Bladder tumor cell strain with squamous differentiation characteristic and application thereof

Abstract

The invention discloses a bladder tumor cell strain with squamous differentiation characteristics and application thereof, and relates to the technical field of biological medicines. Wherein the bladder tumor cell strain is selected from one of a human bladder tumor cell strain MP-MGHU3-LT, a human bladder tumor cell strain UCSD-MGHU3-LT, a human bladder tumor cell strain UCSD-RT4-LT and a mouse bladder tumor cell strain mUCMP, the bladder tumor cell strains are all preserved in the China Center for Type Culture Collection, and the preservation numbers of the bladder tumor cell strains are respectively CCTCC NO: C2025241, CCTCC NO: C2025252, CCTCC NO: C2025253 and CCTCC NO: C2025254. The bladder tumor cell strain provided by the invention has squamous differentiation characteristics, and can be applied to model construction, mechanism research and drug screening of bladder cancer with squamous differentiation characteristics.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a bladder tumor cell line with squamous differentiation characteristics and application thereof. BACKGROUND

[0002] At present, the bladder tumor cell lines on the market are mainly divided into non-muscle invasive bladder tumor cell lines (MB49, RT4, and MGHU3, etc.) and muscle invasive bladder tumor cell lines (MBT2, 5637, UMUC3 and T24, etc.), and the cell types of these cell lines tend to be homogenized (such as being biased towards epithelial or mesenchymal type), which cannot well reflect the heterogeneity of tumor tissues themselves. More importantly, with the tumor phenotype plasticity being valued by more and more people, squamous differentiation as one of the key types of lineage transformation, its key role in tumor drug resistance and development has also been gradually revealed by people. However, at present, there is no bladder tumor cell line with squamous differentiation characteristics on the market, therefore, constructing a bladder tumor cell line with heterogeneity and squamous differentiation characteristics has important scientific value for cancer research. SUMMARY

[0003] In view of the deficiencies of the prior art, the purpose of the present application is to provide a bladder tumor cell line with squamous differentiation characteristics and application thereof, aiming at solving the problem that the prior art lacks a bladder tumor cell line with squamous differentiation characteristics.

[0004] The technical scheme of the present application is as follows: In a first aspect, a bladder tumor cell line with squamous differentiation characteristics is provided, and the bladder tumor cell line is selected from one of the following (1) to (4): (1) a human bladder tumor cell line MP-MGHU3-LT, which is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: C2025241; (2) a human bladder tumor cell line UCSD-MGHU3-LT, which is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: C2025252; (3) a human bladder tumor cell line UCSD-RT4-LT, which is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: C2025253; (4) a mouse bladder tumor cell line mUCMP, which is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: C2025254.

[0005] In a second aspect, the application provides the use of the bladder tumor cell line according to the first aspect in establishing a bladder cancer research model with squamous differentiation characteristics.

[0006] In a third aspect, the bladder tumor cell line according to the first aspect is used in exploring the mechanism of bladder cancer with squamous differentiation characteristics.

[0007] In a fourth aspect, the bladder tumor cell line according to the first aspect is used in screening drugs for treating bladder cancer with squamous differentiation characteristics.

[0008] In a fifth aspect, the bladder tumor cell line according to the first aspect is used in establishing a research model of bladder cancer with chemotherapy resistance and recurrence.

[0009] In a sixth aspect, the bladder tumor cell line according to the first aspect is used in exploring the mechanism of bladder cancer with chemotherapy resistance and recurrence.

[0010] In a seventh aspect, the bladder tumor cell line according to the first aspect is used in screening drugs for treating bladder cancer with chemotherapy resistance and recurrence.

[0011] Optionally, the drug used in the chemotherapy is gemcitabine.

[0012] In an eighth aspect, a method for screening potential bladder cancer squamous differentiation related genes is provided, comprising the steps of: comparing the genes expressed in the bladder tumor cell line according to the first aspect with the genes expressed in normal bladder cells, and screening the genes that are statistically up-regulated or down-regulated in the bladder tumor cell line according to the first aspect, which are potential bladder cancer squamous differentiation related genes.

[0013] Beneficial effects: The bladder tumor cell lines with squamous differentiation characteristics obtained by screening include human bladder tumor cell line MP-MGHU3-LT, human bladder tumor cell line UCSD-MGHU3-LT, human bladder tumor cell line UCSD-RT4-LT, and mouse bladder tumor cell line mUCMP. These bladder tumor cell lines have squamous differentiation characteristics and can be applied to model construction, mechanism research, and drug screening of bladder cancer with squamous differentiation characteristics, which fills the gap in the prior art in terms of bladder tumor cell lines with squamous differentiation characteristics. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 is the related result of Example 1, showing that chemotherapy can induce phenotypic transformation of recurrent tumor cells in transgenic mice; wherein A is a flow chart of bladder tumor recurrence experiment under the background of chemotherapy; and B is a representative pathological graph of mouse tumors in the control group and the chemotherapy group.

