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Isolation and Immortalization of Fibroblasts from Different Tumoral Stages
不同肿瘤阶段成纤维细胞的分离和永生化

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Abstract

Tumour microenvironment and cancer-associated fibroblasts in particular exhibit tumour promoting abilities that are not present in their normal counterparts (Calvo et al., 2013; Hanahan and Coussens, 2012). Therefore, functional and molecular characterization of the modifications occurring in fibroblasts during tumour progression is essential to fully understand their role in tumour progression. Previous studies have addressed this issue using human fibroblasts and comparing normal and adjacent fibroblasts to tumour-associated fibroblasts (Kalluri and Zeisberg, 2006). However, these studies are hampered by the intrinsic variability of human samples (e.g. pairing, age, genomic landscape, etc). In order to overcome these issues, we used a fully characterised mouse breast cancer model, MMTV-PyMT (Guy et al., 1992; Lin et al., 2003). MMTV-PyMT transgenic mice express the Polyoma Virus middle T antigen under the direction of the mouse mammary tumor virus promoter/enhancer. This is a multifocal luminal breast cancer model that goes through well defined and characterised stages (namely, hyperplasia, adenoma, carcinoma and invasive carcinoma). Interestingly, this model has a 100% incidence, is very desmoplastic (presenting high concentration of fibroblasts) and gives raise to spontaneous metastasis in the lung with 80-94% incidence. Importantly, at least for the inguinal mammary glands (glands 4 and 9), the different tumoral stages are well correlated to the age of the mouse: hyperplasia arising at 6 weeks of age, adenoma between 6-8 weeks of age, carcinoma and invasive carcinoma from 8 weeks onwards. This model allowed us to confidently isolate fibroblasts from different tumoral stages and carefully characterise their functional and molecular properties (Calvo et al., 2013).

Materials and Reagents

  1. FVB/n MMTV-PyMT females between 4 and 14 weeks of age and MMTV-PyMT negative siblings for isolation of normal mammary gland fibroblasts (NFs) (The Jackson Laboratory, catalog number: 00 2374 )
  2. Phoenix-Eco packing cells (ATCC, catalog number: CRL-3214 ) (read Note 6 for more information)
  3. pBabe-HPV-E6-puromycin plasmid (or immortalization plasmid of choice, see Note 6)
    Note: This was generated in our laboratory but there are alternatives available (pLenti-puro-HPV-16 E6/E7; Applied Biological Materials, catalog number: G268 ).
  4. Dulbecco’s Modified Eagle’s Medium (DMEM) (high glucose with stable L-glutamine) (Life Technologies, Gibco®, catalog number: 41966-029 )
  5. 50x sodium butyrate (500 mM)
  6. Polybrene® (1,5-dimethyl-1,5-diazaundecamethylene polymethobromide, hexadimethrine bromide) (Sigma-Aldrich, catalog number: AL-118 ) (1000x solution at 2 mg/ml)
  7. Insulin-Transferrin-Selenium (ITS) solution (Life Technologies, catalog number: 51300-044 ).
  8. Calcium phosphate transfection kit (ProFection® Mammalian Transfection System) (Promega Corporation, catalog number: E1200 )
  9. Fetal Bovine Serum (FBS)
  10. Phosphate-buffered saline (PBS)
  11. PBS without Ca2+ and Mg2+
  12. Penicillin/Streptomycin
  13. EDTA (1 mM in PBS without Ca2+ and Mg2+)
  14. 10% formalin solution, neutral-buffered (10% NBF)
  15. 100x Collagenase/dispase (100 mg/ml) (Roche Diagnostics, catalog number: 11097113001 )
  16. 1000x puromycin (2 mg/ml) (Sigma-Aldrich, catalog number: P9620 )
  17. 1000x DNase I (10 mg/ml) (Sigma-Aldrich, catalog number: D4513 )
  18. Trypsin/EDTA
  19. Virkon (DuPont Rely+On Virkon, catalog number: 1235-8667 )
  20. Complete media (see Recipes)

Equipment

  1. 0.22 µm, 0.45 µm, 3 mm filters
  2. 20 mm coverslip
  3. Tissue culture plastic-ware
  4. 37 °C shaker
  5. Tissue culture laminar-flow hood
  6. 10 cm tissue culture dish
  7. Centrifuge
  8. 37 °C, 5% CO2, cell culture incubator
  9. Scalpels
  10. Surgical scissors
  11. Dissecting instruments

Procedure

  1. Before starting
    1. Please read Notes section for guidance.
    2. Obtain MMTV-PyMT positive females between 5 and 14 week of age.
      Note: Correlation between age and tumoral stage may vary in other tumor models. Read Notes section for more information.
    3. Make sure you have all the materials, reagents and equipment.

