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3D Co-culture System of Tumor-associated Macrophages and Ovarian Cancer Cells
肿瘤相关巨噬细胞和卵巢癌细胞的3D共培养系统   

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Jia Li
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The Journal of Clinical Investigation
Nov 2016

Abstract

Ovarian cancer is fairly unique in that ovarian carcinoma cells can detach and spread directly through peritoneal cavity. It has been unclear, however, how detached cancer cells survive in the peritoneum and form spheroid structure. We have recently reported that there is a strong correlation between Tumor-associated macrophages (TAMs)-associated spheroid and clinical pathology of ovarian cancer, and that TAMs promote spheroid formation and tumor growth at early stages of transcoelomic metastasis in orthotopic mouse models. We have established an in vitro spheroid formation assay using a 3D co-culture system in which mouse GFP+F4/80+CD206+ TAMs isolated from spheroids of ovarian cancer-bearing donor tomatolysM-cre mice were mixed with ID8 cells (TAM:ID8 at a ratio of 1:10) in medium containing 2% Matrigel and seeded onto the 24-well plate precoated with Matrigel. As transcoelomic metastasis is also associated with many other cancers such as pancreatic and colon cancers, TAM-mediated spheroid formation assay would provide a useful approach to define the molecular mechanism and therapeutic targets for ovarian cancer and other transcoelomic metastasis cancers.

Keywords: Ovarian cancer (卵巢癌), Tumor-associated macrophage (肿瘤相关巨噬细胞), 3D co-culture system (3D共培养系统), Spheroid formation (球体形成), Transcoelomic metastasis (种植性转移)

Background

Ovarian cancer (OC) is the second most common gynecological cancer and the leading cause of death in the United States (Jemal et al., 2009; Siegel et al., 2012). The major reason for the poor prognosis of OC is intraperitoneal and pelvic extensive implantation metastasis, which is usually unable to be removed completely by surgery. The most widely ascribed explanation for the phenomenon of peritoneal metastasis is that tumor cells become detached from the primary tumor after extension into the peritoneal surface and are transported throughout the peritoneal cavity by peritoneal fluid before seeding intraperitoneally. It has been suggested that the process of transcoelomic metastasis could be divided into several steps: 1) cell detachment, survival and resistance of anoikis; 2) evasion of immunological surveillance; 3) epithelial-mesenchymal transition; 4) spheroid formation; 5) ascites formation; and 6) peritoneal implantation (Tan et al., 2006; Peart et al., 2015; Rafehi et al., 2016). However, it remains unclear how free detached tumor cells survive in transcoelomic environment and form spheroids at initial steps of transcoelomic metastasis. Our recent study reveals that TAMs play an essential role in the survival and proliferation of free cells detached from the primary tumor in transcoelomic environment and spheroid formation at early stages of transcoelomic metastasis (Yin et al., 2016).

One critical method in this study is an in vitro spheroid formation assay using a 3D co-culture system to determine how TAMs facilitate spheroid formation. In this assay, mouse GFP+F4/80+CD206+ TAMs isolated from spheroids of ovarian cancer-bearing donor tomatolysM-cre mice were mixed with ID8 cells (TAM:ID8 at a ratio of 1:10) in medium containing 2% Matrigel and seeded onto the 24-well plate precoated with Matrigel. Similarly, we use human CD14+ TAMs isolated from OC patients and human ovarian cancer SKOV3 cells. In this model, we detect spheroid formation at 48 h of co-culture (Figure 1).

Here, we summarize our detailed protocols for 3D spheroid formation assay.


Figure 1. TAMs and OC cells in vitro 3D co-culture system were showed by Immunofluorescence. A. TAMs and OC cells form spheroids in an in vitro 3D co-culture system. GFP+F4/80+CD206+ TAMs isolated from spheroids of ovarian cancer-bearing donor tomatolysM-cre mice and ID8 cells were co-cultured in the Matrigel-precoated 24-well plate for 48 h. The spheroids were subjected to immunofluorescent staining for E-cadherin for tumor cells. Images for GFP+ TAMs, E-Cadherin+ OC cells and DAPI for all cells in the spheroids are shown. B. Human TAMs were isolated and infected with lentivirus expressing RFP. RFP-expressing TAMs were incubated with SKOV3 human ovarian cancer cells followed by 3D co-culture for 72 h. Spheroids were immunostained with keratin-14. Scale bars = 10 μm.