[0015] Figure 2are the relevant results of Example 2, showing that chemotherapy induces phenotypic transformation of recurrent tumor cells in a xenograft model; wherein, A is a representative pathology of MGHU3-LT tumors in control and chemotherapy groups; B is a representative pathology of RT4-LT tumors in control and chemotherapy groups.

[0016] Figure 3 are the representative organoid images and their size statistics of each cell line in the application example; wherein, A is a representative organoid image and its corresponding organoid size statistics of MGHU3-LT tumor-derived cells; B is a representative organoid image and its corresponding organoid size statistics of MHUC and mUCMP cell lines; C is a representative organoid image and its corresponding organoid size statistics of RT4-LT tumor-derived cells; scale: 20 mm.

[0017] Figure 4 are the cell characterization of control and recurrent MGHU3-LT tumor-derived cells in the application example; wherein, A is the UMAP plot of MGHU3-LT tumor-derived cells, the left side is the UMAP plot of samples, and the right side is the UMAP plot of Leiden clusters; B is the gene signature of each Leiden cluster, the color depth represents the average expression level of the gene, and the size of the point indicates the percentage of cells expressing the gene; C is the hierarchical clustering heat map of all high-variation genes and their corresponding gene modules of MGHU3-LT tumor-derived cells, the left side shows the gene modules of different color bands, and the column chart represents the tree chart connecting the genes; D is the UMAP visualization of the enrichment score of the four gene modules (module 1, module 2, module 3, and module 4) characteristics of MGHU3-LT tumors; E is the z-score heat map of each Leiden cluster in the corresponding module in MGHU3-LT tumors; F is the heat map of the Pearson correlation coefficient between the selected genes and the module score in MGHU3-LT tumors; Squ: squamous; EMT: epithelial-to-mesenchymal transition.

[0018] Figure 5A is the UMAP plot of RT4-LT tumor-derived cells, the left side is the UMAP plot of the sample, and the right side is the UMAP plot of the Leiden cluster; B is the gene marker of each Leiden cluster, the darkness of the color represents the average expression level of the gene, and the size of the point represents the percentage of cells expressing the gene; C is the hierarchical clustering heat map of all high-variation genes of RT4-LT tumor-derived cells and their corresponding gene modules, the left side shows the gene modules of different color bands, and the column chart represents the tree chart connecting the genes; D is the enrichment score of the UMAP visualization of the 2 gene modules (module 1 and module 2) characteristics of the RT4-LT tumor; E is the z-score heat map of each Leiden cluster in the corresponding module in the RT4-LT tumor; F is the heat map of the Pearson correlation coefficient between the selected genes and the module score in the RT4-LT tumor; G is the heat map showing the relative expression level of the squamous differentiation-related genes detected by RNA sequencing in the MHUC (n=3) and mUCMP (n=3); H is the biological process enrichment map of the up-regulated genes in the mUCMP cells compared with the MHUC cells. DETAILED DESCRIPTION

[0019] The present application provides a bladder tumor cell strain with squamous differentiation characteristics and its application. In order to make the purpose, technical scheme and effect of the present application more clear and explicit, the present application is further described in detail as follows.

[0020] The present application provides a bladder tumor cell strain with squamous differentiation characteristics, which is selected from one of the following (1)~(4): (1) Human bladder tumor cell strain (Homo sapiens) MP-MGHU3-LT, which is preserved in China Center for Type Culture Collection, preserved in Wuhan University, preserved on August 19, 2025, and preserved with the number CCTCC NO: C2025241; (2) Human bladder tumor cell strain (Homo sapiens) UCSD-MGHU3-LT, which is preserved in China Center for Type Culture Collection, preserved in Wuhan University, preserved on August 19, 2025, and preserved with the number CCTCC NO: C2025252; (3) Human bladder tumor cell strain (Homo sapiens) UCSD-RT4-LT, which is preserved in China Center for Type Culture Collection, preserved in Wuhan University, preserved on August 19, 2025, and preserved with the number CCTCC NO: C2025253; (4) A bladder tumor cell line (Mus musculus) mUCMP, which is preserved in the China Center for Type Culture Collection, preserved in Wuhan University on August 19, 2025, and has a preservation number of CCTCC NO: C2025254.