  2. Dissecting samples – tumor material
    1. Mammary tissue from the 4th and 9th inguinal glands is dissected using surgical instruments. Tissue is kept in ice-cold PBS until further use.
      Note: For normal mammary gland isolation, mammary tissue of MMTV-PyMT negative females of 10 weeks of age is dissected. For isolation of hyperplastic tumors, mammary tissue of MMTV-PyMT positive females of 6 weeks of age is dissected. For isolation of adenoma, carcinoma, and invasive carcinoma, mammary tissue of MMTV-PyMT positive females of 7, 8 and 10 weeks of age, respectively, is dissected (Figure 1). These time points may vary. For more information about the determination of the correct age and tumoural stage, please read Note 2 at the end of the protocol.
    2. Samples are moved into a laminar flow tissue culture hood under sterile conditions.
    3. Tissue samples are carefully analysed checking for signs indicative of their tumoral stage. As a rough guide, normal tissue appears fatty and soft. Hyperplastic tissue appears considerably enlarged but still fatty and soft. Adenoma, carcinoma, and invasive carcinoma appear enlarged, stiff and multimodular to variable degrees (Figure 1).
    4. Isolate single modules on each tumor (2-3 per tumour is recommended.) before proceeding to the next stage.
      Note: As a result of the multimodular aspect of this type of tumour, special care must be taken in order to use and analyse only single modules. This is highly important if you want to make sure that you are isolating cells from a specific stage as individual modules may have progressed to different tumoral stages. The size of the modules varies with tumour size, age, localization and between individual mice. Expert pathological advice is recommended (see Notes 2-3).
    5. For each single module, take one part for histopathology analysis and one part for isolation of fibroblasts. For histopathological analysis, the sample is fixed in 10% NBF overnight at 4 °C. See Figure 1 for representative hematoxylin and eosin staining of tissues.
      Note: Histopathological analysis procedures will not be discussed here. Please refer to other protocols.
    6. For isolation of fibroblasts, the rest of the sample is transferred to a 10 cm tissue culture dish and processed into smaller pieces using a scalpel and/or surgical scissors.


      Figure 1. Representative tissue samples and hematoxylin and eosin staining of tissues from normal FVB/n mammary glands and progressive breast tumoural stages (hyperplasia, adenoma and carcinoma) from the MMTV-PyMT model. Scale bars represent 100 µm.

  3. Processing normal mammary tissue samples
    1. For normal mammary tissue, the smaller tissue pieces are placed into dishes where they are compressed under a 20 mm coverslip to prevent the fatty tissue floating.
      Note: If normal mammary tissue is processed using the collagenase/disparse approach (see below), very low numbers of fibroblasts are isolated.
    2. Fresh complete media DMEM is then added slowly to prevent samples from floating.
      Note: Samples need to be in contact with the plastic and the coverslip in order for the fibroblasts to come out of the sample.
    3. Media is replaced daily with fresh complete media for 7 days.
      Note: After 7 days, fibroblasts have come out of the tissue into the coverslip and the dish and can be transferred into a new dish.
    4. Original tissue is removed. Coverslips are transferred to a new dish (side with cells facing up).
    5. The original dish and the dish with coverslips are washed once with PBS and fibroblasts trypsinized using Trypsin/EDTA for 10 min at 37 °C.
    6. Cells are resuspended and seeded into a new dish.
      Note: Cell numbers can be expanded minimally, always taking into account that primary cells enter senescence after prolonged culture in vitro.
    7. Cells are now ready for immortalization (starting at step E30).

  4. Processing mammary hyperplasia, adenoma and carcinoma tissue samples
    1. For hyperplasia, adenoma and carcinoma samples smaller tissue pieces are placed into dishes where they are further disgregatted using scalpels.
      Note: Alternatively, for extremely fatty hyperplasia samples normal tissue isolation approach (steps C10-16) can be used.
    2. Collagenase/dispase solution (100 mg/ml) is diluted 1:100 in sterile PBS Mg2+ Ca2+ free and filtered before use with 22 µm filter (final concentration 1 mg/ml).
      Note: Collagenase/dispase solution is a tissue dissociation solution that degredes collagen and reticular fibers without affecting the integrity of the plasma membrane.
    3. Disgregatted tissues are placed in a sterile tube with the appropriate amount of collagenase/dispase. Pipette up and down several times to help disaggregate. Samples are then incubated for 60 min at 37 °C and 180 rpm in an orbital shaker.
      Note: Generally use 2-3 ml per sample (250-500 mg of tissue). Adjust to sample size.
    4. One volume of PBS 1 mM EDTA is added to each sample. Pass it through the pipette several times.
      Note: EDTA stops the protease reaction as it is a collagenase inhibitor.
    5. Samples are filtered using the 3 mm filters.
      Note: This step removes undigested pieces of tissue. Alternative methods are also available (rubber syringe plunger, etc).
    6. Samples are centrifuged at 100 x g for 5 min at room temperature (RT).
    7. Supernatant is discarded. Pellet containing cells is washed with complete media and centrifuged at 100 x g for 5 min at RT again.
    8. Supernatant is discarded. Pellet containing cells is incubated for 10 min at RT with complete media plus DNase 1 (final concentration 10 µg/ml).
      Note: Often as a result of cell damage, DNA leaks into the dissociation medium increasing viscosity and causing handling problems. Purified DNase is included in cell isolation procedures to digest the nucleic acids without damaging the intact cells.
    9. Samples are centrifuged at 100 x g for 5 min at room temperature (RT).
    10. Repeat step D23 twice.
    11. Media is removed and cells are resuspended in complete media and seeded in a dish. Place samples in the incubator.
    12. After 30 min, media in all samples is replaced with fresh complete media and place back in the incubator.
      Note: After 30 min, most of the fibroblasts would have adhered to the dish, whilst other cell types will remain in suspension. Using this tip will help you enrich for the fibroblastic population.
    13. After 2-3 days, isolated cells can be enriched for the fibroblastic population using the enhanced adherence to plastic of fibroblasts.
      1. Media is removed.
      2. Cells are washed with PBS once.
      3. Cells are covered with Trypsin/EDTA and placed in the incubator for 10 min.
      4. Cells are resuspended in complete media, seeded into a new dish and placed back in the incubator for 30 min.
      5. Media is replaced with fresh new complete media.

