Tongue squamous cell carcinoma in-situ PDX model construction method based on SERINC3 high expression and application of SERINC3

Abstract

The invention discloses a tongue squamous cell carcinoma in-situ PDX model construction method based on SERINC3 high expression and application of SERINC3, and belongs to the technical field of tumor biology and animal model construction. The method comprises the following steps: segmenting small tissues from cancer tissues of a patient suffering from tongue squamous cell carcinoma, implanting the small tissues into the armpit of an NCG mouse to culture tumor tissues, and then subculturing the tumor tissues; the subcultured tumor tissue is taken and implanted into the lingual margin of an NCG mouse, and after the tumor is cultured, lentivirus carrying sh-NC / sh1-SERINC3 / sh2-SERINC3 plasmids is injected into the tumor at multiple points to obtain the tongue squamous cell carcinoma in-situ PDX model. The model constructed by the invention can better simulate the natural occurrence and development process of TSCC, reconstructs a tumor microenvironment closer to clinical practice, and particularly has remarkable advantages in the aspect of simulating head and neck lymph node metastasis of tongue squamous cell carcinoma.

Description

Technical Field

[0001] This invention belongs to the field of tumor biology and animal model construction technology, specifically involving a method for constructing an orthotopic PDX model of tongue squamous cell carcinoma based on high expression of SERINC3 and the application of SERINC3. Background Technology

[0002] Tongue squamous cell carcinoma (TSCC) is a highly aggressive head and neck malignancy that easily metastasizes through the head and neck lymph nodes. Clinically, the 5-year survival rate for TSCC patients in the intermediate and advanced stages is only 20-50%. The best way to reduce TSCC mortality and disability rates is through early diagnosis and intervention. However, to date, the etiology and pathogenesis of TSCC remain unclear, posing a significant challenge to primary prevention and early diagnosis. Currently, a major bottleneck limiting TSCC research is the lack of animal models that can accurately simulate the occurrence and development of TSCC.

[0003] Existing TSCC animal models mainly rely on subcutaneous xenograft (PDX) models. While these models can be used for drug evaluation in some cases, traditional PDX models cannot effectively simulate key factors in the tumor microenvironment such as extracellular matrix, immune cell infiltration, and angiogenesis, and cannot fully reflect the real situation of tumor immune escape.

[0004] SERINC3, also known as Tumor Differentially Expressed Protein 1 (TDE1), is a key transmembrane protein in cell membrane biosynthesis and a member of the serine integrator family. It is widely expressed in epithelial cells and other cells. Its main function is to facilitate the integration of polar serine into the cell membrane lipid bilayer and promote the synthesis of phosphatidylserine and sphingolipid derivatives. In recent years, SERINC3 has been found to have pro-cancer effects in various tumors, and its expression level in TSCC is negatively correlated with patient survival. However, the mechanism of action of SERINC3 in the development of TSCC, whether it can serve as a therapeutic target for TSCC, and how to mimic its role in mouse models remain areas of research uncertainty. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a method for constructing an orthotopic PDX model of tongue squamous cell carcinoma based on high expression of SERINC3 and the application of SERINC3. The model of this invention can better simulate the natural occurrence and development process of TSCC, and at the same time reconstruct a tumor microenvironment that is closer to clinical reality, especially in simulating head and neck lymph node metastasis of tongue squamous cell carcinoma, which has significant advantages.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for constructing an orthotopic PDX model of tongue squamous cell carcinoma based on high SERINC3 expression, comprising the following steps:

[0008] S1. Small pieces of tissue were separated from the cancerous tissue of a patient with squamous cell carcinoma of the tongue and implanted into the axilla of NCG mice to culture tumor tissue, and then the tumor tissue was passaged for culture.

[0009] S2. Passaged tumor tissue was implanted into the lateral edge of the tongue of NCG mice. After the tumor was cultured, lentiviruses carrying sh-NC / sh1-SERINC3 / sh2-SERINC3 plasmids were injected into the tumor at multiple points to obtain an orthotopic PDX model of tongue squamous cell carcinoma.