[0021] The embodiment of the present application provides application of the bladder tumor cell line as described above in establishment of a bladder cancer research model with squamous differentiation characteristics.

[0022] The embodiment of the present application provides application of the bladder tumor cell line as described above in exploration of an action mechanism of the bladder cancer with squamous differentiation characteristics.

[0023] The embodiment of the present application provides application of the bladder tumor cell line as described above in screening of a drug for treating the bladder cancer with squamous differentiation characteristics.

[0024] The embodiment of the present application provides application of the bladder tumor cell line as described above in establishment of a bladder cancer research model with chemotherapy resistance and recurrence.

[0025] The embodiment of the present application provides application of the bladder tumor cell line as described above in exploration of an action mechanism of the bladder cancer with chemotherapy resistance and recurrence.

[0026] The embodiment of the present application provides application of the bladder tumor cell line as described above in screening of a drug for treating the bladder cancer with chemotherapy resistance and recurrence.

[0027] In some embodiments, the drug used in the chemotherapy is gemcitabine.

[0028] The embodiment of the present application provides a method for screening a potential bladder cancer squamous differentiation related gene, comprising the following steps: The gene expressed in the bladder tumor cell line is compared with the gene expressed in normal bladder cells, and a gene that is statistically up-regulated or down-regulated in the bladder tumor cell line is screened out, and the gene is a potential bladder cancer squamous differentiation related gene.

[0029] The present application is further described below through specific embodiments.

[0030] Embodiment 1 The embodiment provides a mouse bladder tumor cell line mUCMP with squamous differentiation characteristics, and a construction process thereof is specifically as follows: In order to establish a bladder tumor cell line with squamous differentiation characteristics, Trp53 flox / flox ; Pten flox / flox ; Akaluc-IRES-tdTomato genetically engineered mouse (obtained from a commercially available Akaluc-IRES-tdTomato mouse model) Trp53 flox / flox mouse, Ptenflox / flox The mice were crossed with Akaluc-IRES-tdTomato mice (strain C57BL / 6J) to induce bladder tumors. In this embodiment, an adenovirus expressing Cre recombinase driven by the KRT5 promoter (AdenoKRT5-Cre; Hanheng Biotechnology Co., Ltd (Shanghai, China), reference DOI:10.1038 / s41591-019-0499-y) was surgically delivered to the bladder. Trp53 flox / flox ; Pten flox / flox In Akaluc-IRES-tdTomato genetically engineered mice, specific knockout on basal cells was performed within the bladder cavity. Trp53 and Pten, Simultaneously, Akaluciferase and tdTomato were knocked in. The specific method is as follows: Virus (25 μL, 1.1 x 10⁻⁶) was added. 11 The virus (pfu / mL) was mixed at a 1:1 (v / v) ratio with DMEM medium (Gibco, 2263286) containing polybrene (10 μg / mL, MedChemExpress, HY-112735) and Evans blue dye (0.02% (w / v), Sigma-Aldrich, 314-13-6). The diluted virus was injected intravesically into the bladder of the genetically engineered mice at a volume of 5-10 μL. Once microtumor formation was confirmed by in vivo imaging (approximately seven weeks after virus injection), the mice were treated. Treatment was administered via bladder instillation of 50 μL of physiological saline or gemcitabine hydrochloride (240 mg / kg, Sigma-Aldrich, 122111-03-9), once a week for a total of four times. Treatment was then discontinued, allowing the tumor or microresidual lesions (if any) to continue growing. Figure 1 (A)

[0031] The construction method of a novel mouse bladder tumor cell line is as follows: In the saline control group, bladder tumors were collected at the experimental endpoint and then digested into a single-cell suspension. TdTomato-positive cells were further screened using fluorescence-activated cell sorting to serve as the MHUC (Mouse Heterogeneous Urothelial Carcinoma) cell line. Figure 1(A). To obtain cell lines from chemotherapy-resistant recurrent tumors, when the bioluminescent signal of the chemotherapy group mice was close to that of the control group mice, the same procedure as that performed on the control mice treated with saline was performed to establish mUCMP (Mouse Urothelial Carcinoma of Mixed Phenotypes). Notably, the pathological results of the recurrent bladder tumors in mice showed that the tumors had obvious keratinized pearls (squamous features) and other pathological features, such as sarcomatoid ( Figure 1 (B)

[0032] The novel mouse bladder tumor cell line mUCMP has been deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, on August 19, 2025, with accession number CCTCC NO: C2025254.