  5. Immortalizing primary fibroblasts from different tumoral stages
    1. Read Notes 4-5 before start.
    2. Growing Phoenix-ECO cells are seeded to reach 80% confluency by the time of transfection (see Note 6).
      Note: Phoenix-Eco cells are used to produce ecotropic viruses to infect mouse fibroblasts. To immortalize human fibroblasts, please use alternative approaches (see Notes 4 and 6). Optimization of volumes and titration of retroviruses is highly recommended but will not be discussed here. Using easy-to-spot positive controls (e.g. transfection of retroviral plasmids encoding fluorescent proteins) is also recommended. This allows for rapid evaluation of your infection efficiency (e.g. scoring for fluorescently positive cells).
    3. Once they reach 80% confluency, Phoenix-Eco cells are transfected using the calcium phosphate protocol. Usually for 10 cm plate.
      1. Replace media with complete media 2 h before transfection.
      2. In Tube A, mix pBabe-HPV-E6-puromycin (10 µg), 2 M CaCl2 (67 µl) and Nuclease-free water (up to 500 µl).
      3. In Tube B, add 500 µl of 2x Hepes.
      4. While bubbling the content in Tube A, slowly add content of Tube B into it.
      5. Leave 30 min at RT.
      6. Add transfection mix dropwise into Phoenix-Eco cells.
    4. After 18 h, in order to burst transcription, Sodium Butyrate is added to the cells (final concentration 10 mM).
    5. After 4 h, media is replaced with fresh complete media.
    6. After 48-72 h, media containing viruses is collected and filtered through a 0.45 µm filter. Virus can be aliquoted and stored at -80 °C until used.
      Note: All used material must be left o/n in virkon before disposal (see Note 5).
    7. Primary fibroblasts are infected with HPV-E6-puromycin retroviruses.
      1. Media on fibroblast cultures is removed.
      2. The virus-containing media is mixed 1:1 with complete fresh media and the resulting solution added to the fibroblasts.
      3. Polybrene (final concentration 2 µg/ml) is also added to your cells.
        Note: Infection efficiency may increase if receptor cells are in suspension (e.g. after trypsinization). Polybrene (hexadimethrine bromide) is a cationic polymer used to increase the efficiency of infection of certain cells with a retrovirus in cell culture.
    8. After 5 h, media is replaced with fresh media.
      Note: Alternatively, media can be replaced after 18 h.
    9. After 24-48 h, infected cells are selected by addition of puromycin (2 µg/ml final concentration) in the complete culture media.
      Note: Puromycin subsceptibility varies depending on the cell type. Final concentration needs to be optimized prior to final use.
    10. After 7-10 days, resistant clones expressing HPV-E6 (i.e. immortalized cell lines) are visible. Clones or pools can be expanded in culture of frozen down for subsequent use.
      Note: Eventually, characterization of the nature of the isolated and immortalized populations must be performed. This can be performed by analysis of expression of certain markers by immunofluorescence, FACS, etc. Recommended fibroblast markers are CD140A/PDGFRA, CD90/Thy-1, Vimentin, and Fibronectin. Fibroblasts should also be negative for immune cell markers (CD45 negative), endothelial markers (CD31 negative) and epithelial markers (E-Cadherin, EpCAM negative). For cancer-associated fibroblast markers we recommend αSMA, FAP and FSP1/S100A4.