[0010] Preferably, the diameter of the small tissue in S1 is 0.8-1.5 mm.

[0011] Preferably, in step S1, the tumor tissue is cultured to a volume of 1000–1500 mm³. 3 Timely generation and propagation.

[0012] Preferably, the number of generations in S1 is 2.

[0013] Preferably, the tumor tissue in S2 is 3-4 mm in size. 3 .

[0014] Preferably, the tumor in S2 is cultured to 8-9 mm. 3 Injection should be performed at the appropriate time.

[0015] Preferably, the injection dose in S2 is 1×10 8 PFU / 100mL / mouse; administer once every 4 days, for a total of 4-6 injections.

[0016] This invention provides the application of SERINC3 expression in regulating the development of oral squamous cell carcinoma.

[0017] Preferably, inhibiting the development of oral squamous cell carcinoma by suppressing SERINC3 expression has at least the following beneficial technical effects:

[0018] Currently, commonly used TSCC mouse models, such as subcutaneous PDX models and chemically induced models, have limitations in accurately reflecting head and neck lymph node metastasis. Because the mouse tongue is located within the confined space of the oral cavity, conventional subcutaneous PDX models cannot simulate the natural development and metastasis of tongue squamous cell carcinoma in the tongue. To overcome these problems, the purpose of this invention is to propose an in situ PDX model for tongue squamous cell carcinoma based on high SERINC3 expression. In clinical TSCC patients, high SERINC3 expression exhibits stronger proliferative capacity, higher invasiveness, and higher mitochondrial metabolic activity, and is associated with clinical characteristics such as poor prognosis, short survival time, and rapid tumor development. Therefore, this model can better simulate the natural occurrence and development of TSCC, while reconstructing a tumor microenvironment that more closely resembles clinical reality, especially showing significant advantages in simulating head and neck lymph node metastasis in tongue squamous cell carcinoma. By establishing this model, this invention aims to further investigate the role of SERINC3 in the occurrence and development of TSCC and explore its feasibility as a potential therapeutic target. Attached Figure Description

[0019] Figure 1 This is a flowchart illustrating the construction process of an in situ PDX model for tongue squamous cell carcinoma.

[0020] Figure 2 The expression of SERINC3 in TECC clinical samples.

[0021] Figure 3 Construction and evaluation of the subcutaneous PDXSERINC3 High model.

[0022] Figure 4 To target and inhibit the effect of SERINC3 on the growth of TSCC tissue.

[0023] Figure 5 Results of constructing PDX and tail vein transfer models for knocking down SERINC3 cell lines.

[0024] Figure 6 Results of constructing PDX and tail vein transfer models for knocking down SERINC3 cell lines. Detailed Implementation

[0025] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. The invention will now be described with reference to specific embodiments. It should be noted that these embodiments are merely descriptive and do not limit the invention in any way.

[0026] Example 1

[0027] 1. Establishing an orthotopic PDX model of tongue squamous cell carcinoma based on SERINC3 expression level

[0028] To simulate the clinical development of TSCC, a 1mm piece of tissue was first separated from the cancerous tissue removed from a TSCC patient and implanted into the axilla of an NCG mouse within 2 hours (P0 xenograft).

[0029] Transplanted tumor tissues were scored using SERINC3 immunohistochemical staining and divided into three groups according to their expression levels:

[0030] G1 (low expression group): Immunohistochemical score 0-1 point

[0031] G2 (medium expression group): Immunohistochemical score of 2 points

[0032] G3 (high expression group): Immunohistochemistry score of 3 points

[0033] Six mice were selected in each group, and tumor growth characteristics under different expression levels were evaluated by observing tumor growth, measuring tumor volume and weight.

[0034] Based on the immunohistochemical score, the SERINC3 high expression and low expression groups can be quantitatively separated, which facilitates subsequent observation of the experimental performance of the SERINC3 high expression group.

