currently, the conventional treatment for locally recurrent NPC is a second course of radiotherapy. However, this treatment often carries significant toxicities and side effects, and the survival prognosis for patients with recurrent nasopharyngeal carcinoma (rNPC) is poor, with a reported 5-year overall survival rate of only 44.9%. Immune checkpoint blockade (ICB) has demonstrated clinical efficacy by enhancing immune activation to counteract immune evasion and has become an important salvage treatment for rNPC, although response rates remain suboptimal.

[0003] TME and immune escape mechanisms can affect the therapeutic efficacy of ICB, but the mechanism of immune escape in recurrent NPC remains unclear. Therefore, finding biomarkers that are highly correlated with immune escape in patients with recurrent NPC can provide prognostic assessment and treatment targets for patients with recurrent NPC. Summary of the Invention

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

[0005] To achieve the above-mentioned purpose, the present invention adopts the following technical solutions.

[0006] A first aspect of the present invention provides use of the COL4A1 gene as a biomarker in the prognosis assessment of patients with recurrent nasopharyngeal carcinoma.

[0007] A second aspect of the present invention provides the use of type IV collagen as a biomarker in the prognosis assessment of patients with recurrent nasopharyngeal carcinoma.

[0008] The third aspect of the present invention provides the use of a reagent for detecting COL4A1 gene expression in the preparation of a product for evaluating the prognosis of patients with recurrent nasopharyngeal carcinoma.

[0009] In some embodiments, the reagents include reagents for PCR detection, fluorescent quantitative PCR detection, and sequencing detection.

[0010] A fourth aspect of the present invention provides the use of a reagent for detecting type IV collagen content in the preparation of a product for evaluating the prognosis of patients with recurrent nasopharyngeal carcinoma.

[0011] In some embodiments, the reagents include reagents for immunohistochemistry detection and Western Blot detection.

[0012] In some embodiments, the agent is an antibody specific for type IV collagen.

[0013] In some embodiments, the specific antibody is a specific monoclonal antibody.

[0014] In some embodiments, the product is a kit.

[0015] A fifth aspect of the present invention provides use of an agent for inhibiting type IV collagen expression in the preparation of a drug for inhibiting immune escape of nasopharyngeal carcinoma.

[0016] A sixth aspect of the present invention provides use of an agent for inhibiting type IV collagen expression in the preparation of a medicament for treating nasopharyngeal carcinoma.

[0017] The present 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. Compared with patients with low expression of type IV collagen, patients with high expression of type IV collagen have worse LRRFS (p = 0.036) and OS (p = 0.031). The expression level of type IV collagen encoding gene COL4A1 is significantly correlated with worse PFS in patients with recurrent nasopharyngeal carcinoma. Therefore, type IV collagen and its encoding gene COL4A1 can be used as specific prognostic assessment biomarkers for patients with recurrent nasopharyngeal carcinoma, and can be used to assess the prognosis of patients with recurrent nasopharyngeal carcinoma.

[0018] Furthermore, inhibiting the expression of type IV collagen can promote T cell infiltration, inhibit the immune escape of nasopharyngeal carcinoma, and effectively inhibit the growth of nasopharyngeal carcinoma. Therefore, agents that inhibit the expression of type IV collagen can be used as drugs to inhibit the immune escape of nasopharyngeal carcinoma and are used to treat nasopharyngeal carcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the detection result of type IV collagen and its encoding gene COL4A1 in Example 1, which can be used for prognosis evaluation of nasopharyngeal carcinoma patients.

[0020] Figure 2These are the research results on the therapeutic effect of COL4A1 gene knockout on nasopharyngeal carcinoma. DETAILED DESCRIPTION

[0021] Experimental procedures in the following examples, where specific conditions are not specified, generally followed conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. All commonly used chemical reagents used in the examples were commercially available.

[0022] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

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

[0024] The following describes the method in conjunction with specific embodiments.

[0025] Example 1

[0026] We collected fresh tissue samples from 11 newly diagnosed NPC patients and 13 patients with recurrent NPC for single-cell sequencing. We also collected samples from 8 newly diagnosed NPC patients and 7 patients with recurrent NPC for spatial transcriptome sequencing. Both single-cell sequencing and spatial transcriptome sequencing were performed by Guangzhou Yuanxin Biotechnology Co., Ltd.

[0027] Based on the sequencing results, by constructing a global tumor ecosystem map, we found that samples from patients with recurrent nasopharyngeal carcinoma had higher immune surveillance and immune escape characteristics ( Figure 1 A), in which we found that genes significantly associated with immune escape, such as CD47, were significantly increased in tumor cells in samples from patients with recurrent nasopharyngeal carcinoma, suggesting that they can inhibit the infiltration of dendritic cells and antigen presentation ( Figure 1 B).

[0028] We found that the immune microenvironment of recurrent nasopharyngeal carcinoma was significantly altered, and recurrent nasopharyngeal carcinoma had a high ability to evade immune-mediated cell killing. Among them, we found that collagen can regulate lymphocyte infiltration in tumors.