Notes

  1. This procedure works exceptionally well because it is optimized for this tumour model. Importantly, MMTV-PyMT breast cancer cells do not grow on tissue culture plastic dishes. On the other hand, fibroblasts grow very well. Therefore, just by culturing your extracts in plastic, you are enriching for the fibroblast population and any contamination derived from cancer cells is avoided. However, this procedure does not work if established cancer cell lines are used, as those cells are adapted to grow in plastic and will eventually overtake the fibroblast population. In order to prevent this, other approaches need to be designed (FACS sorting, use of suicide genes in cancer cells, etc).
  2. This procedure needs a well-characterized model and much familiarization with the different tumoral stages of its development. Therefore, it is essential that, prior to its application, the researcher gets familiar with it. Expert pathological evaluation is essential. We recommend thorough pathological analysis of multiple tumours of different ages, sizes, aspects and sites. This will eventually allow the researcher to confidently predict the stage of each tumour. Even then, retrospective pathological analysis of the tumoral stage of the tumours used for fibroblast isolation must always be performed.
  3. Histopathology and immunocytochemistry approaches will not be discussed in this protocol. Tumour progression is highly variable depending on type, tissue, mouse strain, etc. In order to characterise each tumoral stage, consult a pathologist to get advice about the best approaches, techniques and markers to clearly define each stage.
  4. Primary cells and specially fibroblasts can be maintained in culture for few passages before they become senescent. Senescence induces a pletora of transcriptional and functional changes that may affect profoundly the phenotypes under study. Therefore, if long term culture is desired, immortalization is necessary. Ideally, primary and immortalized cultures should be compared in order to check that the immortalization procedure has not induced any change in the phenotypes under study. For immortalizing mouse fibroblasts, we express the human papillomavirus “early gene” E6 that prevents replicative senescence by suppressing p53 activity. As a result, other “off-target” consequences in specific functions and signalling pathways are to be expected. For cells that are most affected by telomere length (e.g. human), an alternative approach is recommended. In those cases, cell immortalization can be achieved by expression of Telomerase Reverse Transcriptase protein (hTERT). This protein is inactive in most somatic cells, but when hTERT is exogenously expressed, the cells are able to maintain sufficient telomere lengths to avoid replicative senescence. Analysis of several telomerase-immortalized cell lines has verified that the cells maintain a stable genotype and retain critical phenotypic markers.
  5. Immortalization is achieved by retroviral infection of target fibroblasts. Thus, handling and disposal of viruses and contaminated material must be always performed in accordance with you institution regulations.
  6. Phoenix-ECO were originally established in Garry P. Nolan lab (Stanford University, USA) and are available at ATCC (Pear et al., 1993). They are a retrovirus producer line for the generation of helper free ecotropic retroviruses. The line is based on the 293T cell line (a human embryonic kidney line transformed with adenovirus E1a and carrying a temperature sensitive T antigen co-selected with neomycin). This cell line is highly transfectable with either calcium phosphate mediated transfection or lipid-based transfection protocols. The line was created by placing into 293T cells constructs capable of producing gag-pol, and envelope protein for ecotropic viruses. Phoenix-Ampho is a similar cell line that instead produces amphotropic retroviruses.

Recipes

  1. Complete media (always to be used warm 37 °C)
    Dulbecco’s Modified Eagle’s Medium (DMEM) high glucose with stable L-glutamine
    1% Insulin-Transferrin-Selenium solution (ITS)
    10% Fetal Bovine Serum (FBS)
    Penicillin/Streptomycin

Acknowledgments

This protocol is an extended version of the one described in Calvo et al. (2013). FC, SH and ES were supported by a Cancer Research UK grant CRUK_A5317. We thank lab members for help and advice throughout this work.

References

  1. Calvo, F., Ege, N., Grande-Garcia, A., Hooper, S., Jenkins, R. P., Chaudhry, S. I., Harrington, K., Williamson, P., Moeendarbary, E., Charras, G. and Sahai, E. (2013). Mechanotransduction and YAP-dependent matrix remodelling is required for the generation and maintenance of cancer-associated fibroblasts. Nat Cell Biol 15(6): 637-646.
  2. Guy, C., Cardiff, R. and Muller, W. (1992). Induction of mammary tumors by expression of polyomavirus middle T oncogene: a transgenic mouse model for metastatic disease. Mol  Cellr Biol 12(3): 954-961.
  3. Hanahan, D. and Coussens, L. M. (2012). Accessories to the crime: functions of cells recruited to the tumor microenvironment. Cancer Cell 21(3): 309-322.
  4. Kalluri, R. and Zeisberg, M. (2006). Fibroblasts in cancer. Nat Rev Cancer 6(5): 392-401. 
  5. Lin, E. Y., Jones, J. G., Li, P., Zhu, L., Whitney, K. D., Muller, W. J. and Pollard, J. W. (2003). Progression to malignancy in the polyoma middle T oncoprotein mouse breast cancer model provides a reliable model for human diseases. Am J Pathol 163(5): 2113-2126.
  6. Pear, W. S., Nolan, G. P., Scott, M. L. and Baltimore, D. (1993). Production of high-titer helper-free retroviruses by transient transfection. Proc Natl Acad Sci U S A 90(18): 8392-8396.

