Human sebaceous gland cancer cell line and use thereof

Abstract

A human sebaceous gland cancer cell line and a use thereof, wherein the human sebaceous gland cancer cell line SHNPH-SeC is preserved in the China Center for Type Culture Collection (CCTCC) on 31 August 2023, and the preservation number thereof is CCTCC NO: C2023113. The human sebaceous gland cancer cell line SHNPH-SeC has a TP53 mutation and is identified as a novel single cell strain by means of STR; the cell line has good adherence and stable cell traits, and can be stably passed down over multiple generations; and the cell line has the ability of rapid proliferation and migration, and meets the features of malignant tumor cells. an SHNPH-SeC cell, its tumor formation in a nude mouse and a clinical human eyelid sebaceous gland cancer tissue specimen from which the SHNPH-SeC cell is derived form a corresponding relationship, so that a novel test material can be provided for researching correlation of the sensitivity and drug resistance of in-vitro, in-vivo, and clinical anti-cancer drugs, as well as the occurrence, development, metastasis and biomarkers of human sebaceous gland cancer.

Description

Human sebaceous cancer cell line and its application Technical Field

[0001] The invention relates to a human sebaceous gland cancer cell line and an application thereof, belonging to the technical field of cell lines. Background Art

[0002] Eyelid sebaceous carcinoma is the second most common eyelid malignancy, accounting for nearly 40% of all eyelid malignancies in China. Patients with stage T4 disease account for 6.25% of all eyelid sebaceous carcinoma cases and are four times more likely to metastasize and approximately 15 times more likely to die from the disease than those with earlier-stage disease. There is an urgent need to identify targeted drug therapies for aggressive, poorly differentiated eyelid sebaceous carcinoma, yet the etiology and pathogenesis of eyelid sebaceous carcinoma remain unclear. The establishment of a human eyelid sebaceous carcinoma cell line provides a rich source of experimental material for understanding the biological characteristics of the tumor, studying its carcinogenesis, molecular genetics, and metastatic evolution, and can contribute to research on tumor mechanisms and chemoresistance mechanisms.

[0003] Currently, the most common mutation in eyelid sebaceous gland carcinoma occurs in TP53, found in approximately 70% of patients. TP53 is a classic tumor suppressor gene, and its mutations play a key role in the development and progression of many tumors. However, currently available sebaceous gland carcinoma cell lines lack TP53 mutations. Therefore, establishing new sebaceous gland carcinoma cell lines harboring TP53 mutations is crucial to enrich and complete the sebaceous gland carcinoma cell line library, provide suitable in vitro models for studying tumor heterogeneity, and promote basic and clinical research on sebaceous gland carcinoma.

[0004] Summary of the Invention

[0005] The purpose of the present invention is to address the low biodiversity of existing human sebaceous cancer cell lines, which have been unable to meet research needs in recent years. The present invention provides a new human sebaceous cancer cell line and its use. The human sebaceous cancer cell line is different from the existing human sebaceous cancer cell lines in typing, carries a TP53 mutation, is more consistent with common clinical tumor types, can be stably passaged, has good tumorigenicity, can be amplified in large quantities and cultured in vitro for a long time, and is suitable for establishing animal models.

[0006] In order to achieve the above-mentioned purpose, the technical solution adopted by the present invention is to provide a human sebaceous gland cancer cell line, which was deposited in the China Center for Type Culture Collection on August 31, 2023, with the deposit number CCTCC NO: C2023113.

[0007] The present invention also provides the use of the human sebaceous gland cancer cell line in preparing a human sebaceous gland cancer animal model.

[0008] The present invention also provides the use of the human sebaceous gland cancer cell line in screening anti-sebaceous gland cancer drugs.

[0009] The present invention also provides the use of the human sebaceous gland cancer cell line in studying the occurrence, development, and metastasis mechanism of human sebaceous gland cancer and screening human sebaceous gland cancer biomarkers.

