Visualization of Macropinocytosis in Prostate Fibroblasts
前列腺成纤维细胞中巨胞饮的可视检测   

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The Journal of Clinical Investigation
Oct 2018

 

Abstract

Macropinocytosis has emerged as an important mechanism for non-selective route to internalize extracellular fluids and dissolved molecules in eukaryotic cell. As fundamental cellular behavior, macropinocytosis plays specific and distinct roles in many physiological and pathological processes, such as nutrients uptake, antigen presentation, pathogen capture, and tumorigenesis. It supports tumorigenesis by providing metabolic needs to dividing cells in Ras driven cancer. In recent years, macropinocytosis has gained considerable interest in physiology and various diseases, including cancer, neurodegenerative diseases and atherosclerosis, which in turn has led to the discovery of new endocytic recycling systems. Approaches to assess macropinocytosis will provide insight into its underlying regulatory molecular mechanisms and enable the physiological control of macropinocytosis for controlled drug delivery and targeted cancer therapy. Macropinocytosis is an important phenomenon in Ras-expressing cancer cells and, recently, we have revealed a functional role for macropinocytosis in cancer associated fibroblasts (CAFs) fueling cancer cell growth. Here, we describe a protocol for detection of macropinocytosis in prostatic fibroblasts in vitro by utilizing fluorescently-labeled, lysine-fixable, 70 kDa high molecular weight dextran. Macropinosomes are visualized as fluorescent intracellular puncta either by confocal or fluorescent microscopy. To follow, subsequent intracellular events and their underlying mechanisms after macropinosomes formation, we perform co-localization of quenched BSA (DQTM-BSA) along with dextran labeling in cancer associated fibroblasts. Our protocol provides a consistent way to understand macropinocytosis in wild type or genetic manipulated prostatic fibroblast.

Keywords: Macropinosomes (巨胞饮), Prostate cancer (前列腺癌), Fibroblasts (成纤维细胞), Glutamine (谷氨酰胺), Ras (Ras), 5-(n-ethyl-n-isopropyl)-amiloride (EIPA) (5-(N-乙基-N-异丙基)-阿米洛利)

Background

Macropinocytosis or ‘cell drinking’ is a type of endocytosis that involves the nonspecific uptake of extracellular fluid into large intracellular vesicles known as macropinosomes (Swanson and Watts, 1995). The macropinosomes are heterogeneous in size and shape, with their diameters ranging between 0.2 and 5 micrometers (Lim and Gleeson, 2011). The Src and Ras are prominent oncogenes which stimulate macropinocytosis in various type of cancer (Bar-Sagi and Feramisco, 1986). Macropinocytosis has been prominently described in pancreatic cancer cells, recognized for having active Ras signaling (Commisso et al., 2013; Davidson et al., 2017). We determined that epigenetic activation of Ras signaling mediates macropinocytosis in prostatic cancer associated fibroblasts resulting in albumin uptake, lysosomal degradation, and release of constituent amino acids (Mishra et al., 2018). These amino acids, predominantly glutamine, were found to support the metabolism and differentiation of the prostate cancer epithelial cells. Therefore, macropinocytosis is an important nutrient processing pathway to support the energetic needs of cancer associated fibroblasts (CAFs) and may represent a predictive biomarker for cancer therapy. The quantification of macropinocytosis can be achieved through confocal laser scanning microscopy.