简介

肿瘤微环境和癌相关成纤维细胞特别表现出其正常对应物中不存在的肿瘤促进能力(Calvo等人,2013; Hanahan和Coussens,2012)。因此,在肿瘤进展期间发生在成纤维细胞中的修饰的功能和分子表征是完全理解它们在肿瘤进展中的作用所必需的。以前的研究已经解决了这个问题使用人类成纤维细胞和比较正常和相邻成纤维细胞与肿瘤相关的成纤维细胞(Kalluri和Zeisberg,2006)。然而,这些研究受到人类样品的内在变异性(例如配对,年龄,基因组景观,等)的阻碍。为了克服这些问题,我们使用了完全表征的小鼠乳腺癌模型MMTV-PyMT(Guy等人,1992; Lin等人,2003)。 MMTV-PyMT转基因小鼠在小鼠乳腺肿瘤病毒启动子/增强子的指导下表达多瘤病毒中T抗原。这是一个多焦点管腔乳腺癌模型,经历良好定义和特征阶段(即增生,腺瘤,癌和浸润性癌)。有趣的是,这种模型有100%的发病率,是非常结构性的(呈现高浓度的成纤维细胞),并引起肺自发转移80-94%的发病率。重要的是,至少对于腹股沟乳腺(腺4和9),不同的肿瘤阶段与小鼠的年龄很好相关:在6周龄时出现的增生,6-8周龄之间的腺瘤,癌和侵袭性癌症从8周开始。该模型使我们可靠地分离来自不同肿瘤阶段的成纤维细胞,并仔细表征其功能和分子性质(Calvo等人,2013)。

材料和试剂

  1. 4周龄和14周龄的FVB/n MMTV-PyMT雌性和用于分离正常乳腺成纤维细胞(NF)的MMTV-PyMT阴性兄弟姐妹(The Jackson Laboratory,目录号:002374)
  2. Phoenix-Eco包装细胞(ATCC,目录号:CRL-3214)(阅读注释6了解更多信息)
  3. pBabe-HPV-E6-嘌呤霉素质粒(或选择的永生化质粒,参见注释6)
    注意:这是在我们的实验室生成的,但有替代品(pLenti-puro-HPV-16 E6/E7;应用生物材料,目录号:G268)。
  4. Dulbecco改良的Eagle培养基(DMEM)(具有稳定的L-谷氨酰胺的高葡萄糖)(Life Technologies,Gibco ,目录号:41966-029)
  5. 50x丁酸钠(500mM)
  6. (1,5-二甲基-1,5-二氮杂十一亚甲基聚甲溴化物,溴化己二甲铵)(Sigma-Aldrich,目录号:AL-118)(1000x溶液,2mg/ml)
  7. 胰岛素 - 转铁蛋白 - 硒(ITS)溶液(Life Technologies,目录号:51300-044)。
  8. 磷酸钙转染试剂盒(ProFection Mammalian Transfection System)(Promega Corporation,目录号:E1200)
  9. 胎牛血清(FBS)
  10. 磷酸盐缓冲盐水(PBS)
  11. 没有Ca 2+ 2 + 和Mg 2+ 2 +
    的PBS
  12. 青霉素/链霉素
  13. EDTA(1mM,在不含Ca 2+和Mg 2+的PBS中)
  14. 10%福尔马林溶液,中性缓冲(10%NBF)
  15. 100x胶原酶/分散酶(100mg/ml)(Roche Diagnostics,目录号:11097113001)
  16. 1000x嘌呤霉素(2mg/ml)(Sigma-Aldrich,目录号:P9620)
  17. 1000x DNA酶I(10mg/ml)(Sigma-Aldrich,目录号:D4513)
  18. 胰蛋白酶/EDTA
  19. Virkon(DuPont Rely + On Virkon,目录号:1235-8667)
  20. 完成媒体(见配方)

设备

  1. 0.22μm,0.45μm,3mm过滤器
  2. 20毫升盖玻片
  3. 组织培养塑料制品
  4. 37℃摇床
  5. 组织培养层流罩
  6. 10厘米组织培养皿
  7. 离心机
  8. 37℃,5%CO 2,细胞培养箱
  9. 解释器
  10. 外科剪刀
  11. 解剖仪器

程序

  1. 开始之前
    1. 请阅读注释部分以获取指导。
    2. 获得5和14周龄之间的MMTV-PyMT阳性雌性。
      注意:年龄和肿瘤阶段之间的相关性在其他肿瘤模型中可能不同。有关详情,请阅读说明部分。
    3. 确保您拥有所有材料,试剂和设备。