[0010] Compared with the prior art, the present invention has the following beneficial effects:

[0011] (1) The human sebaceous gland cancer cell line SHNPH-SeC of the present invention was identified as a new single cell line by STR. The cell line has good adherence, stable cell properties, and can be stably passaged multiple times. It also has rapid proliferation and migration capabilities, which are consistent with the characteristics of malignant tumor cells.

[0012] (2) The human sebaceous gland cancer cell line SHNPH-SeC of the present invention has a TP53 mutation, which is significantly different from the sebaceous gland cancer cell lines (TP53 negative) in the existing cell bank. Since TP53 is the most common mutated gene in human sebaceous gland cancer, more than 60% of patients have TP53 mutations. Therefore, the present invention provides new experimental materials for the study of human sebaceous gland cancer that are close to the biological characteristics of clinical tumors, further enriching the cell bank;

[0013] (3) Compared with the prior art, the human sebaceous gland cancer cell line SHNPH-SeC of the present invention is more sensitive to chemotherapy and can be used as a research cell line for effective drug screening; the human sebaceous gland cancer cell line of the present invention has a very strong in vitro tumor-forming ability and can successfully prepare a sebaceous gland cancer model for basic research and drug screening; in the present invention, the tumor volume reached 467 mm in 21 days. 3 . BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 shows the optical morphology of cells at generations P0 (A), P5 (B), P30 (C), and P80 (D) of the SHNPH-SeC cell line;

[0015] FIG2 shows the results of karyotype analysis and STR analysis of the SHNPH-SeC cell line;

[0016] Figure 3 shows the cell proliferation ability detected by CCK-8 assay and the invasion and migration ability of SHNPH-SeC cells detected by Transwell invasion assay; A: Growth curve of SHNPH-SeC cell line drawn by CCK-8 assay; B: Crystal violet staining of Transwell invasion assay;

[0017] FIG4 is a comparison of the immunohistochemical staining results of clinical human sebaceous gland carcinoma surgical tissue specimens and the immunofluorescence results of SHNPH-SeC cells;

[0018] FIG5 is a tumor growth curve after SHNPH-SeC cells were xenotransplanted into mice;

[0019] FIG6 shows the HE staining results of the original tumor tissue (A) and the tumor tissue formed by SHNPH-SeC cells in nude mice (B);

[0020] FIG7 is the immunofluorescence analysis results of tumor tissue formed by SHNPH-SeC cells in nude mice;

[0021] Figure 8 shows mutation analysis of SHNPH-SeC; A: Whole exome sequencing analysis of mutation types in exon regions of SHNPH-SeC and original tumors; B: Mutation substitution analysis of mutation characteristics of SHNPH-SeC and original tumors;

[0022] FIG9 shows the drug sensitivity test results of the SHNPH-SeC cell line to different clinical anti-eyelid sebaceous gland carcinoma drugs; A: drug sensitivity test results of mitogen-activated protein kinase C (MMC); B: drug sensitivity test results of 5-fluorouracil (5-FU).

[0023] Preservation Instructions

[0024] The human sebaceous carcinoma cell line SHNPH-SeC was deposited in the China Center for Type Culture Collection (CCTCC) on August 31, 2023, with the deposit number CCTCC NO: C2023113, and the deposit address is Wuhan University, Wuhan, China. DETAILED DESCRIPTION

[0025] To make the present invention more clearly understood, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

[0027] Example 1 Preparation of human sebaceous carcinoma cell line (SHNPH-SeC cell line)

[0028] 1.1 Source of specimens

[0029] On August 31, 2021, a postoperative specimen was obtained from an 87-year-old patient with stage T4a sebaceous carcinoma at the Ninth People's Hospital affiliated with Shanghai Jiao Tong University School of Medicine. The patient presented with a 2-year history of a mass in the inner canthus of his left eye, which had grown rapidly for six months, causing swelling of the entire eyelid, exotropia, and inability to move the eyeball. The pathological diagnosis was sebaceous carcinoma. The tissue measured 4 x 2.5 x 2.2 cm. The patient signed an informed consent form before surgery.