Materials and Reagents

  1. Pipette tips
  2. 15 cm Petri dish
  3. 10 ml serological pipette
  4. 50 ml sterile conical tube (Corning, Falcon®, catalog number: 352070 )
  5. 18-gauge needle
  6. 10 ml syringe
  7. FalconTM cell strainers,100 μm (Corning, Falcon®, catalog number: 352360 )
  8. 1.5 ml microcentrifuge tubes
  9. Greiner cellstar multiwall culture plates (24 wells, Greiner Bio One International, catalog number: 662102 )
  10. Cell lines: ID8 ovarian cancer cell line (Yin et al., 2016) was a gift from Jack Lawler and Carmelo Nucera at Beth Israel Deaconess Medical Center (Harvard Medical School, Boston, Massachusetts, USA).
    Note: ID8 cells are mouse epithelial OC line derived from C57BL/6 background; Passage under 30 and less than 1 week culture before injections.
  11. C57BL/6 mice, female, age: 8 weeks
  12. Tomato reporter transgenic mice: ID8 OC cells were labeled by stably expressing mCherry fluorescence protein while LysMCre mice crossed to the tomato reporter mT/mG
  13. Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, GibcoTM, catalog number: 10567014 )
  14. D-glucose
  15. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 26140079 )
  16. Penicillin-streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140 )
  17. 0.25% Trypsin-EDTA (Thermo Fisher Scientific, GibcoTM, catalog number: 25200056 )
  18. Ketamine
  19. Bovine serum albumin (BSA) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: B14 )
  20. Collagenase (Thermo Fisher Scientific, GibcoTM, catalog number: 17104019 )
  21. F4/80 monoclonal antibody, APC conjugate for flow cytometry (Thermo Fisher Scientific, Invitrogen, catalog number: MF48005 )
  22. PE anti-mouse CD206 (MMR) antibody (BioLegend, catalog number: 141705 )
  23. DAPI (Vector Laboratories, catalog number: H-1200 )
  24. Anti-mouse E-Cadherin antibody (BD, PharmingenTM, catalog number: 610404 )
  25. Donkey anti-mouse (Alexa Flour 594) (Thermo Fisher Scientific, InvitrogenTM, catalog number: A-21203 )
  26. Live cell tracker CMFDA (Thermo Fisher Scientific, InvitrogenTM, catalog number: C2925 )
  27. EGFP (abm, catalog number: LV011-a )
  28. Na2HPO4
  29. NaCl
  30. KH2PO4
  31. KCl
  32. Corning Matrigel (Basement membrane matrix, Corning, catalog number: 356234 )
  33. Tween 20 (Sigma-Aldrich, catalog number: P7949-500ML )
  34. Paraformaldehyde (Sigma-Aldrich, catalog number: 16005 )
  35. PBS (see Recipes)
  36. PBST (see Recipes)
  37. AC buffer (0.5% BSA) (see Recipes)
  38. 2% Matrigel (see Recipes)
  39. 3.7% paraformaldehyde (see Recipes)

Equipment

  1. Safety cabinet
  2. Pipette-aid
  3. Centrifuge with swinging-bucket rotor and adaptors for 50-ml conical tubes
  4. Water bath set at 37 °C
  5. Humidified cell culture incubator set to 37 °C and 5% CO2
  6. Zeiss Axiovert 200 fluorescence microscope (Carl Zeiss, model: Axiovert 200 )
  7. Upright microscope with 10x objective
  8. Cell sorter and the scale (BD, model: FACSAriaTM II ); sorting at a rate of 80,000 cell/h

Software

  1. Quantitation of the average distance between the geometrical center of nuclei of adjacent cells can be measured using Openlab3 software (Improvision, Lexington, MA) or other commercially available image analysis software
  2. SAS software (version 9.1.4, SAS Institute, Cary, NC)

Procedure

Note: All animal studies were approved by the Institutional Animal Care and Use Committee of Yale University.

  1. Macrophage isolation and labeling
    1. Cross Lysozyme Cre mice with tomato reporter C57BL/6 background mice and isolate EGFP-positive macrophages from peritoneal cavity by FACS sorting (Yin et al., 2016).
    2. As a positive control for spheroids, ID8 cells (1 x 106/ml) are injected into the abdominal cavity of Lysozyme Cre C57BL/6 mice (Age: 8 weeks) in 100 μl of DMEM. Sacrifice the mice after nine weeks according to procedures approved by Yale’s Institutional Animal Care and Use Committee, and analyze and sort the primary mouse tumor-associated macrophages (TAMs-GFP) using FACS (Yin et al., 2016).

  2. Preparation of ID8 cell cultures
    1. Expand the ID8 mouse ovarian cancer cells (Number of starting cells: 1 x 106 ) in DMEM (4.5 g/L D-glucose, 10% FBS, 100 IU penicillin and 100 μg/ml streptomycin) on a 15 cm Petri dish and culture ID8 cells until reaching 70-80% confluence.
      1. Preparation: Prepare a safety cabinet. Switch on and allow the cabinet to reach working airflow pressure for approximately 15 min before use.
      2. Warm medium. To avoid unnecessary stress to the cells, pre-equilibrate the medium in the incubator. If you are going to passage adherent cells, also thaw and warm an aliquot of trypsin.
      3. Checking cells: Cells should be checked microscopically daily to ensure they are healthy and growing as expected. Attached cells should be mainly attached to the bottom of the Petri dish, round and plump or elongated in shape and refracting light around their membrane.
    2. Upon reaching 70-80% confluence, aspirate the culture medium from the dish and add 10 ml of fresh DMEM.
    3. Harvest the ID8 cells by washing them from the Petri dish with the DMEM (10 ml) using a pipette-aid and 10 ml serological pipette.
    4. Transfer the ID8 cell suspension from the plate to a 50 ml conical tube, and pellet the cells by centrifugation at 200 x g for 4 min (room temperature).
    5. Suspend the ID8 cell in DMEM and count the cells and prepare a suspension of the ID8 in DMEM at a density of 1 x 106 cells per ml and ID8 cells should be kept on ice for injection. 