Materials and Reagents

  1. Pipette tips (Fisher Scientific, catalog number: 02707404)
  2. Sterile 10 ml serological pipette (Santa Cruz Biotechnology Inc., catalog number: sc-200281)
  3. Sterile 15 ml conical tubes (BD Falcon, catalog number: 669993)
  4. 12-well cell culture plate (Fisher Scientific, catalog number: 64976)
  5. Cover glass (Fisher Scientific, catalog number: 22050221)
  6. Aluminum foil (Fisher healthcare, catalog number: 01213101)
  7. Kimwipe (lint-free paper towel; Fisher healthcare, catalog number: S47299)
  8. Human prostatic fibroblasts and RasV12 (Mishra et al., 2018)
    Note: These cell types have active Ras activity compared to their normal counterpart.
  9. Fetal Bovine Serum (Atlanta Biologicals, catalog number: S11550) 
  10. DMEM/F12 medium (Fisher Scientific, catalog number: SH300404)
  11. Tetramethylrhodamine-conjugated high molecular weight dextran (TMR-dextran, 75 kDa), lysine fixable (Invitrogen Molecular Probes, catalog number: D1818)
  12. DQ-BSA (Thermo Fisher Scientific, InvitrogenTM, catalog number: D12050)
  13. LysoTracker® Green DND-26 (Life Technologies, catalog number: L7526)
  14. 5-(N-ethyl-N-isopropyl) amiloride (EIPA) (Invitrogen Molecular Probes, catalog number: e-3111) 
  15. Vectashield Antifade Mounting Medium with DAPI (Vector Laboratories, catalog number: H-1200)
  16. Formaldehyde solution, ACS reagent grade, 37% (vol/vol) (Sigma, catalog number: 252549) 
  17. NuSerum (Fisher Scientific, catalog number: 355500)
  18. Insulin, human recombinant, zinc solution (Invitrogen, catalog number: 12585-014)
  19. Sodium chloride (NaCl) (Fisher Biosciences, catalog number: BP358-1)
  20. Dimethyl sulfoxide (DMSO) (Fisher Biosciences, catalog number: D128-1)
  21. Sodium dihydrogen phosphate (NaH2PO4) (Fisher Biosciences, catalog number: ICN19550080) 
  22. Sodium Phosphate, Dibasic, Anhydrous, Na2HPO4 (Fisher Biosciences, catalog number: AC424375000)
  23. 10-8 M testosterone (Nacalai Tesque, catalog number: 32811-61)
  24. Poly-L-Lysine (Sigma, catalog number: P4707)
  25. EIPA (5-(N-ethyl-N-isopropyl) amiloride) (Sigma, catalog number: A3085) 
  26. Sterile distilled water
  27. Stromal complete medium (see Recipes)
  28. PBS 1x, pH 7.4 (see Recipes)
  29. Dextran stock solution (see Recipes)
  30. EIPA stock solution (see Recipes)
  31. LysoTracker green DND-26 (see Recipes)
  32. DQ-Green BSA stock solution (see Recipes)
  33. Dextran cell culture incubation medium (see Recipes)
  34. Fixation buffer (see Recipes)

Equipment

  1. Pipette-aid
  2. Tissue culture hood
  3. Centrifuge with adaptors for 15 ml conical tubes
  4. Humidified cell culture incubator set to 37 °C and 5% CO2
  5. Confocal Laser Scanning Microscope (Leica, Germany)
  6. Fine-point forceps
  7. 4 °C refrigerator
  8. -20 °C refrigerator

Procedure

  1. Growing fibroblasts on coverslips
    1. Place the sterile cover slips in a 12-well plate; rinse the cover slips with Phosphate Buffered Saline (PBS), followed by a quick rinse with culture media. Plate the cells on the cover slips at a density of ~3,000/well in 500 µl of media.
    2. The labeling of macropinosomes involves cell growth under serum-starved conditions. Therefore, prior optimization is needed to find out suitable time point for getting 50%-75% confluency of cells. The confluency of the cell is very critical for proper labeling of macropinosomes and spreading on coverslip.

  2. Macropinosomes labeling
    1. Cells are cultured in a 5% CO2 incubator at 37 °C and all subsequent treatments are performed at 37 °C using pre-warmed media.
    2. Cells grown on cover slips in 12-well plates were serum starved for 18 h, followed by incubation with 1 mg/ml TMR-dextran for 30 min in serum free DMEM/F12 medium in CO2 incubator.
      Note: Dextran is available in different colors and molecular weights and it can be selected as per requirement and desired goals of the experiment.
    3. Remove culture medium as much as possible and wash the cells with 500 µl of ice cold PBS for 5 times. Fix the cells with 150 µl of 3.7% formaldehyde in PBS. Incubate the cells at room temperature for 20 min in the dark by covering it with aluminum foil.
      Note: Proper rinsing of the samples is very crucial to avoid fluorescent background signal.
    4. Remove the coverslip from the 12-well plates using forceps. Hold the coverslip vertical, with the bottom rim touching a paper towel to absorb remaining PBS and dry the coverslip completely.
    5. For nuclear staining and mounting, apply one drop of DAPI mounting medium to each glass slide. Take the cover slip and set it at an angle to the slide so that one edge of it touches DAPI mounting media, then carefully lower it over the drop so that the cover slip covers the medium without trapping air bubbles underneath. Use the corner of a paper towel to blot-up any excess DAPI mounting media at the edges of the cover slip. 
    6. Set the coverslip down on DAPI mounting medium with the cells facing down. Make sure, the cells become dry if not, then it can be left at room temperature for more time. Then carefully seal with clear fingernail polish. Dry sealing in the dark at room temperature for 2 h.