[0029] Compared with newly diagnosed NPC patients, the expression of type IV collagen was significantly increased in patients with recurrent NPC.

[0030] Furthermore, we performed multiple immunohistochemistry on 40 paired tissue samples from patients with newly diagnosed and recurrent NPC in validation cohort 1 and found that high expression of type IV collagen was associated with lower T cell infiltration. All samples were tested for type IV collagen expression, 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 1 D).

[0031] In addition, because type IV collagen is mainly encoded by genes COL4A1 and COL4A2, single-cell sequencing results also found that the expression levels of COL4A1 and COL4A2 were significantly increased in patients with recurrent nasopharyngeal carcinoma ( Figure 1 E and F).

[0032] These results indicate that the expression levels of type IV collagen and its encoding gene COL4A1 are associated with the immune escape characteristics of patients with recurrent nasopharyngeal carcinoma, suggesting that the expression levels of type IV collagen and COL4A1 genes can be used as prognostic indicators for patients with recurrent nasopharyngeal carcinoma.

[0033] Example 2

[0034] To verify the correlation between type IV collagen expression level and the prognosis of patients with recurrent nasopharyngeal carcinoma, we collected 86 newly diagnosed nasopharyngeal carcinoma samples that had received radical radiotherapy (validation cohort 2) for validation.

[0035] Multiple immunohistochemical staining was used to detect the type IV collagen content in all NPC patient tissue samples in the validation cohort 2 to determine the expression level of type IV collagen.

[0036] Based on the test results, patients in the top 50% of type IV collagen expression were defined as the high type IV collagen expression group, and those in the bottom 50% were defined as the low type IV collagen expression group. Based on this standard, in validation cohort 2, 43 patients were in the high type IV collagen expression group and 43 patients were in the low type IV collagen expression group.

[0037] LRRFS and OS were analyzed for patients in the type IV collagen high expression group and the type IV collagen low expression group. The results showed that in the validation cohort 2, the type IV collagen high expression group was associated with worse LRRFS (p = 0.036) and OS (p = 0.031) compared with the type IV collagen low expression group ( Figure 1G and 1H). These results indicate that the expression of type IV collagen is significantly correlated with the prognosis of patients with recurrent nasopharyngeal carcinoma and can be used as a prognostic marker for patients with recurrent nasopharyngeal carcinoma.

[0038] Example 3

[0039] To verify the correlation between the expression level of type IV collagen encoding gene COL4A1 and the prognosis of patients with recurrent nasopharyngeal carcinoma, we re-enrolled 88 patients with newly diagnosed nasopharyngeal carcinoma (validation cohort 4) for verification.

[0040] Bulk RNA sequencing (commissioned by Guangzhou Yuanxin Biotechnology Co., Ltd.) was used to detect the expression level of COL4A1 in 88 patients with newly diagnosed nasopharyngeal carcinoma. Based on the expression level of COL4A1, the patients were divided into COL4A1 high expression group and COL4A1 low expression group using surv_cutpoint in R language. PFS analysis was performed on patients in the COL4A1 high expression group and the COL4A1 low expression group, and it was found that the PFS of patients in the COL4A1 high expression group was significantly worse than that in the COL4A1 low expression group ( Figure 1 Middle I).

[0041] 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 1 J). We also found that, based on ST data, COL4A1 expression was negatively correlated with the CD8+T ratio (r=-0.554, p-value=0.0323) and CD4+T ratio (r=-0.886, p-value<0.0001) ( Figure 1 K).

[0042] The above results indicate that the expression level of type IV collagen encoding gene COL4A1 is significantly correlated with the prognosis of patients with recurrent nasopharyngeal carcinoma, and patients with recurrent nasopharyngeal carcinoma with high expression of COL4A1 gene have a worse prognosis.

[0043] Example 4

[0044] Since type IV collagen and its encoding gene COL4A1 are significantly associated with immune escape and prognosis of recurrent nasopharyngeal carcinoma, we further investigated whether they could serve as therapeutic targets to inhibit immune escape of nasopharyngeal carcinoma.

[0045] 1. Experimental Methods

[0046] To better explore the relationship between type IV collagen and immune escape in nasopharyngeal carcinoma, we constructed a humanized mouse model. MCAM+ tumor-associated fibroblasts with or without COL4A1 knockdown were co-injected with C666 tumor cells into mice. The PBS and C666 co-injection groups served as controls. The specific procedures were as follows: To construct a humanized nasopharyngeal carcinoma (CDX) xenograft model, NCG mice were irradiated with a dose of 80-100 cGy. 24 hours after irradiation, 1×10 5 Fresh human volunteer CD34 + Hematopoietic stem cells (HSCs) are injected intravenously into mice to generate various hematopoietic or immune cells. + Mice with HSCs content exceeding 15% were identified as peripherally humanized. Ten weeks after transplantation of hematopoietic stem cells, mice were bled and humanized chimerism was confirmed by flow cytometry before establishing NPC xenograft models. Mice were randomly divided into C666+COL4A1 KO MCAM + mCAFs group, C666+MCAM + mCAFs group and C666+PBS group (6 mice in each group). C666 cells (1×10 6 ) and COL4A1 KO MCAM + mCAFs or MCAM + mCAFs were mixed at a ratio of 3:1 or with PBS as a negative control and injected subcutaneously into the buttocks of mice. On day 10, when the tumors of mice were measurable, the tumor volume (V) and weight of each mouse were measured every 2 days.