[0030] 1.2 Specimen processing

[0031] Fresh sebaceous carcinoma specimens were obtained after surgical resection, rinsed with PBS three times, and the tumor tissue was separated from the normal tissue.

[0032] Under sterile conditions, sebaceous gland carcinoma specimens were cut into small tissue blocks and immersed in RPMI-1640 culture medium (purchased from Gibco) (containing 2% fetal bovine serum, 200 mg / ml streptomycin, and 200 IU / ml penicillin) at room temperature.

[0033] Under sterile conditions, sebaceous gland carcinoma specimens were cut into tissue blocks of approximately 1 mm*1 mm in size using a scalpel. After tissue shearing, the tissues were placed in a 15 ml sterile centrifuge tube and RPMI-1640 containing 0.5% collagenase I (purchased from Invitrogen) was added. The tubes were shaken and digested at 37°C for 4 h.

[0034] After digestion, the tissue was centrifuged at 800 rpm for 4 minutes at room temperature. After treatment with trypsin-EDTA (0.53 mM EDTA, 0.05% trypsin, purchased from Gibco) for 2 minutes, the cells were neutralized and precipitated in RPMI-1640 medium (containing 10% fetal bovine serum, 100 mg / ml streptomycin, and 100 IU / ml penicillin). After further centrifugation, the cells were harvested and cultured in RPMI-1640 medium (containing 10% fetal bovine serum, 100 mg / ml streptomycin, and 100 IU / ml penicillin) in a 37°C, 5% CO2 incubator for 2 weeks.

[0035] 1.3 Monoclonal Purification

[0036] Select the cell areas with strong vitality and prepare a single cell suspension of 10 cells / mL. Take 100 μL of each suspension and culture it in a 96-well plate to make a single cell proliferation well for monoclonal purification.

[0037] Single cell clones were obtained and expanded after trypsin digestion for 1 minute. The culture medium was RPMI-1640 medium. The cells were incubated at 37°C and 5% CO2 in an incubator and passaged once a week. The cells are currently growing well with a relatively uniform morphology and have been passaged for more than 80 generations.

[0038] The above-mentioned human sebaceous gland cancer cells were named human sebaceous gland cancer cell line SHNPH-SeC. The cell line was deposited in the China Center for Type Culture Collection (CCTCC) on August 31, 2023, with the deposit number CCTCC NO: C2023113.

[0039] Example 2 Biological Characteristics and Applications of SHNPH-SeC Cell Line

[0040] 2.1 Morphological observation of SHNPH-SeC cell line

[0041] Subcultured SHNPH-SeC cells were observed under an optical microscope (100×) for viable cell growth. The results are shown in Figures 1A to 1D, which show optical morphology images of cells at passages P0, P5, P30, and P80 of the SHNPH-SeC cell line, respectively. As can be seen from Figures 1A to 1D, the SHNPH-SeC cell line exhibited vigorous growth, a clear background, minimal impurities, and spindle-shaped adherent cells.

[0042] 2.2 STR analysis of genetic material in the SHNPH-SeC cell line

[0043] Short tandem repeats (STRs), also known as microsatellite DNA, are a type of DNA sequence on chromosomes consisting of a core unit of several base pairs (2-6 base pairs) that repeats in tandem (10 to 60 times, with gene segments under 400 base pairs). The number of repeats of each core unit varies between individuals, resulting in alleles of varying lengths. Therefore, the number of repeats of a set of STR sequences is virtually unique across individuals, serving as a hallmark of their genetic identity and a primary method used in cell biology to identify cell identity and origin.

[0044] Genomic DNA was extracted using a QIAGEN genomic extraction kit (QIAamp PowerFecal DNA Kit, purchased from QIAGEN) and analyzed for 21 STR loci and the sex gene Amelogenin on an ABI 3730XL genetic analyzer. The results are shown in Figure 2.

[0045] STR sequence searches in the databases of the German Collection of Microorganisms (DSMZ), the American Type Culture Collection (ATCC), the Japanese Cell Collection (JCRB), and the RIKEN Institute of Physical and Chemical Research (RIKEN) revealed no identical STR results. This means no matching sites were found, indicating a novel cell line. The absence of multiple, equal sites indicates a single cell line, free of contamination from other cells.