  3. ID8 tumor implantation and metastasis model
    1. Pathogen-free C57BL/6 background mice are purchased from THE JACKSON LABORATORY.
    2. ID8 cells (1 x 106/ml in 100 μl of DMEM) will be injected into the abdominal cavity of C57BL/6 background mice.
    3. Mice body weight gains will be measured weekly.
    4. The mice will be sacrificed at nine weeks prior to obvious signs of distress, pain or death.
    5. Ascitic fluid volume and tumor weight will be measured, and the tumor spheroids will be analyzed by histology and FACS.
      Ascitic fluid may begin to build up within 8-10 weeks following the injection of the ID8 cells. Tap the fluid when the mouse is noticeably large, but before the mouse has difficulty moving. Carefully withdraw as much fluid as possible with an 18-gauge needle attached to 10 ml syringe.
      Note: Sedating the mouse will make the collection of the ascitics fluid easier: ketamine 2 ng/mouse.

  4. Collect TAMs from tumor spheroids (Figure 3)


    Figure 3. The steps of collecting TAMs from spheroids

    1. Collect tumor spheroids from 3 ml ascitic fluid by using FalconTM cell strainers.
    2. Transfer the tumor spheroids to a 50 ml conical centrifuge tube, add wash medium (0.5% BSA with PBS 10 ml) for 2 min and discard it, and then add wash medium, repeat this washing step 3 times.
    3. At the end of the washing cells, centrifuge the cell suspension at 200 x g for 3 min (room temperature).
    4. Discard the wash medium and add 10 ml 500 U/ml collagenase in DMEM solution to the tube containing the tumor spheroids.
    5. Incubate the tube at 37 °C for 1 h, and swirl the tube vigorously every 15 min. At the end of the incubation period, spheroid should be almost completely digested and no longer visible.
      Note: If spheroids are still visible after 1 h, continue incubation, checking every 15 min, until spheroids are no longer visible (do not exceed 2.5 h).
    6. At the end of the collagenase digestion, centrifuge the cell suspension at 200 x g for 10 min (room temperature).
    7. Remove the supernatant from the tube carefully without dislodging the pellet.
    8. Add 2 ml FACS buffer (0.5% BSA with PBS) to the 50 ml tube and suspend the cell pellet, and then transfer the suspension to a FACS tube through filters (Corning Falcon 5 ml round bottom tube with cell strainer snap cap, A 35 μm nylon mesh).
      Note: It is necessary to obtain a single cell suspension.
    9. Centrifuge the cell suspension again at 200 x g for 10 min (room temperature).
    10. Remove the supernatant from the tube carefully without dislodging the pellet.
    11. Resuspend the pellet in 500 μl FACS buffer.
    12. Add 2.5 μl F4/80 (APC, 0.2 mg/ml) and 5 μl CD206 (PE, 0.2mg/ml) antibody to stain TAMs.
    13. Sort GFP+ F4/80+ and CD206+ TAMs through FACS analysis (Figure 2).


    Figure 2. FACS sorting of M2 TAMs. Spheroids were collected from ovarian cancer-bearing donor mice and dispersed to single cells. Cells labeled with anti-F4/80 and anti-CD206 were isolated by FACS sorting on FACSAriaII (BD Biosciences).

  5. Precoat 24-well plate with Matrigel 
    A 3D culture model was examined to determine the suitable cell culture system for our study.
    1. In the 3D-base model, a 24-well plate is precoated with Matrigel as basement membrane by adding 160 μl of Matrigel to each well.
    2. Incubate at 37 °C for 30 min to let the Matrigel solidify.
    3. Plate the cells (TAMs from step D7, ID8 cells or SKOV-3) onto the gel in 1 ml regular medium containing 2% Matrigel (3D-base and embedded, containing laminin as a major component, collagen type IV, heparin sulfate proteoglycan, entactin, and other minor components).

  6. 3D co-culture system of TAM and ID8 cells
    1. Isolate mouse GFP+ F4/80+ and CD206+ TAMs by FACS sorting from spheroids of ovarian cancer-bearing donor tomatolysM-cre mice.
    2. Precoat the 24-well plates with Matrigel as described above.
    3. Seed the mixtures of TAMs and ID8 cells (at a ratio of 1:10 but with a fixed total cell number as 40,000 cells/well) directly onto the Matrigel-precoated 24-well plate with 300 μl DMEMs in each well.
    4. Incubate the cells were incubated at 37 °C for up to 48 h to allow the aggregates spheroids to form.
      Note: The wells without cells but containing medium are used as negative control.

  7. E-Cadherin staining in ID8 cells by immunofluorescent method
    1. Gently wash the wells 2 times each with 500 μl PBS to remove DMEM.
    2. After PBS removal, add 300 μl of buffered paraformaldehyde (3.7% paraformaldehyde, 10 nM, pH 7.4) for 10 min.
    3. Wash the wells 2 times with PBS for removing the PFA and add 100 μl E-Cadherin antibody (1:200 with 1% BSA) to incubate at 4 °C overnight (staining protocol see Yin et al., 2016).
    4. On the second day wash spheroids 3 times with 500 μl PBST for about 15 min/time.
    5. Add 100 μl PE anti-mouse second antibody (1:500 with 1% BSA) into the 24-well plate with spheroids and incubate for 1-h at RT.
    6. Capture fluorescent microscopic images by using Openlab 3 software (Improvision, Lexington, MA) under Zeiss Axiovert 200 fluorescence microscope (Carl Zeiss MicroImaging; Thornwood, NY). The captured images are used for analyzing the cell morphology.