Data analysis

Our protocol describes the visualization of macropinosomes in prostatic fibroblasts. These macropinosomes further fused into lysosome and get degraded by their acidic enzymes (lysozymes). To rule out the background signal possibility in labeling macropinosomes, we also included negative control group by treating Ras overexpressing mouse fibroblasts with macropinocytosis inhibitors. In addition to negative control, experimental methods were set up to observe macropinosomes fusion in lysosomes and its degradation by lysozymes. To track lysosomes, LysoTracker green and for degradation DQTM-BSA was used. The steps of the analysis are summarized below:

  1. We follow four criteria to identify macropinosomes:
    1. Dextran positive.
    2. Excluded from the nucleus.
    3. Surrounded by GFP-positive cytoplasm.
    4. Fluorescence intensities of macropinosomes are comparable with or higher than that of the extracellular space. 
  2. We have had the best results imaging these samples using a Confocal Laser Scanning Microscope (Leica, Germany). 
  3. Images were acquired with a 60x 1.4 NA oil immersion lens. Different lasers were used that span the blue (457, 477, and 488 nanometers), green (514 and 543 nanometers), and red (633 and 647 nanometers) spectral regions. Fluorescence was recorded in individual channels acquired in a sequential mode to avoid cross-talk using a highly sensitive 32-channel gallium arsenide phosphide detector. The pinhole was set to 1 Airy unit.
  4. As an example of the in vitro implementation of this protocol, we have assessed the extent of macropinocytosis in Ras-overexpressing mouse fibroblast. A representative example of a confocal image is shown in Figure 1.


    Figure 1. Monitoring of macropinosomes. Representative images show TMR-dextran–positive macropinosomes (arrowheads) in RasV12 prostatic mouse fibroblasts (expressing GFP). Scale bar = 7.5 μm.

  5. Validation controls were analyzed as described below:
    1. Negative control: Grow the RasV12 mouse prostatic fibroblasts on coverslip as described in Procedure A. Treat cells with EIPA at 25 μM for 30 min prior to adding dextran in serum-free stromal media. EIPA is a clinical inhibitor of the Na+/H+ exchanger situated in the plasma membrane. Label the cells with TMR-dextran as described in Procedure B. Cells were fixed and analyzed by confocal microscopy. A representative example of a confocal image is shown in Figure 2.


      Figure 2. Reduction of macropinocytosis by EIPA. Macropinosomes visualization in RasV12 prostatic mouse fibroblasts was carried out with 30 min pretreatment with 5-(N-ethyl-N-isopropyl) amiloride (EIPA). Scale bar = 24 μm.

    2. Monitoring of macropinosomes lysosome fusion: Grow the RasV12 mouse prostatic fibroblasts on coverslip as described in Procedure A. Cells are incubated with a mixture of Lyso tracker green DND-26 (75 nM) and TMR-dextran (1 mg/ml each) for 30 min at 37 °C. After 30 min, cells are placed on ice, washed for 10 min in ice-cold dye-free medium for three times, and fixed with 3.7% formaldehyde for 20 min on ice. The fluorescent signal emanating from Lysotracker with TMR-dextran–positive staining indicates lysosomal degradation of macropinosomes. Image was obtained using microscope (Figure 3).


      Figure 3. Monitoring internalization of macropinosomes in lysosomes. Acidification of macropinosomes was monitored by co-localization of LysoTracker (green) with TMR-Dextran suggesting fusion of macropinosomes with lysosomes (see orange puncta in the merged image). Images show representative microscopic images. Scale bar, 30 μm.

    3. Test for albumin uptake by macropinosomes and lysosomal localization: Macropinocytosis substantially elevated uptake of proteins such as bovine serum albumin (BSA) from the extracellular fluid (Commisso et al., 2013). We choose DQTM-BSA to confirm albumin uptake and lysosomal function. DQTM-BSA is a quenched fluorogenic compound that requires enzymatic cleavage in an acidic intracellular compartment (i.e., lysosomes) for strong fluorescence. Grow the Cancer associated prostatic fibroblasts on coverslip as described in Procedure A. Cells are incubated with TMR-dextran and DQTM-BSA (10 μg/ml in serum free medium) for 1 h at 37 °C to assure macropinosomes formation and that the reagent reaches the lysosomal compartment. Cells were fixed and analyzed by confocal microscopy. A representative example of a confocal image is shown in Figure 4.