[0046] 2.3 Growth kinetics and invasiveness studies of the SHNPH-SeC cell line

[0047] Take the logarithmically growing cells and make 2×10 4Cells / ml cell suspension was plated in 96-well plates at a volume of 100 μL per well, and cell proliferation was detected on days 0, 1, 2, 3, and 4 using the Cell Counting Kit-8 (CCK-8) assay. The CCK-8 assay was performed according to the kit instructions. The sample was incubated with 10 μL of CCK-8 solution in a 37°C, 5% CO2 incubator for 2 hours, and the OD value was obtained at 450 nm. The growth curve of the SHNPH-SeC cell line is shown in Figure 3. As shown in Figure 3A, the growth curve of SHNPH-SeC cells doubled in approximately 36 hours, indicating strong proliferation capacity.

[0048] The invasion assay is to evaluate the migration ability of cells. A Transwell chamber is placed in a culture plate. The upper chamber is filled with a culture medium containing 2% PBS, and the lower chamber is filled with 900 μL of complete culture medium. The upper and lower layers are separated by a polycarbonate membrane (8 μm pores). 5×10 4 After incubation for 24 hours, the cells were stained with 0.25% crystal violet and observed and recorded under a microscope (see Figure 3B). It showed that most cells migrated through the membrane into the lower chamber within 24 hours, indicating that SHNPH-SeC cells have invasive behavior.

[0049] 2.4 Identification of biological characteristics of the SHNPH-SeC cell line

[0050] 2.4.1 SHNPH-SeC cells and clinical human sebaceous gland carcinoma surgical tissue specimens were subjected to immunofluorescence and immunohistochemical staining according to the following steps:

[0051] SHNPH-SeC cell treatment procedure: Cells were plated in 24-well plates and cultured for 24 hours before being fixed with 4% paraformaldehyde for 30 minutes. The cells were then treated with 0.5% Triton X-100 for 15 minutes and blocked with 5% goat serum (Invitrogen) for 1 hour. The cells were then treated with the following primary antibodies (anti-CK5, anti-EMA, anti-Adipophilin, anti-p53, and anti-Ki-67) at 4°C overnight, followed by treatment with secondary antibodies for 1 hour at room temperature.

[0052] Human sebaceous carcinoma tissue processing procedure: Tissue was cut into approximately 1 cm × 1 cm × 0.3 cm sections, placed in a tissue embedding cassette, and fixed in 10% neutral buffered formalin. Dehydrated for 2 hours using 75%, 80%, 95%, 95%, 100%, and 100% ethanol, respectively. Treated with xylene for 30 minutes twice, immersed in paraffin at 60°C for 2 hours, embedded and sectioned at 4 μm thickness, and baked in a 60°C oven for 1 hour. Following sectioning, sections were deparaffinized with xylene and then treated with 0.1% Triton X-100 and 0.3% H2O2. Tissues were blocked with 4% goat serum and incubated with primary antibodies (anti-CK5, anti-EMA, anti-adipophilin, anti-p53, and anti-Ki-67) for 60 minutes at room temperature.

[0053] 2.4.2 Microscopic photography was performed under a Nikon Eclipse microscope in Japan to compare the biomarker staining of clinical sebaceous gland carcinoma tissue specimens and SHNPH-SeC cells: Immunofluorescence staining showed that SHNPH-SeC cells positively expressed CK5, EMA, Adipophilin, p53, and Ki-67 with a labeling index of 60%, which was consistent with the immunohistochemical staining results of tumor tissues, as shown in Figure 4.

[0054] 2.5 Tumorigenicity of cells

[0055] SHNPH-SeC cells in the logarithmic growth phase were collected and prepared into a cell suspension. 1×10 6 The cells were implanted in the right subcostal skin of male NOD / SCID mice. Tumor formation was observed and recorded, and tumor volume was assessed every 3 days to plot a tumor growth curve. Tumors were removed 21 days after implantation.