Data analysis

  1. Spheroids were visualized directly under a fluorescence microscope or were subjected to Line were subjected to immunostaining with anti-CD68, anti-K14 and DAPI followed by confocal imaging (see Figure 1).
  2. The number (per well) and size (area) of spheroids at 48 h were quantified.
  3. Statistical analyses: The differences of results of spheroid formation were analyzed by Student’s t-test. Statistical analyses in this study were performed using SAS software (version 9.1.4, SAS Institute, Cary, NC). All statistical tests were two-tailed, and P-values less than 0.05 were considered statistically significant. All data are presented as means ± SEM, n = 5, *, P < 0.05; **, P < 0.01; ***, P < 0.001. 

Notes

  1. For Matrigel preparation, freeze pipette tips to better handle Matrigel (which should be used at 4 °C).
  2. Alternatively, mouse peritoneal macrophages from C57BL/6 background mice were isolated followed by pre-staining with live cell tracker CMFDA (Qin et al., 2014) or by transduction with lentivirus expressing EGFP as we have shown for vascular endothelial cells by FACS sorting (Zhou et al., 2016).
  3. Euthanasia: Mice were euthanized with an overdose of ≥ 100 mg/kg bw pentobarbital and exsanguinated.
  4. SKOV3 human ovarian cancer cells: (human ovarian adenocarcinoma cell line) were obtained from ATCC. SKOV3 cells are resistant to tumor necrosis factor and several cytotoxic drugs, including diphtheria toxin, cisplatinum, and adriamycin.

Recipes

  1. PBS
    8 mM Na2HPO4
    136 mM NaCl
    2 mM KH2PO4
    2.6 mM KCl
  2. PBST
    500 ml 1x PBS
    1 ml Tween-20
  3. AC buffer (0.5% BSA)
    Add 50 mg BSA into 10 ml PBS
  4. Regular medium
    Add 50 ml fetal bovine serum and 5 ml penicillin and streptomycin into 500 ml DMEM medium
  5. 2% Matrigel
    Add 200 μl Matrigel into 10 ml regular medium
    Note: 2% Matrigel contains 8-12 mg/ml protein which includes laminin (major component), collagen type IV, heparin sulfate proteoglycan, entactin, and other minor components. Addition of Collagen type IV to the gel increases polymerization.
  6. 3.7% paraformaldehyde
    Add 37 mg paraformaldehyde into 1 ml PBS

Acknowledgments

Conflict of interest disclosure: None.
This work was partly supported by National Key Research and Development Program of China (2016YFC1300600), National Natural Science Foundation of China (No. 91539110), and Scientific Grants of Guangdong (Nos. 2015B020225002 and 2015A050502018) to WM, R01 HL109420 and R01 HL115148, and CT Stem Cell Innovation Award (Established Investigator Grant) 14-SCB-YALE-17 to WM.

References

  1. Jemal, A., Siegel, R., Ward, E., Hao, Y., Xu, J. and Thun, M. J. (2009). Cancer statistics, 2009. CA Cancer J Clin 59(4): 225-249.
  2. Peart, T., Ramos Valdes, Y., Correa, R. J., Fazio, E., Bertrand, M., McGee, J., Prefontaine, M., Sugimoto, A., DiMattia, G. E. and Shepherd, T. G. (2015). Intact LKB1 activity is required for survival of dormant ovarian cancer spheroids. Oncotarget 6(26): 22424-22438.
  3. Qin, L., Huang, Q., Zhang, H., Liu, R., Tellides, G., Min, W. and Yu, L. (2014). SOCS1 prevents graft arteriosclerosis by preserving endothelial cell function. J Am Coll Cardiol 63(1): 21-29.
  4. Rafehi, S., Ramos Valdes, Y., Bertrand, M., McGee, J., Prefontaine, M., Sugimoto, A., DiMattia, G. E. and Shepherd, T. G. (2016). TGFβ signaling regulates epithelial-mesenchymal plasticity in ovarian cancer ascites-derived spheroids. Endocr Relat Cancer 23(3): 147-159.
  5. Siegel, R., Naishadham, D. and Jemal, A. (2012). Cancer statistics, 2012. CA Cancer J Clin 62(1):10-29.
  6. Tan, D. S., Agarwal, R. and Kaye, S. B. (2006). Mechanisms of transcoelomic metastasis in ovarian cancer. Lancet Oncol 7(11): 925-934.
  7. Yin, M., Li, X., Tan, S., Zhou, H. J., Ji, W., Bellone, S., Xu, X., Zhang, H., Santin, A. D., Lou, G. and Min, W. (2016). Tumor-associated macrophages drive spheroid formation during early transcoelomic metastasis of ovarian cancer. J Clin Invest 126(11): 4157-4173.
  8. Zhou, H. J., Qin, L., Zhang, H., Tang, W., Ji, W., He, Y., Liang, X., Wang, Z., Yuan, Q., Vortmeyer, A., Toomre, D., Fuh, G., Yan, M., Kluger, M. S., Wu, D. and Min, W. (2016). Endothelial exocytosis of angiopoietin-2 resulting from CCM3-deficiency contributes to the progression of cerebral cavernous malformation. Nat Med 22(9): 1033-42.