[0047] On day 28, tumors were removed from the mice for measurement.

[0048] During the experiment, the following indicators were tested:

[0049] (1) Tumor volume: The volume of mice was measured every 2 days. The mice were sacrificed on the 28th day, and the tumors were dissected and measured. The calculation formula was V = (length × width2) / 2.

[0050] (2) Tumor weight: After the mice were sacrificed on day 28, the tumors were dissected and weighed.

[0051] (3)CD8 +T cell activation index detection: Tumors of hu-NCG mice were dissected, washed, minced and digested at 37°C using the Tumor Dissociation Kit according to the protocol of Miltenyi Biotec, China. The cell suspension was incubated with RBC lysis buffer three times for 10 minutes each time. Subsequently, cells were first stained with surface antibodies according to the manufacturer's protocol, washed with a fixation / permeabilization kit (BD Pharmingen), blocked with Human BD Fc Block (Fc1.3216), and stained with the listed antibodies. Live CD3 + T cells were sorted from the suspension by FACS (Beckman Coulter) and stained with the following antibodies: BB515 Mouse Anti-Human CD3 (UCHT1) (BD Pharmingen), 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 (G44-26), FLEXAPC Mouse Anti-Human CD62L (DREG-56).

[0052] 2. Experimental Results

[0053] The results showed that MCAM + The experimental group after knockdown of COL4A1 in tumor-associated fibroblasts (COL4A1 KO ) had weaker tumor formation ability, and the tumor volume and tumor weight were significantly lower than those of the COL4A1 non-knockout group (Vector). Figure 2 A and Figure 2 As shown in Figure 2, COL4A1 expression promotes immune escape in NPC, thereby promoting its growth. Knockdown of COL4A1 expression inhibits immune escape in NPC, restricts its growth, and significantly reduces tumor volume and weight.

[0054] Moreover, in IF staining and flow cytometry analysis, CD4 + and CD8 + The proportion of T cells was significantly lower than that of COL4A1 KO group and PBS group ( Figure 2 C). This indicates that IV collagen significantly inhibits T cell infiltration and promotes immune escape.

[0055] In addition, we observed that higher type IV collagen scores were associated with lower CD4 + T cell infiltration (r = -0.523, p = 0.026) and CD8+ T cell infiltration (r = -0.752, p = 0.0003) were significantly correlated ( Figure 2 D). This study demonstrates the important role and therapeutic potential of type IV collagen in inhibiting T cell infiltration in nasopharyngeal carcinoma.

[0056] These results demonstrate the potential role of type IV collagen in suppressing T cell infiltration in nasopharyngeal carcinoma. Inhibiting the expression of type IV collagen can inhibit the immune escape of nasopharyngeal carcinoma, thereby playing a therapeutic role in nasopharyngeal carcinoma. Therefore, type IV collagen and its encoding gene COL4A1 may serve as new targets for inhibiting the immune escape of nasopharyngeal carcinoma and for its treatment.

[0057] The technical features of the above-described embodiments can be combined arbitrarily. To make the description concise, 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.

[0058] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. Application of COL4A1 gene as a biomarker in the prognostic evaluation of patients with recurrent nasopharyngeal carcinoma.

2. Application of type IV collagen as a biomarker in the prognostic evaluation of patients with recurrent nasopharyngeal carcinoma.

3. Application of reagents for detecting COL4A1 gene expression in the preparation of products for prognosis assessment of patients with recurrent nasopharyngeal carcinoma.

4. The use according to claim 3, characterized in that The reagents include reagents for PCR detection, fluorescent quantitative PCR detection, and sequencing detection.

5. Application of reagents for detecting type IV collagen content in the preparation of products for prognosis assessment of patients with recurrent nasopharyngeal carcinoma.

6. The use according to claim 5, characterized in that The reagents include reagents for immunohistochemistry detection and Western Blot detection.

7. The use according to claim 6, characterized in that The reagent is a specific antibody against type IV collagen; preferably, the specific antibody is a specific monoclonal antibody.

8. The use according to any one of claims 3 to 7, characterized in that The product is a test kit.

9. Use of an agent that inhibits the expression of type IV collagen in the preparation of a drug for inhibiting the immune escape of nasopharyngeal carcinoma.

10. Use of an agent for inhibiting the expression of type IV collagen in the preparation of a drug for treating nasopharyngeal carcinoma.

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