[0035] After approximately 12 weeks, the tumor volume reached 1500 mm. 3 At this stage, tumors were dissected from host mice, cut into solid tumors with a diameter of 3-5 mm, and implanted into the backs of next-generation mice. The mice were then passaged sequentially in P1 and P2 generations, and in the P3 generation, the tumor tissue was recut into 4 mm sections. 3 Small, round tissue fragments were surgically implanted into the lateral edge of the tongue of NCG mice to create an orthotopic PDX model of tongue squamous cell carcinoma. The P3 xenograft tumor was then implanted until it reached 9 mm. 3 At the same time, multiple injections were performed into the tongue cancer cells of mice. This was achieved by injecting lentivirus carrying the sh-NC / sh1-SERINC3 / sh2-SERINC3 plasmids into the mouse tumors at a dose of 1×10⁻⁶. 8 The PFU / 100mL / mouse was administered every 4 days for a total of 4-6 injections to complete the construction of an orthotopic PDX model of tongue squamous cell carcinoma.

[0036] The passage method is as follows: donor mice are euthanized, the skin is opened, and tumor tissue is dissected, avoiding the inclusion of necrotic or connective tissue. The tumor tissue is then placed in a sterile dish containing culture medium to maintain humidity. Under sterile conditions, the tumor is cut into 3-5 mm sections. 3 Small pieces. Recipient mice were anesthetized, and the tumor piece was implanted into the back of the recipient mouse. The incision was then sutured or clamped.

[0037] The construction method of plasmids sh-NC / sh1-SERINC3(5'-CCACTTGTTCTTACAAATCGT-3') / sh2-SERINC3(5'-CCAAGTCTACTAAGCATAATT-3') is as follows:

[0038] 1. Vector digestion: Digest at 37℃ for 1 hour, recover by gel electrophoresis, and identify.

[0039] 2. Amplification system:

[0040] Oligos Mix Template: 1μL;

[0041] F(10P) 1μL;

[0042] R(10P) 1μL;

[0043] 2x PCR Master Mix 10μL;

[0044] PCR enhancer 4μL;

[0045] dd H2O 3μL;

[0046] Total 20μL.

[0047] 3. Amplification procedure:

[0048] 94℃, 2 min 30 s; 94℃, 20 s; 20 cycles; 62℃, 30 s; 68℃, 2 min 30 s; 68℃, 5 min; 15℃ Forever.

[0049] The full-length gene PCR product was purified and recovered.

[0050] 4. Transformation

[0051] Add 5 μL of ligation solution to 50 μL of competent cells, incubate on ice for 30 min, heat shock at 42 °C for 45 s, incubate at 0 °C for 2 min, and after recovery for 1 h, spread on 2xYT(Kan) plates.

[0052] 5. Screening of positive clones

[0053] Six single colonies were picked and placed in 4 mL of 2xYT(Kan) medium and cultured at 37℃ and 250 rpm for 10-12 h. Plasmids were extracted and selected at appropriate heights for sequencing.

[0054] 6. Sequencing

[0055] Select plasmids with accurate sequences based on sequencing results.

[0056] The construction process of the orthotopic PDX model of tongue squamous cell carcinoma is as follows: Figure 1 .

[0057] Example 2

[0058] Based on the model established in Example 1, the tumor volume in mice was monitored every 4 days to assess its growth rate and the feasibility of treatment. Simultaneously, plans are underway to screen small molecule chemical drugs that target and inhibit SERINC3 to further explore their potential in the treatment of TSCC.

[0059] 1. Clinical databases and tissue samples confirm that SERINC3 is significantly overexpressed in TSCC epithelium.