  2. 解剖样本 - 肿瘤材料
    1. 使用外科器械解剖来自第4和第9腹股沟腺的乳房组织。将组织保持在冰冷的PBS中直至进一步使用。
      注意:对于正常的乳腺分离,解剖10周龄的MMTV-PyMT阴性雌性的乳腺组织。为了分离增生性肿瘤,解剖6周龄的MMTV-PyMT阳性雌性的乳腺组织。为了分离腺瘤,癌和侵袭性癌,分别分析7,8和10周龄的MMTV-PyMT阳性雌性的乳腺组织(图1)。这些时间点可能不同。有关确定正确年龄和肿瘤分期的更多信息,请阅读方案结尾处的注2。
    2. 将样品在无菌条件下移入层流组织培养罩中
    3. 仔细分析组织样品,检查指示其肿瘤阶段的征兆。作为粗略的指导,正常组织出现脂肪和软。增生组织看起来相当大,但仍是脂肪和软。腺瘤,癌和侵袭性癌出现放大,僵硬和多模态到不同程度(图1)。
    4. 在进行下一阶段之前,隔离每个肿瘤上的单个模块(推荐每个肿瘤2-3个)。
      注意:由于这种类型的肿瘤的多模态方面,必须特别注意,以便仅使用和分析单个模块。这是非常重要的,如果你想确保你是从特定阶段分离细胞,因为单个模块可能已经进展到不同的肿瘤阶段。模块的大小随着肿瘤大小,年龄,定位和各个小鼠之间而变化。推荐专家病理建议(见注2-3)。
    5. 对于每个单个模块,取一部分用于组织病理学分析,一部分用于分离成纤维细胞。对于组织病理学分析,将样品在4℃下在10%NBF中固定过夜。参见图1,组织的代表性苏木精和曙红染色 注意:这里不讨论组织病理学分析程序。请参阅其他协议。
    6. 为了分离成纤维细胞,将样品的其余部分转移到10cm组织培养皿中,并使用手术刀和/或手术剪刀加工成较小的片。


      图1.来自MMTV-PyMT模型的来自正常FVB/n乳腺和进行性乳腺肿瘤阶段(增生,腺瘤和癌)的组织的代表性组织样品和苏木精和伊红染色。 比例尺表示100μm。

  3. 处理正常乳腺组织样本
    1. 对于正常乳房组织,将较小的组织片放置在盘中,在其中在20mm盖玻片下将其压缩以防止脂肪组织漂浮。
      注意:如果使用胶原酶/疏散方法(见下文)处理正常乳腺组织,则分离非常少量的成纤维细胞。
    2. 然后缓慢加入新鲜的完全培养基DMEM以防止样品漂浮。
      注意:样品需要与塑料和盖玻片接触,以便成纤维细胞从样品中出来。
    3. 每天用新鲜的完全培养基更换培养基7天。
      注意:7天后,成纤维细胞从组织中出来进入盖玻片和皿中,并且可以转移到新的皿中。
    4. 去除原始组织。 将盖片转移到新的皿(细胞面朝上的一侧)。
    5. 将原始皿和具有盖玻片的皿用PBS洗涤一次,并使用胰蛋白酶/EDTA在37℃下胰蛋白酶化成纤维细胞10分钟。
    6. 将细胞重悬浮并接种到新培养皿中。
      注意:细胞数目可以最小化扩展,总是考虑到原代细胞在体外延长培养后进入衰老。
    7. 细胞现在准备永生化(从步骤E30开始)。

  4. 加工乳腺增生,腺瘤和癌组织样品
    1. 对于增生,腺瘤和癌样品,将较小的组织片放置在盘中,在那里它们使用手术刀进一步分离。
      注意:或者,对于极度脂肪性增生样品,可以使用正常的组织分离方法(步骤C10-16)。
    2. 将胶原酶/分散酶溶液(100mg/ml)在无菌PBS Mg 2 +缓冲液中稀释1:100,并在使用前用22μm过滤器过滤(终浓度1mg/ml) 注意:胶原酶/分散溶液是一种组织分解溶液,其在不影响质膜完整性的情况下使胶原蛋白和网状纤维降解。
    3. 将离析的组织置于具有适量胶原酶/分散酶的无菌管中。吸移上下数次以帮助分解。然后将样品在轨道摇床上在37℃和180rpm下孵育60分钟 注意:一般每个样品使用2-3ml(250-500mg的组织)。调整样品大小。
    4. 向每个样品中加入一体积的PBS 1mM EDTA。让它通过移液器几次。
      注意:EDTA停止蛋白酶反应,因为它是一种胶原酶抑制剂。
    5. 使用3mm过滤器过滤样品。
      注意:此步骤可去除未消化的组织。也可以使用替代方法(橡胶注射器柱塞等)。
    6. 将样品在室温(RT)下以100×g离心5分钟。
    7. 丢弃上清液。含有细胞的沉淀用完全培养基洗涤并在100×g下再次在室温下离心5分钟。
    8. 弃去上清液。含有细胞的细胞在室温下用完全培养基加DNA酶1(终浓度10μg/ml)温育10分钟。
      注意:通常由于细胞损伤,DNA泄漏到解离介质中,增加粘度并引起处理问题。纯化的DNase包括在细胞分离程序中以消化核酸而不损伤完整细胞。
    9. 将样品在室温(RT)下以100×g离心5分钟。
    10. 重复步骤D23两次。
    11. 除去培养基,将细胞重悬于完全培养基中并接种在培养皿中。 将样品放在培养箱中。
    12. 30分钟后,所有样品中的培养基用新鲜的完全培养基替换并放回培养箱中 注意:30分钟后,大多数成纤维细胞将粘附在培养皿上,而其他细胞类型将保持悬浮状态。 使用此提示将帮助您丰富成纤维细胞群体。
    13. 2-3天后,可以使用增强的对成纤维细胞塑料的粘附性来富集分离的细胞用于成纤维细胞群。
      1. 媒体已删除。
      2. 用PBS洗涤细胞一次
      3. 细胞用胰蛋白酶/EDTA覆盖并置于培养箱中10分钟。
      4. 将细胞重悬于完全培养基中,接种到新培养皿中并放回培养箱中30分钟
      5. 用新的新的完全培养基替换培养基。