[0056] The tumor growth curve from 0 to 21 days after implantation is shown in Figure 5. It can be seen that the tumor diameter increases rapidly from 0 to 21 days after implantation, and the tumor volume reaches 467 mm on day 21. 3 It can be seen that SHNPH-SeC cells exhibit very strong in vitro tumorigenic ability.

[0057] 2.6 Pathological identification of tumors

[0058] The tumor formed by the subcutaneous inoculation of SHNPH-SeC cells in nude mice 21 days after step 2.5 above was paraffin-embedded, sectioned, and stained with H&E according to the following steps: the tissue was cut into pieces of approximately 1 cm × 1 cm × 0.3 cm, placed in a tissue embedding box, and fixed with 10% neutral buffered formalin; dehydrated with 75%, 80%, 95%, 95%, 100%, and 100% ethanol for 2 hours in sequence; transparentized with xylene for 30 minutes, twice; immersed in 60°C paraffin for 2 hours; embedded with a German Leica JUNG embedding machine; sliced ​​with a German Leica 2135 paraffin microtome with a thickness of 4 μm; baked in a 60°C oven for 1 hour; stained with a German Leica ST5020 stainer; and sealed with a German Leica CV5030 sealing machine.

[0059] Microscopic photography was performed under an optical microscope to observe the morphology of tumor cells formed by SHNPH-SeC cells in nude mice by HE staining, as shown in Figure 6B. Figure 6A is a HE staining slide of the original tumor, showing that the histological characteristics of the mouse xenografted SeC are consistent with the original tumor.

[0060] 2.7 Immunofluorescence analysis of nude mouse tumor tissue

[0061] Nude mouse tumor tissues were taken for immunofluorescence analysis (the experimental process was the same as the immunofluorescence analysis method in the above 2.4). The results of immunofluorescence analysis of nude mouse tumor tissues are shown in Figure 7. It can be seen that the immunofluorescence display of xenografted SeC was the same as that of the original tumor and SHNPH-SeC cells, showing CK5, EMA, Adipophilin, p53 and Ki-67 positivity (Figure 4).

[0062] 2.8 Mutational analysis of SHNPH-SeC

[0063] Whole-exome sequencing can comprehensively reflect the genetic information of biological samples and reveal genomic mutational characteristics, making it a commonly used method for identifying the presence of gene mutations. Genomic DNA was extracted from primary tumor tissue and SHNPH-SeC cells using a DNA extraction kit (purchased from Beyotime). DNA quantification and integrity were analyzed using a Thermo Fisher spectrophotometer and 1% agarose gel electrophoresis, respectively. Genomic DNA was collected using the Agilent SureSelect Human All Exon V6 library according to the manufacturer's instructions and sequenced on the HiSeq X10 sequencing platform. Analysis included single nucleotide polymorphisms, insertions and deletions, copy number variations, and structural variations.

[0064] SHNPH-SeC and the original tumor have similar mutation types in the exon region, as shown in Figure 8A. Mutation substitution analysis also shows that SHNPH-SeC and the original tumor have consistent characteristics, as shown in Figure 8B. The top ten genes associated with sebaceous gland carcinoma in the COSMIC database are TP53, RB1, CTNNB1, LEF1, ERBB2, KMT2C, PIK3CA, PBRM1, KMT2D, and IL6ST, among which the mutation rate of TP53 is approximately 70%. There are 7 shared protein-altering mutations in SHNPH-SeC and tumors, all of which are associated with the top ten genes associated with sebaceous gland carcinoma. Among them, TP53, the gene most frequently mutated in sebaceous gland carcinoma, has a missense mutation (c.C215G: p.P72R), as shown in Table 1 below:

[0065] Table 1:

[0066] According to prior art, all sebaceous cancer cell lines in existing cell banks do not have TP53 mutations. However, the SHNPH-SeC cell line in the present invention does have a TP53 mutation, demonstrating that the SHNPH-SeC sebaceous cancer cell line of the present invention is distinct from the cell lines in existing cell banks. Furthermore, because TP53 mutations are characteristic of approximately 70% of sebaceous cancers, the present invention can further improve sebaceous cancer cell banks and facilitate the development of basic experiments.