简介

卵巢癌相当独特,因为卵巢癌细胞可以通过腹膜腔直接分离和扩散。然而,目前还不清楚癌细胞如何在腹膜中存活并形成球状结构。我们最近报道,肿瘤相关巨噬细胞(TAMs)相关的球状体与卵巢癌的临床病理学之间存在很强的相关性,并且TAMs在原位小鼠模型中促进球体形成和肿瘤生长在转移瘤体转移的早期阶段。我们已经建立了使用3D共培养系统的体外球体形成测定法,其中小鼠GFP + F4 / 80 + CD206 F 从含有卵巢癌的供体番茄lysM-cre小鼠的球状体中分离的TAM与在含有2%基质胶的培养基中的ID8细胞(TAM:ID8比例为1:10)混合并接种到用Matrigel预包被的24孔板上。由于transcoelomic转移也与许多其他癌症,如胰腺癌和结肠癌,TAM介导的球体形成实验将提供一个有用的方法来定义卵巢癌和其他transcoelomic转移癌症的分子机制和治疗目标。

【背景】在美国,卵巢癌(OC)是第二常见的妇科癌症和主要死亡原因(Jemal等人,2009; Siegel等人,2012年) )。 OC预后不良的主要原因是腹腔内和盆腔广泛植入转移,通常手术无法完全切除。对腹膜转移现象最广泛的解释是肿瘤细胞在延伸到腹膜表面后与原发肿瘤分离,并在腹膜内播种之前通过腹膜液输送到整个腹腔。有人提出,转移瘤的转移过程可分为几个步骤:1)细胞脱落,失巢凋亡的存活和抵抗; 2)逃避免疫监视; 3)上皮 - 间充质转变; 4)球体形成; 5)腹水形成;和6)腹膜植入(Tan等人,2006; Peart等人,2015; Rafehi等人,2016)。然而,目前还不清楚自由分离的肿瘤细胞如何在跨体腔环境中存活并且在转移性囊肿转移的初始阶段形成球状体。我们最近的研究表明,TAMs在跨体线性环境中从原发性肿瘤分离的游离细胞的存活和增殖以及在转移性胶质细胞瘤转移的早期阶段形成球体中起着至关重要的作用(Yin等人,2016) 。

该研究中的一个关键方法是使用3D共培养系统进行体外球体形成测定以确定TAM如何促进球体形成。在该测定中,从含有卵巢癌的供体番茄lysM-1的球状体中分离出小鼠GFP + F4 / 80 + CD206 + TAM, cre小鼠在含有2%Matrigel的培养基中与ID8细胞(TAM:ID8比率为1:10)混合,并接种到用Matrigel预包被的24孔板上。同样,我们使用从OC患者和人卵巢癌SKOV3细胞分离的人CD14 + TAM。在这个模型中,我们在48小时的共培养中检测到球体形成(图1)。

在这里,我们总结了我们关于3D球体形成测定的详细方案。


图1. TAM和OC细胞体外3D共培养系统通过免疫荧光显示A.TAM和OC细胞在体外形成球状体三维联合文化系统。从含有卵巢癌的供体番茄lysMcre小鼠的球状体分离的GFP + F4 / 80 + CD206 + TAM和将ID8细胞在基质胶预包被的24孔板中共培养48小时。球状体进行E-钙粘蛋白免疫荧光染色以获得肿瘤细胞。显示了球体中所有细胞的GFP <+> TAM,E-钙粘蛋白+ OC细胞和DAPI的图像。 B.分离人TAM并用表达RFP的慢病毒感染。将表达RFP的TAM与SKOV3人卵巢癌细胞孵育,然后进行3D共培养72小时。球状体用角蛋白-14免疫染色。比例尺=10μm。

关键字:卵巢癌, 肿瘤相关巨噬细胞, 3D共培养系统, 球体形成, 种植性转移

材料和试剂

  1. 移液器吸头
  2. 15厘米培养皿
  3. 10毫升血清移液器
  4. 50毫升无菌锥形管(Corning,Falcon ,目录号:352070)
  5. 18号针
  6. 10毫升注射器
  7. Falcon TM细胞过滤器,100μm(Corning,Falcon ,目录号:352360)
  8. 1.5 ml微量离心管
  9. Greiner cellstar多壁培养板(24孔,Greiner Bio One International,目录号:662102)
  10. 细胞系:ID8卵巢癌细胞系(Yin等,2016)是Beth Israel Deaconess医学中心(美国马萨诸塞州波士顿哈佛医学院)的Jack Lawler和Carmelo Nucera的礼物。
    注:ID8细胞是来源于C57BL / 6背景的小鼠上皮OC系;通过注射前30和不到1周培养。