[0033] Example 2 This embodiment provides two human bladder tumor cell lines, MP-MGHU3-LT and UCSD-MGHU3-LT, which have squamous differentiation characteristics. The specific construction process is as follows: First, following the method described in the reference (DOI: 10.1126 / science.abc1944), human non-muscle invasive bladder cancer cell lines MGHU3 (MGHU3-LT) and RT4-LT with lineage tracing (LT) capabilities were constructed. These lines were then injected into the bladder cavity of immunodeficient mice (NCG) to construct MGHU3-LT xenograft models and RT4-LT xenograft models, respectively. Next, human bladder tumor cell lines were constructed using the method for generating novel cell lines from genetically engineered mice as described in Example 1.

[0034] The construction method of a novel human bladder tumor cell line is as follows: Since xenograft cells with lineage tracing capabilities simultaneously possess GFP, mCherry, and BFP, triple-positive (GFP+; mCherry+; BFP+) cells can be sorted using fluorescence-activated cell sorting to construct the cell line. In the chemotherapy group of the MGHU3-LT xenograft model, two phenotypes different from those in the control group were observed: mixed-phenotype (MP) bladder cancer tumors and urothelial carcinoma of squamous differentiation (UCSD). Figure 2Surprisingly, metastases were also found in mice with mixed phenotype tumors. The MP-MGHU3-LT cell line was obtained from a mixed phenotype bladder tumor by fluorescence-activated cell sorting, the UCSD-MGHU3-LT cell line was obtained from a urothelial tumor with squamous differentiation, and the Mets-MGHU3-LT cell line was obtained from metastases. Similarly, the CTR-MGHU3-LT cell line was obtained from a tumor in the control group. In addition, in the RT4-LT xenograft model, the UCSD-RT4-LT cell line was obtained from a tumor in the chemotherapy group using the same treatment method as described above, and the CTR-RT4-LT cell line was obtained from a tumor in the control group. Figure 2 Example 3. Characterization of the cell lines Figure 2 Specifically, pathological results showed that the MP-MGHU3-LT tumor had multiple characteristics such as urothelial, squamous differentiation (keratin pearl or intercellular bridge), and sarcomatoid Figure 2 In summary, this example successfully constructed a new type of heterogeneous human bladder tumor cell line with squamous differentiation characteristics.

[0035] The human bladder tumor cell line MP-MGHU3-LT has been deposited with the China Center for Type Culture Collection, located at Wuhan University, on August 19, 2025, and has the deposit number CCTCC NO: C2025241.

[0036] The human bladder tumor cell line UCSD-MGHU3-LT has been deposited with the China Center for Type Culture Collection, located at Wuhan University, on August 19, 2025, and has the deposit number CCTCC NO: C2025252.

[0037] The human bladder tumor cell line UCSD-RT4-LT has been deposited with the China Center for Type Culture Collection, located at Wuhan University, on August 19, 2025, and has the deposit number CCTCC NO: C2025253.

[0038] Application Example The cell lines constructed in Examples 1 and 2 were used to explore the relationship between squamous differentiation and the malignant potential of tumors.

[0039] First, tumor organoids were constructed using the aforementioned cells and their corresponding control cells (reference DOI:10.1016 / j.cell.2018.03.017). Results for MGHU3-LT tumor organoids showed that MP-MGHU3-LT tumor organoids grew significantly faster than control tumor organoids. Organoids derived from UCSD tumors and metastases were between the sizes of those derived from MP tumors and control tumor organoids. Organoids derived from metastases grew faster than UCSD-MGHU3 tumor organoids. Figure 3 (A). Similarly, the growth rate of mUCMP tumor organoids was significantly faster than that of their control tumor organoids (A). Figure 3 (B). However, the size of the UCSD-RT4-LT tumor organoids was comparable to that of its control organoids (B). Figure 3 (C). In summary, these data indicate that the recurrent MGHU3-LT tumors and mUCMP tumors are more malignant than their control tumors. Although there was no significant difference between RT4-LT tumor organoids and control tumors, these data suggest that the squamous differentiation type described in this invention may not affect the malignancy of the tumor.

[0040] To further elucidate the relationship between squamous differentiation and tumor malignancy potential, this invention integrates scRNA-seq data of MGHU3-LT tumors. A total of 10 distinct subsets were detected, including squamous cell subsets characterized by SPRR3, LCE3D, S100A2, CRCT, and SPRR2G expression, and mesenchymal cell clusters characterized by RHOH, SNAI2, DGKI, CDH2, and IGFBP5 expression. Figure 4 (A and B). These 10 MGHU3-LT tumor subpopulations can be divided into four gene modules based on all highly variable genes (A and B). Figure 4 In module C: the cell clusters in module 1 are mainly composed of UCSD-MGHU3 and metastatic tumors; module 2 is dominated by common cell clusters; the cell clusters in module 3 originate from CTR-MGHU3 tumors; the cell clusters in module 4 mainly originate from MP-MGHU3 tumors. Figure 4 (D and E). Consistent with histopathological findings, high expression of classic luminal feature-related genes (PPARG, GATA3, GRHL2, UPK1B, and FOXA1) was observed in module 3, supporting the luminal characteristics of CTR-MGHU3 tumors. Compared to module 3, module 1 showed changes in basal and squamous lineage-specific gene expression, while module 4 showed changes in EMT / malignant progression lineage-specific gene expression. Notably, module 2, containing a common cell subset, showed the highest squamous differentiation-related gene expression among the four modules ( Figure 4 (Middle F).