[0067] 2.9 Drug sensitivity test of SHNPH-SeC cell line

[0068] Take the logarithmically growing cells and make 2×10 4 Cells / ml cell suspension was seeded in a 96-well plate at a volume of 100 μL per well. When the cell density reached about 80%, mitogen-activated protein cytokines (MMC) at 0, 0.15625, 0.3125, 0.625, 1.25, 2.5, 5, 10, and 20 μmol / L or 5-fluorouracil (5-FU) at 0, 5, 10, 20, 40, 80, 160, 320, and 640 μmol / L was added to each well in sequence. After 48 hours, the cell viability of each well was detected using the CCK-8 method. For specific methods, refer to 2.3. The results of the drug sensitivity test of MMC and 5-FU are shown in Figures 9A and 9B, respectively. The half-maximal effective doses (EC50) of MMC and 5-FU in the SHNPH-SeC cell line were 1.13 μM and 83.05 μM, respectively. It was previously reported that the EC50 of MMC in the sebaceous cancer cell line BP50 without TP53 mutation is 10.4 μM, and the EC50 of 5-FU is 80 mM. Therefore, it can be seen that SHNPH-SeC is more sensitive to MMC and 5-FU than the BP50 cell line.

[0069] Based on the above experimental observations and validation, SHNPH-SeC cells grown in vitro exhibited good adherence and stable cell characteristics, allowing for multiple passages. They also exhibited malignant growth. STR analysis confirmed that the cells were a novel single cell line. Positive staining for sensitive and reliable biomarkers for eyelid sebaceous carcinoma, including CK5, EMA, Adipophilin, and p53, confirmed the cell line as a sebaceous carcinoma cell line. The SHNPH-SeC cells were capable of large-scale expansion, exhibited robust proliferative activity, and were able to form tumors in nude mice. Concordant staining of the original tumor, SHNPH-SeC cells, and xenografts indicated that SHNPH-SeC cells were derived from a patient with eyelid sebaceous carcinoma. Similar H&E staining between the original tumor and xenografts further confirmed that SHNPH-SeC was an eyelid sebaceous carcinoma cell line. Furthermore, the shared mutational profile between the original tumor and SHNPH-SeC cells was confirmed, with SHNPH-SeC cells harboring the most common TP53 mutation in sebaceous carcinoma. In terms of malignant characteristics, the SHNPH-SeC cell line exhibits a high Ki-67 positivity rate, abundant sebaceous carcinoma-related gene mutations, rapid in vitro proliferation and migration, and in vivo tumorigenicity. Drug sensitivity also demonstrates that SHNPH-SeC cells respond well to chemotherapy, even outperforming sebaceous carcinoma cells without TP53 mutations. The SHNPH-SeC cells, their tumorigenicity in nude mice, and the clinical human eyelid sebaceous carcinoma tissue specimens from which they are derived form a corresponding relationship, providing new experimental material for studying the correlation between in vitro, in vivo, and clinical anticancer drug sensitivity and resistance, as well as the occurrence, development, metastasis, and biomarkers of human sebaceous carcinoma.

[0070] The above embodiments are only preferred embodiments of the present invention and are not intended to limit the present invention in any form or substance. It should be noted that a person skilled in the art may make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be considered as the scope of protection of the present invention.

Claims

1. A human sebaceous gland cancer cell line, characterized in that The human sebaceous cancer cell line was deposited in the China Center for Type Culture Collection on August 31, 2023, with the deposit number CCTCC NO: C2023113.

2. Use of the human sebaceous gland cancer cell line according to claim 1 in preparing a human sebaceous gland cancer animal model.

3. Use of the human sebaceous gland cancer cell line according to claim 1 in screening anti-sebaceous gland cancer drugs.

4. Use of the human sebaceous gland cancer cell line according to claim 1 in studying the occurrence, development and metastasis mechanism of human sebaceous gland cancer and screening human sebaceous gland cancer biomarkers.

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