  5. 永生化不同肿瘤阶段的原代成纤维细胞
    1. 开始之前请阅读说明4-5。
    2. 生长Phoenix-ECO细胞接种以在转染时达到80%汇合(参见注释6)。
      注意:Phoenix-Eco细胞用于产生亲嗜性病毒以感染小鼠成纤维细胞。为了使人成纤维细胞永生化,请使用替代方法(参见注释4和6)。强烈建议优化逆转录病毒的体积和滴定,但不在此讨论。还推荐使用易于点阳性对照(例如转染编码荧光蛋白的逆转录病毒质粒)。这允许快速评估您的感染效率(例如对荧光阳性细胞评分)。
    3. 一旦它们达到80%汇合,使用磷酸钙方案转染Phoenix-Eco细胞。通常为10厘米板。
      1. 在转染前2小时用完全培养基更换培养基。
      2. 在管A中,混合pBabe-HPV-E6-嘌呤霉素(10μg),2M CaCl 2(67μl)和无核酸酶水(高达500μl)。
      3. 在管B中,加入500μl的2x Hepes
      4. 在鼓泡A中的内容物时,慢慢地向其中加入管B的内容物
      5. 在室温下放置30分钟。
      6. 将转染混合物滴加到Phoenix-Eco细胞中
    4. 18小时后,为了爆发转录,将丁酸钠加入细胞(终浓度10mM)。
    5. 4小时后,用新鲜的完全培养基替换培养基
    6. 48-72小时后,收集含有病毒的培养基并通过0.45μm过滤器过滤。 病毒可以等分并保存在-80℃直至使用。
      注意:所有使用的材料在处置前必须留在virkon中(见注5)。
    7. 原代成纤维细胞用HPV-E6-嘌呤霉素逆转录病毒感染。
      1. 去除成纤维细胞培养物上的培养基
      2. 将含有病毒的培养基与完全新鲜培养基1:1混合,将所得溶液加入成纤维细胞中。
      3. 聚凝胺(终浓度2μg/ml)也加入到细胞中。
        注意:如果受体细胞悬浮(例如在胰蛋白酶消化后),感染效率可能增加。 聚凝胺(溴化己二甲铵)是一种阳离子聚合物,用于提高某些细胞在细胞培养中用逆转录病毒感染的效率。
    8. 5小时后,用新鲜培养基更换培养基 注意:或者,可以在18小时后更换介质。
    9. 24-48小时后,通过在完全培养基中加入嘌呤霉素(2μg/ml终浓度)选择感染的细胞。
      注意:根据细胞类型的不同,嘌呤霉素的可接受性不同。最终浓度需要在最终使用前进行优化。
    10. 在7-10天后,表达HPV-E6(即永生化细胞系)的抗性克隆是可见的。克隆或池可以在冷冻培养物中扩增用于随后使用。
      注意:最终,必须进行分离和永生化群体的性质的表征。这可以通过通过免疫荧光,FACS等分析某些标志物的表达来进行。推荐的成纤维细胞标志物是CD140A/PDGFRA,CD90/Thy-1,波形蛋白和纤连蛋白。成纤维细胞对于免疫细胞标记(CD45阴性),内皮标记(CD31阴性)和上皮标记(E-钙粘着蛋白,EpCAM阴性)也应是阴性的。对于癌症相关的成纤维细胞标记,我们推荐αSMA,FAP和FSP1/S100A4。