  11. C57BL / 6小鼠,女性,年龄:8周
  12. 番茄报道基因转基因小鼠:通过稳定表达mCherry荧光蛋白标记ID8 OC细胞,而LysMCre小鼠与番茄报道分子mT / mG
    杂交
  13. Dulbecco改良的Eagle培养基(DMEM)(Thermo Fisher Scientific,Gibco TM,产品目录号:10567014)
  14. D-葡萄糖
  15. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:26140079)
  16. 青霉素 - 链霉素(Thermo Fisher Scientific,Gibco TM,目录号:15140)
  17. 0.25%胰蛋白酶-EDTA(Thermo Fisher Scientific,Gibco TM,目录号:25200056)
  18. 氯胺酮
  19. 牛血清白蛋白(BSA)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:B14)
  20. 胶原酶(Thermo Fisher Scientific,Gibco TM,目录号:17104019)
  21. F4 / 80单克隆抗体,用于流式细胞术的APC缀合物(Thermo Fisher Scientific,Invitrogen,目录号:MF48005)
  22. PE抗小鼠CD206(MMR)抗体(BioLegend,目录号:141705)
  23. DAPI(Vector Laboratories,目录号:H-1200)
  24. 抗小鼠E-钙黏着蛋白抗体(BD,Pharmingen TM,目录号:610404)
  25. 驴抗小鼠(Alexa面粉594)(Thermo Fisher Scientific,Invitrogen TM,目录号:A-21203)
  26. 活细胞追踪器CMFDA(Thermo Fisher Scientific,Invitrogen TM,目录号:C2925)
  27. EGFP(abm,目录号:LV011-a)
  28. Na 2 HPO 4 4/2
  29. NaCl
  30. KH <2> PO <4>
  31. KCl
  32. 康宁Matrigel(地下室膜基质,康宁,目录号:356234)
  33. 吐温20(Sigma-Aldrich,目录号:P7949-500ML)
  34. 多聚甲醛(Sigma-Aldrich,目录号:16005)
  35. PBS(见食谱)
  36. PBST(见食谱)
  37. AC缓冲液(0.5%BSA)(见食谱)
  38. 2%Matrigel(见食谱)
  39. 3.7%多聚甲醛(见食谱)

设备

  1. 安全柜
  2. 移液助剂
  3. 用摆动转子离心机和适用于50-ml锥形管的适配器

  4. 水浴温度设定为37°C
  5. 加湿的细胞培养箱设置为37℃和5%CO 2
  6. 蔡司Axiovert 200荧光显微镜(卡尔蔡司,型号:Axiovert 200)
  7. 10倍物镜的直立显微镜
  8. 细胞分选仪和秤(BD,型号:FACSAria TM II);按每小时80,000个细胞排序

软件

  1. 可以使用Openlab3软件(Improvision,Lexington,MA)或其他商业上可获得的图像分析软件来测量相邻细胞核的几何中心之间的平均距离的定量。
  2. SAS软件(版本9.1.4,SAS Institute,Cary,NC)

程序

注意:所有动物研究均由耶鲁大学的动物管理和使用委员会批准。

  1. 巨噬细胞分离和标记
    1. 用番茄报道分子C57BL / 6背景小鼠交叉溶菌酶Cre小鼠,并通过FACS分选从腹膜腔分离EGFP阳性巨噬细胞(Yin等人,2016)。
    2. 作为球体的阳性对照,在100μlDMEM中将ID8细胞(1×10 6 / ml)注射到溶菌酶Cre C57BL / 6小鼠(年龄:8周)的腹腔中。根据耶鲁大学机构动物护理和使用委员会批准的程序在9周后牺牲小鼠,并使用FACS分析和分选原发性小鼠肿瘤相关巨噬细胞(TAMs-GFP)(Yin等, 2016)。

  2. ID8细胞培养物的制备
    1. 在DMEM(4.5g / L D-葡萄糖,10%FBS,100IU青霉素和100μg/ ml链霉素)中展开ID8小鼠卵巢癌细胞(起始细胞数:1×10 6)置于15cm培养皿中培养ID8细胞直至达到70-80%汇合。
      1. 准备:准备一个安全柜。
        开机并让机柜在使用前达到工作气流压力约15分钟。
      2. 温暖的媒介。为了避免对细胞造成不必要的压力,在培养箱中平衡培养基。如果你要传代贴壁细胞,也要解冻并加热胰蛋白酶等分试样。
      3. 检查细胞:细胞应每天进行显微镜检查,以确保它们健康,并按预期增长。附着的细胞应该主要附着在培养皿的底部,圆形,丰满或拉长形状并在其膜周围折射光线。
    2. 达到70-80%汇合后,从培养皿中吸出培养基并加入10ml新鲜DMEM。

    3. 使用移液管辅助器和10毫升血清移液管通过用DMEM(10毫升)从培养皿中清洗它们来收获ID8细胞。
    4. 将ID8细胞悬浮液从平板转移到50ml锥形管中,并通过以200μgxg离心4分钟(室温)来沉淀细胞。
    5. 将ID8细胞悬浮在DMEM中并计数细胞并以1×10 6细胞/ ml的密度在DMEM中制备ID8的悬浮液,并将ID8细胞保存在冰上用于注射。

  3. ID8肿瘤植入和转移模型
    1. 无病原体的C57BL / 6背景小鼠购自THE JACKSON LABORATORY。
    2. 将ID8细胞(在100μlDMEM中1×10 6 / ml)注射到C57BL / 6背景小鼠的腹腔内。