      Figure 4. Monitoring of extracellular albumin internalization in macropinosomes. CAF that were co-incubated with fluorescent DQ-BSA (green) and TMR-dextran (red) then fixed after 1-h chase. The fluorescent signal emanating from DQ-BSA with TMR-dextran–positive staining (arrowheads) indicates albumin uptake by macropinosomes and subsequent lysosomal breakdown. Scale bar = 7.5 μm.

Recipes

  1. Stromal complete medium
    DMEM/F12 media
    5% FBS
    5% NuSerum (HyClone)
    0.1% Insulin
  2. PBS 1x, pH 7.4 (1 L)
    NaCl 9 g
    NaH2PO4 0.23 g
    Na2HPO4 1.15 g
    Sterile distilled water, up to 1 L
  3. Dextran stock solution
    Dissolve 25 mg of fluorescently labeled 70-kDa dextran in 1.25 ml of PBS to obtain a final concentration of 20 mg/ml dextran stock solution
    Store stock solution in 100-μl aliquots in the dark at -20 °C
    Note: Make sure that dextran solution becomes clear and transparent. Dextran takes 10-15 min to dissolve completely by pipetting up and down.
  4. EIPA stock solution
    EIPA stock solutions (10 mM) are prepared in Dimethyl sulfoxide (DMSO) and used at a working concentration of 25 mM
  5. LysoTracker green DND-26 Green
    1. Allow a vial of 1 mM LysoTracker DND-26 solution (50 μl in DMSO) (Invitrogen L7526) to warm to room temperature
    2. Briefly centrifuge to deposit the solution at the bottom of the vial before opening
    3. From the 1 mM solution, prepare a 500 nM stock solution using molecular grade H2O
    4. Cover in aluminum foil to minimize light exposure
    5. Store stock solution below -20 °C
    6. Immediately before use, prepare a staining solution of roughly 75 nM LysoTracker Green by combining 1.5 μl of the 500 nM stock solution with 8 μl of molecular-grade H2
  6. DQ-Green BSA stock solution
    1. Dissolve 1 mg of lyophilized powder of DQ-BSA in 500 μl of PBS to make a stock of 2 mg/ml of DQ-Green BSA
    2. Mix thoroughly by vortexing and pipetting
    3. After reconstitution wrap it with aluminum foil to protect it from light and store at 4 °C 
  7. Fixation buffer
    Add 37% (vol/vol) ACS reagent-grade formaldehyde solution to PBS to a final concentration of 3.7% (vol/vol)
    Note: Use freshly prepared solution.

Acknowledgments

This work was supported by grants from the National Cancer Institute (CA108646 to NAB) and Veterans Affairs (BX001040 to NAB). Support by Enhanced Seed grants EF/2018-19/QE04-11 (to R.M.) from Manipal University Jaipur, Rajasthan, India is gratefully acknowledged.

Competing interests

The authors declare that they have no conflict of interest.

Ethics

In accordance with institutional animal care and use committee approval, primary mouse fibroblasts were harvested and grown using approved procedures.

References

  1. Bar-Sagi, D. and Feramisco, J. R. (1986). Induction of membrane ruffling and fluid-phase pinocytosis in quiescent fibroblasts by ras proteins. Science 233(4768): 1061-1068.
  2. Commisso, C., Davidson, S. M., Soydaner-Azeloglu, R. G., Parker, S. J., Kamphorst, J. J., Hackett, S., Grabocka, E., Nofal, M., Drebin, J. A., Thompson, C. B., Rabinowitz, J. D., Metallo, C. M., Vander Heiden, M. G. and Bar-Sagi, D. (2013). Macropinocytosis of protein is an amino acid supply route in Ras-transformed cells. Nature 497(7451): 633-637.
  3. Davidson, S. M., Jonas, O., Keibler, M. A., Hou, H. W., Luengo, A., Mayers, J. R., Wyckoff, J., Del Rosario, A. M., Whitman, M., Chin, C. R., Condon, K. J., Lammers, A., Kellersberger, K. A., Stall, B. K., Stephanopoulos, G., Bar-Sagi, D., Han, J., Rabinowitz, J. D., Cima, M. J., Langer, R. and Vander Heiden, M. G. (2017). Direct evidence for cancer-cell-autonomous extracellular protein catabolism in pancreatic tumors. Nat Med 23(2): 235-241.
  4. Lim, J. P. and Gleeson, P. A. (2011). Macropinocytosis: an endocytic pathway for internalising large gulps. Immunol Cell Biol 89(8): 836-843.
  5. Mishra, R., Haldar, S., Placencio, V., Madhav, A., Rohena-Rivera, K., Agarwal, P., Duong, F., Angara, B., Tripathi, M., Liu, Z., Gottlieb, R. A., Wagner, S., Posadas, E. M. and Bhowmick, N. A. (2018). Stromal epigenetic alterations drive metabolic and neuroendocrine prostate cancer reprogramming. J Clin Invest 128(10): 4472-4484.
  6. Swanson, J. A. and Watts, C. (1995). Macropinocytosis. Trends Cell Biol 5(11): 424-428.