笔记

  1. 这个程序工作异常好,因为它是针对这种肿瘤模型进行了优化。重要的是,MMTV-PyMT乳腺癌细胞不在组织培养塑料盘上生长。另一方面,成纤维细胞生长非常好。因此,只需将您的提取物在塑料中培养,即可富集成纤维细胞群,避免任何来自癌细胞的污染。然而,如果使用已建立的癌细胞系,则该方法不起作用,因为那些细胞适于在塑料中生长并且将最终超过成纤维细胞群体。为了防止这种情况,需要设计其他方法(FACS分选,在癌细胞中使用自杀基因,等)。
  2. 这个程序需要一个良好的特征模型,并很多熟悉其发展的不同肿瘤阶段。因此,重要的是,在其应用之前,研究人员熟悉它。专家病理评估至关重要。我们建议对不同年龄,大小,方面和部位的多种肿瘤的彻底病理分析。这将最终允许研究者自信地预测每个肿瘤的阶段。即使如此,必须始终对用于成纤维细胞分离的肿瘤的肿瘤阶段进行回顾性病理分析。
  3. 组织病理学和免疫细胞化学方法不会在本协议中讨论。根据类型,组织,小鼠应变等,肿瘤进展是高度可变的。为了表征每个肿瘤阶段,请咨询病理学家以获得关于最佳方法,技术和标记的建议以清楚地定义每个阶段。
  4. 原代细胞和特别地成纤维细胞可以在它们变得衰老之前在培养中维持几次传代。衰老诱导多种转录和功能变化,这可能深刻影响所研究的表型。因此,如果需要长期培养,永生化是必要的。理想地,应当比较初级和永生化培养物以检查永生化程序是否未诱导所研究的表型的任何变化。对于永生化小鼠成纤维细胞,我们表达人乳头瘤病毒"早期基因"E6,通过抑制p53活性防止复制衰老。因此,预期在特定功能和信号传导途径中的其它"脱靶"后果。对于受端粒长度(例如人类)影响最大的细胞,推荐一种替代方法。在这些情况下,细胞永生化可以通过端粒酶逆转录酶蛋白(hTERT)的表达来实现。这种蛋白质在大多数体细胞中是无活性的,但是当hTERT外源表达时,细胞能够保持足够的端粒长度以避免复制衰老。对几种端粒酶 - 永生化细胞系的分析已证实细胞维持稳定的基因型并保留关键的表型标记物。
  5. 永生化通过靶成纤维细胞的逆转录病毒感染来实现。因此,处理和处置病毒和受污染材料必须始终根据您的机构法规进行。
  6. Phoenix-ECO最初在Garry P.Nolan实验室(美国斯坦福大学)建立,并且可在ATCC(Pear等人,1993)获得。它们是用于产生无辅助性的亲嗜性逆转录病毒的逆转录病毒生产线。该系基于293T细胞系(用腺病毒E1a转化并携带与新霉素共同选择的温度敏感性T抗原的人胚胎肾系)。该细胞系可用磷酸钙介导的转染或基于脂质的转染方案高度转染。通过将能够产生gag-pol的构建体和用于亲嗜性病毒的包膜蛋白置于293T细胞中产生该系。 Phoenix-Ampho是一种类似的细胞系,而不是产生双嗜性逆转录病毒

食谱

  1. 完整的介质(始终使用温暖37°C)
    Dulbecco改良的Eagle培养基(DMEM)高葡萄糖与稳定的L-谷氨酰胺
    1%胰岛素 - 转铁蛋白 - 硒溶液(ITS) 10%胎牛血清(FBS)
    青霉素/链霉素

致谢

此协议是Calvo 等人(2013)中描述的扩展版本。 FC,SH和ES由Cancer Research UK grant CRUK_A5317支持。 我们感谢实验室成员在整个工作中提供帮助和建议。

参考文献

  1. Calvo,F.,Ege,N.,Grande-Garcia,A.,Hooper,S.,Jenkins,RP,Chaudhry,SI,Harrington,K.,Williamson,P.,Moeendarbary,E.,Charras, Sahai,E。(2013)。 机械转导和YAP依赖性基质重塑是癌症相关成纤维细胞的产生和维持所必需的。/a> Nat Cell Biol 15(6):637-646。
  2. Guy,C.,Cardiff,R。和Muller,W。(1992)。 通过多瘤病毒中间T癌基因表达诱导乳腺肿瘤:转移性疾病的转基因小鼠模型。 Mol Cellr Biol 12(3):954-961。
  3. Hanahan,D。和Coussens,L.M。(2012)。 犯罪附件:招募到肿瘤微环境的细胞的功能。 Cancer Cell 21(3):309-322。
  4. Kalluri,R。和Zeisberg,M。(2006)。 癌症中的成纤维细胞。 Nat Rev Cancer 6(5 ):392-401。
  5. Lin,E.Y.,Jones,J.G.,Li,P.,Zhu,L.,Whitney,K.D.,Muller,W.J.and Pollard,J.W。(2003)。 在多形性中层T恶性肿瘤 癌蛋白小鼠乳腺癌模型为人类疾病提供了可靠的模型。美国病理学杂志163(5):2113-2126。
  6. Pear,W.S.,Nolan,G.P.,Scott,M.L。和Baltimore,D。(1993)。 通过瞬时转染产生高滴度无辅助病毒的逆转录病毒。 Proc Natl Acad Sci USA 90(18):8392-8396。
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引用:Calvo, F., Hooper, S. and Sahai, E. (2014). Isolation and Immortalization of Fibroblasts from Different Tumoral Stages. Bio-protocol 4(7): e1097. DOI: 10.21769/BioProtoc.1097.
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