    3. 每周测量小鼠体重增加量

    4. 在出现明显的痛苦,痛苦或死亡迹象之前的九周,将处死小鼠
    5. 测量腹水量和肿瘤重量,并通过组织学和FACS分析肿瘤球状体。
      在注射ID8细胞后8-10周内腹水可能开始积聚。当鼠标明显较大时,但在鼠标移动困难之前点击液体。用10号注射器连接的18号针小心取出尽可能多的液体。
      注意:镇静鼠标会使麻醉液的收集更容易:氯胺酮2 ng /鼠标。

  4. 从肿瘤球体收集TAMs(图3)

    “”src
    图3.从球体收集TAM的步骤


    1. 使用Falcon™细胞过滤器从3毫升腹水中收集肿瘤球体。
    2. 将肿瘤球体转移到50ml锥形离心管中,加入洗涤培养基(含有10ml PBS的0.5%BSA)2分钟并丢弃它,然后加入洗涤培养基,重复该洗涤步骤3次。
    3. 在洗涤细胞结束时,将细胞悬浮液以200μgxg离心3分钟(室温)。
    4. 丢弃洗涤介质,并在DMEM溶液中加入10ml 500U / ml胶原酶到含有肿瘤球体的管中。
    5. 37°C孵育1小时,每15分钟剧烈搅拌一次。在孵化期结束时,球体应该几乎完全消化,不再可见。
      注意:如果球体在1小时后仍然可见,则继续培养,每15分钟检查一次,直到球体不再可见(不超过2.5小时)。
    6. 在胶原酶消化结束时,将细胞悬浮液在200×g下离心10分钟(室温)。

    7. 。小心取出管中的上清液,不要将颗粒移出。
    8. 加入2ml FACS缓冲液(含PBS的0.5%BSA)到50ml管中并悬浮细胞沉淀,然后通过过滤器将悬浮液转移到FACS管中(Corning Falcon 5ml带细胞滤网咬合盖的圆底管,A35 μm尼龙网)。
      注意:有必要获得单细胞悬液。

    9. 再次将细胞悬液以200×g离心10分钟(室温)。

    10. 。小心取出管中的上清液,不要将颗粒移出。

    11. 在500μlFACS缓冲液中重悬沉淀
    12. 加入2.5μlF4 / 80(APC,0.2mg / ml)和5μlCD206(PE,0.2mg / ml)抗体染色TAMs。
    13. 通过FACS分析(图2)分类GFP <+> F4 / 80 <+>和 + <+> TAM。


    图2. M2TAMs的FACS分选从携带卵巢癌的供体小鼠收集球状体并分散到单细胞中。通过在FACSAriaII(BD Biosciences)上进行FACS分选来分离用抗-F4 / 80和抗-CD206标记的细胞。

  5. 带Matrigel的预涂24孔板
    检查3D培养模型以确定适合我们研究的细胞培养系统。
    1. 在基于3D的模型中,通过将160μlMatrigel添加到每个孔中,用Matrigel作为基底膜预涂24孔板。

    2. 在37°C孵育30分钟以使Matrigel凝固。
    3. 将细胞(来自步骤D7,ID8细胞或SKOV-3的TAM)在含有2%Matrigel(3D-碱基和嵌入的,含有层粘连蛋白作为主要的)的1ml常规培养基中铺板到凝胶上组分,IV型胶原蛋白,硫酸肝素蛋白聚糖,entactin和其他次要成分)。

  6. TAM和ID8细胞的3D共培养系统
    1. 通过FACS从含有卵巢癌的供体番茄lysM-1的球状体中分选分离小鼠GFP + F4 / 80 +和CD206 + TAM。 cre 老鼠。
    2. 如上所述,预涂24孔板用基质胶预包被。
    3. 将TAM和ID8细胞的混合物(比例为1:10,但具有固定的总细胞数量为40,000个细胞/孔)直接接种到每孔中含有300μlDMEM的Matrigel预包被的24孔板上。 >
    4. 培养细胞在37°C孵育48小时,以形成聚集体球状体。
      注:不含细胞但含有培养基的孔用作阴性对照。

  7. 通过免疫荧光方法在ID8细胞中进行E-钙粘蛋白染色

    1. 每次用500μlPBS轻轻冲洗孔2次以除去DMEM。
    2. 去除PBS后,加入300μl缓冲的多聚甲醛(3.7%多聚甲醛,10nM,pH7.4)10分钟。
    3. 用PBS清洗孔2次,除去PFA,加入100μlE-钙粘蛋白抗体(1:200含1%BSA)在4℃过夜孵育(染色方案参见Yin等人 ,2016)。
    4. 第二天用500μlPBST将球状体洗3次,时间约为15分钟。
    5. 将具有球状体的24孔板加100μlPE抗小鼠第二抗体(1:500与1%BSA)并在室温孵育1小时。
    6. 在Zeiss Axiovert 200荧光显微镜(Carl Zeiss MicroImaging; Thornwood,NY)下使用Openlab 3软件(Improvision,Lexington,MA)捕获荧光显微图像。捕获的图像用于分析细胞形态。