[0060] like Figure 2 As shown, immunohistochemical staining and scoring of 85 clinical TSCC specimens revealed that (A) SERINC3 expression in TSCC tissues (TS, carcinoma in situ; I-II, pathological grades I-II; III, pathological grade III) and metastatic lymph nodes (LN) was significantly increased compared to normal tongue epithelial tissue (NL) and precancerous lesions (PC, leukoplakia); (B) Statistical analysis of SERINC3 expression in different disease stages. TCGA database analysis revealed that (CD) SERINC3 expression in TSCC tissues was significantly increased compared to adjacent normal tissues, and was negatively correlated with survival rate; (E) Further analysis of single-cell sequencing data showed that SERINC3 expression was highest in TSCC epithelial cells (red dashed box). In the figure: *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

[0061] High expression of SERINC3 was observed in TSCC tissues using clinical samples (Figure A, B). Further analysis using the TCGA database revealed that SERINC3 expression in TSCC tissues was significantly higher than in adjacent normal tissues (Figure C), and was negatively correlated with survival (Figure D). Further analysis of single-cell sequencing data showed that SERINC3 expression was highest in the epithelial cells of TSCC cells (Figure E), suggesting that abnormal expression of SERINC3 in the epithelium is closely related to the occurrence and development of TSCC.

[0062] 2. A mouse subcutaneous PDXSERINC3 high model clearly demonstrated that TSCC cell spheroids and tissues with high SERINC3 expression grew faster.

[0063] See Figure 3Among them, (A) flowchart of the construction of the TSCC mouse subcutaneous PDXSERINC3 High model; immunohistochemical scores of 0-1 (G1, low expression group, Low), 2 (G2, medium expression group, Medium) and 3 (G3, high expression group, High); (BD) TSCC tissue P0 generation xenografts with high SERINC3 expression grew faster than those with low SERINC3 expression.

[0064] Constructing mouse subcutaneous TSCC PDX SERINC3 High Model observation revealed that TSCC tissues with high SERINC3 expression grew faster, clarifying the pro-cancer role of SERINC3 in TSCC.

[0065] 3. The PDXSERINC3 High model confirmed that targeted inhibition of SERINC3 has an inhibitory effect on the growth of TSCC tissue, suggesting that SERINC3 has the potential to become a new target for the treatment of TSCC.

[0066] See Figure 4 In the figure, (A) shows the treatment of mice with subcutaneous PDX by targeting and inhibiting SERINC3. SERINC3 High Flowchart of the model; (BD)PDX SERINC3 High In the model, targeted inhibition of SERINC3 significantly suppressed the growth of TSCC tissues. *, p<0.05; **, p<0.01.

[0067] To further clarify the clinical significance of SERINC3 and observe its potential as a therapeutic target, subcutaneous PDX was used in mice with tongue squamous cell carcinoma. SERINC3 High The model targeted inhibition of SERINC3, and the results showed that targeting and inhibiting SERINC3 significantly suppressed the growth of human TSCC tissue. Therefore, SERINC3 is a potential therapeutic target for TSCC.

[0068] 4. In the PDX model and tail vein metastasis model, tongue squamous cell carcinoma cell lines that target and knock out the SERINC3 gene in tongue squamous cell carcinoma cells exhibited low proliferation and migration capabilities.

[0069] See Figure 5 In the figure, (A,B) are subcutaneous tumor models after SERINC3 knockdown; (C,D) are tail vein metastatic tumor models after SERINC3 knockdown.

[0070] A stable SERINC3 knockout tongue squamous cell carcinoma cell line was constructed using a lentiviral vector. Subcutaneous in situ and metastatic tumor models in nude mice were established using two SERINC3 knockout cell lines and one control cell line to evaluate the association between SERINC3 and OSCC progression. In the subcutaneous tumor model, SERINC3 knockdown significantly reduced tumor growth compared to the control group (Fig. A, B). Furthermore, SERINC3 inhibition significantly reduced the number, volume, and weight of lung metastases (Fig. C, D). To assess its clinical application potential, a PDX mouse model was treated with lentiviral targeting of SERINC3 (Fig. E). Results showed that SERINC3 targeting significantly inhibited tumor weight and volume in the PDX model (Fig. F). These findings indicate that SERINC3 knockout inhibits the growth and metastasis of OSCC tumors in vivo, highlighting its potential as a therapeutic target for OSCC.

[0071] 5. The SERINC3 knockout (KO) mouse model can inhibit the development of oral squamous cell carcinoma (OSCC) in vivo.