[0041] RT4-LT tumor cells were divided into six subsets, including squamous cells and luminal cells. Figure 5 (A and B). These six RT4-LT tumor subpopulations can be divided into two gene modules based on all highly variable genes (A and B). Figure 5 (C). Cell subpopulations in gene module 1 (Leyden clusters 1, 2, and 3) originated from UCSD-RT4-LT. Cell subpopulations in gene module 2 (Leyden clusters 0, 2, and 4) originated from control tumors (C). Figure 5 (D and E). Notably, there are significant differences in the gene expression profiles of UCSD-RT4-LT and CTR-RT4-LT (D and E). Figure 5 Specifically, the upregulation of squamous differentiation-related genes (SPRR3, KRT14, and TGM1) and other luminal-like markers (GATA3, UPK2, UPK1B, and KRT20) in UCSD-RT4-LT tumors indicates that UCSD-RT4-LT tumors, despite undergoing squamous transformation, still retain luminal characteristics. In contrast, CTR-RT4-LT tumors maintain luminal-like characteristics (PPARG, GRHL2, and FOXA1) and have not undergone squamous differentiation. Figure 5 (F). In summary, these data suggest that chemotherapy-induced lineage shifts (such as luminal-basal shift and EMT) increase the malignant potential of tumors, but squamous differentiation is not associated with malignant potential.

[0042] Furthermore, based on transcriptomic data from mUCMP and MHUC cell lines, this invention found that, compared to MHUC, squamous differentiation-related genes (e.g., Ivl, Crct1, and Sprr2e / h / g) were upregulated in mUCMP cells. Figure 5 Enrichment analysis of biological processes also revealed that, compared to MHUC, angiogenesis, epithelial proliferation, EMT, skin development, and epidermal development were significantly enriched in mUCMP (mUCMP). Figure 5 (H). In summary, these data suggest that these novel cell lines could be a valuable tool for cancer research.

[0043] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A bladder tumor cell line with squamous differentiation characteristics, characterized in that, The bladder tumor cell line is selected from one of (1)-(4) as follows: (1) human bladder tumor cell line MP-MGHU3-LT, which is preserved in China Center for Type Culture Collection, with the preservation number of CCTCC NO: C2025241; (2) human bladder tumor cell line UCSD-MGHU3-LT, which is preserved in China Center for Type Culture Collection, with the preservation number of CCTCC NO: C2025252; (3) human bladder tumor cell line UCSD-RT4-LT, which is preserved in China Center for Type Culture Collection, with the preservation number of CCTCC NO: C2025253; (4) mouse bladder tumor cell line mUCMP, which is preserved in China Center for Type Culture Collection, with the preservation number of CCTCC NO: C2025254.

2. The bladder tumor cell line of claim 1 in the establishment of a research model of bladder cancer with squamous differentiation characteristics.

3. The bladder tumor cell line of claim 1 in the exploration of the mechanism of bladder cancer with squamous differentiation characteristics.

4. The bladder tumor cell line of claim 1 in the screening of drugs for the treatment of bladder cancer with squamous differentiation characteristics.

5. The bladder tumor cell line of claim 1 in the establishment of a research model of bladder cancer with chemotherapy resistance and recurrence.

6. The bladder tumor cell line of claim 1 in the exploration of the mechanism of bladder cancer with chemotherapy resistance and recurrence.

7. The bladder tumor cell line of claim 1 in the screening of drugs for the treatment of bladder cancer with chemotherapy resistance and recurrence.

8. Use according to any one of claims 5 to 7, characterized in that, The drug used in the chemotherapy is gemcitabine.

9. A method of screening for potential genes associated with squamous differentiation in bladder cancer, comprising, The method comprises the following steps: comparing the genes expressed in the bladder tumor cell line of claim 1 with the genes expressed in normal bladder cells, and screening out the genes in the bladder tumor cell line of claim 1 that are statistically up-regulated or down-regulated, which are potential bladder cancer squamous differentiation related genes.

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