数据分析

  1. 在荧光显微镜下直接观察球状体或使其经历Line对抗CD68,抗K14和DAPI进行免疫染色,随后进行共焦成像(见图1)。
  2. 对48小时的球体的数量(每个孔)和大小(面积)进行量化。
  3. 统计分析:通过学生的测试分析球体形成结果的差异。本研究中的统计分析使用SAS软件(版本9.1.4,SAS Institute,Cary,NC)进行。所有统计学检验均为双尾检验,<0.05> <0.05为差异有统计学意义。所有数据均以平均值±SEM表示,n = 5,*, 0.05; **, P 0.01; ***, P &lt; 0.001。

笔记

  1. 对于基质胶的制备,将移液器吸头冷冻以更好地处理Matrigel(应该在4°C下使用)。
  2. 或者,分离来自C57BL / 6背景小鼠的小鼠腹膜巨噬细胞,随后用活细胞追踪器CMFDA(Qin等人,2014)或通过用表达EGFP的慢病毒转导进行预染色,如我们已经表明的通过FACS分选检测血管内皮细胞(Zhou等人,2016)。
  3. 安乐死:小鼠安乐死过量≥100毫克/千克体重戊巴比妥,并放血。
  4. SKOV3人卵巢癌细胞:(人卵巢腺癌细胞系)获自ATCC。 SKOV3细胞对肿瘤坏死因子和几种细胞毒性药物,包括白喉毒素,顺铂和阿霉素都有抗性。

食谱

  1. PBS
    8mM Na 2 HPO 4 4/2 136 mM NaCl
    2mM KH 2 PO 4 4/2 2.6 mM KCl
  2. PBST

    500毫升1x PBS 1毫升Tween-20
  3. AC缓冲液(0.5%BSA)

    加入50毫克BSA到10毫升PBS中
  4. 常规媒体

    加入50 ml胎牛血清和5 ml青霉素和链霉素到500 ml DMEM培养基中
  5. 2%Matrigel

    加入200μlMatrigel到10 ml常规培养基中 注:2%Matrigel含8-12mg / ml蛋白质,其中包括层粘连蛋白(主要成分),IV型胶原蛋白,硫酸肝素蛋白多糖,entactin和其他次要成分。向凝胶中添加IV型胶原增加了聚合。

  6. 3.7%多聚甲醛
    加入37毫克多聚甲醛到1毫升PBS中

致谢

利益冲突披露:无。
这项工作得到了WM,R01 HL109420和R01 HL115148的部分支持,中国国家重点科学研究计划(2016YFC1300600),国家自然科学基金(编号91539110)和广东科学基金(编号2015B020225002和2015A050502018)和西格玛干细胞创新奖(建立研究者资助)14-SCB-YALE-17。

参考

  1. Jemal,A.,Siegel,R.,Ward,E.,Hao,Y.,Xu,J。和Thun,M.J。(2009)。 2009年癌症统计数据 Cancer J Clin 59 (4):225-249。
  2. Peart,T.,Ramos Valdes,Y.,Correa,RJ,Fazio,E.,Bertrand,M.,McGee,J.,Prefontaine,M.,Sugimoto,A.,DiMattia,GE和Shepherd,TG(2015) 。 完整的LKB1活性是休眠卵巢癌球状体生存所必需的。 Oncotarget 6(26):22424-22438。
  3. Qin,L.,Huang,Q.,Zhang,H.,Liu,R.,Tellides,G.,Min,W.和Yu,L。(2014)。 SOCS1通过保护内皮细胞功能防止移植动脉硬化 J Am Coll Cardiol 63(1):21-29。
  4. Rafehi,S.,Ramos Valdes,Y.,Bertrand,M.,McGee,J.,Prefontaine,M.,Sugimoto,A.,DiMattia,G.E。和Shepherd,T.G。(2016)。 TGFβ信号调节卵巢癌腹水衍生球体的上皮 - 间充质可塑性 Endocr Relat Cancer 23(3):147-159。
  5. Siegel,R.,Naishadham,D.和Jemal,A。(2012)。 2012年癌症统计数据 CA Cancer J Clin 62 (1):10-29。
  6. Tan,D.S.,Agarwal,R.和Kaye,S.B。(2006)。 卵巢癌转移瘤的转移机制 Lancet Oncol 7(11):925-934。
  7. 中国科学院上海生命科学研究院生物化学与分子生物学研究所研究人员发现, W.(2016)。 肿瘤相关巨噬细胞驱动卵巢癌早期transcoelomic转移过程中的球体形成 J Clin Invest 126(11):4157-4173。
  8. Zhou,HJ,Qin,L.,Zhang,H.,Tang,W.,Ji,W.,He,Y.,Liang,X.,Wang,Z.,Yuan,Q.,Vortmeyer,A.,Toomre ,D.,Fuh,G.,Yan,M.,Kluger,MS,Wu,D。和Min,W。(2016)。 CCM3缺陷引起的血管生成素-2的内皮细胞胞吐作用有助于大脑海绵状血管畸形的进展。 Nat Med 22(9):1033-42。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Long, L., Yin, M. and Min, W. (2018). 3D Co-culture System of Tumor-associated Macrophages and Ovarian Cancer Cells. Bio-protocol 8(8): e2815. DOI: 10.21769/BioProtoc.2815.
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