[0072] See Figure 6 In the figure, (AD) tumor weight, number, and volume in Serinc3 knockout mice; (E, F) disease progression and survival; (G) HE staining of tumor tissue infiltration; (BD) analysis using a two-tailed t-test; (E, F) analysis using one-way ANOVA. *, p < 0.05, **, p < 0.01, ***, p < 0.001

[0073] To verify the tumor-suppressive effect of SERINC3 in vivo, a Serinc3 knockout (KO) mouse model was established using an OSCC mouse model induced by 4-nitroquinoline 1-oxide (4NQO). The results showed that the tumors in Serinc3 knockout mice exhibited significantly reduced tumor weight, number, and volume (Figures AD). Furthermore, Serinc3 knockout mice demonstrated reduced OSCC tumor progression and improved survival (Figures E, F). H&E staining further revealed that 4NQO-induced mouse tumors breached the basement membrane at an earlier time point and exhibited stronger malignant characteristics (Figure G). These results indicate that Serinc3 knockout inhibits 4NQO-induced OSCC progression.

[0074] 6. Small molecule compounds that specifically target SERINC3

[0075] Using the Autodock molecular docking simulation platform, small molecule compounds with high binding energies to SERINC3 were screened, and their corresponding prices were provided.

[0076] Table 1 Small molecule compounds with high binding energy to SERINC3

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090] Screening for small molecule compounds targeting SERINC3 can provide personalized and targeted treatment strategies for patients with high SERINC3 expression. Ultimately, this can further advance the transformation of SERINC3 from a "biological target" to a "drug development target." The screened small molecules targeting SERINC3 can significantly inhibit SERINC3 expression or its downstream pathway activity (such as the c-MET / MAPK / PGC1α axis) at the cellular or animal level, thereby exhibiting anti-tumor effects (proliferation, invasion and migration, mitochondrial function, etc.) similar to those observed with sh-SERINC3 in experiments.

[0091] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for constructing an orthotopic PDX model of tongue squamous cell carcinoma based on high SERINC3 expression, characterized in that, Includes the following steps: S1. Small pieces of tissue were separated from the cancerous tissue of a patient with squamous cell carcinoma of the tongue and implanted into the axilla of NCG mice to culture tumor tissue, and then the tumor tissue was passaged for culture. S2. Passaged tumor tissue was implanted into the lateral edge of the tongue of NCG mice. After the tumor was cultured, lentiviruses carrying sh-NC / sh1-SERINC3 / sh2-SERINC3 plasmids were injected into the tumor at multiple points to obtain an orthotopic PDX model of tongue squamous cell carcinoma.

2. The method for constructing an orthotopic PDX model of tongue squamous cell carcinoma according to claim 1, characterized in that, The diameter of the small tissue in S1 is 0.8-1.5 mm.

3. The method for constructing an orthotopic PDX model of tongue squamous cell carcinoma according to claim 1, characterized in that, In step S1, the tumor tissue is cultured until its volume reaches 1000-1500 mm³. 3 Timely generation and propagation.

4. The method for constructing an orthotopic PDX model of tongue squamous cell carcinoma according to claim 1, characterized in that, The number of generations in S1 is 2.

5. The method for constructing an orthotopic PDX model of tongue squamous cell carcinoma according to claim 1, characterized in that, The tumor tissue in S2 is 3-4 mm in size. 3 .

6. The method for constructing an orthotopic PDX model of tongue squamous cell carcinoma according to claim 1, characterized in that, The tumor in S2 was cultured to 8-9 mm. 3 Injection should be performed at the appropriate time.

7. The method for constructing an orthotopic PDX model of tongue squamous cell carcinoma according to claim 1, characterized in that, The injection dose in S2 is 1×10 8 PFU / 100mL / mouse; administer once every 4 days, for a total of 4-6 injections.

8. Application of SERINC3 expression in regulating the development of oral squamous cell carcinoma.

9. The application according to claim 8, characterized in that, Inhibiting the development of oral squamous cell carcinoma by suppressing the expression of SERINC3.

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