简介

摘要:巨蛋白细胞增多症已成为非选择性途径内化细胞外液和溶解的分子在真核细胞中的重要机制。作为基本的细胞行为,巨胞饮作用在许多生理和病理过程中起着特定和独特的作用,例如营养摄取,抗原呈递,病原体捕获和肿瘤发生。它通过提供代谢需要在 Ras 驱动的癌症中分裂细胞来支持肿瘤发生。近年来,巨胞饮作用已经在生理学和各种疾病中获得了相当大的兴趣,包括癌症,神经退行性疾病和动脉粥样硬化,这反过来又导致了新的内吞循环系统的发现。评估巨噬细胞增多症的方法将提供对其潜在的调节分子机制的洞察,并且能够实现巨胞饮作用的生理控制,用于受控药物递送和靶向癌症治疗。巨噬细胞增多症是表达Ras的癌细胞中的重要现象,并且最近,我们已经揭示了巨噬细胞增多在癌症相关成纤维细胞(CAF)中促进癌细胞生长的功能性作用。在这里,我们描述了一种通过利用荧光标记的赖氨酸可固定的70kDa高分子量葡聚糖来体外检测前列腺成纤维细胞中的巨胞饮作用的方案。通过共聚焦或荧光显微镜观察巨噬细胞体作为荧光细胞内斑点。为了跟踪随后的细胞内事件及其在巨链球菌形成后的潜在机制,我们进行猝灭的BSA(DQ TM -BSA)的共定位以及癌症相关成纤维细胞中的葡聚糖标记。我们的方案提供了一致的方式来理解野生型或遗传操纵的前列腺成纤维细胞中的巨胞饮作用。


背景:巨胞饮作用或“细胞饮用”是一种内吞作用,其涉及将细胞外液非特异性摄取到称为巨噬细胞的大细胞内囊泡中(Swanson和Watts,1995)。巨噬菌体的大小和形状是不均匀的,其直径在0.2和5微米之间(Lim和Gleeson,2011)。 Src Ras 是突出的致癌基因,其在各种类型的癌症中刺激巨胞饮作用(Bar-Sagi和Feramisco,1986)。巨细胞增多症已在胰腺癌细胞中被突出描述,被认为具有活性Ras信号传导(Commisso 等人,2013; Davidson 等人,2017)。我们确定Ras信号传导的表观遗传激活介导前列腺癌相关成纤维细胞中的巨胞饮作用,导致白蛋白摄取,溶酶体降解和组成氨基酸释放(Mishra 等,,2018)。发现这些氨基酸(主要是谷氨酰胺)支持前列腺癌上皮细胞的代谢和分化。因此,巨胞饮作用是支持癌症相关成纤维细胞(CAF)的能量需求的重要营养物加工途径,并且可代表癌症治疗的预测生物标志物。通过共聚焦激光扫描显微镜可以实现巨胞饮作用的定量。

关键字:巨胞饮, 前列腺癌, 成纤维细胞, 谷氨酰胺, Ras, 5-(N-乙基-N-异丙基)-阿米洛利

材料和试剂

  1. 移液器吸头(Fisher Scientific,目录号:02707404)
  2. 无菌10毫升血清移液管(Santa Cruz Biotechnology Inc.,目录号:sc-200281)
  3. 无菌15毫升锥形管(BD Falcon,目录号:669993)
  4. 12孔细胞培养板(Fisher Scientific,目录号:64976)
  5. 盖玻片(Fisher Scientific,目录号:22050221)
  6. 铝箔(Fisher healthcare,目录号:01213101)
  7. Kimwipe(不起毛的纸巾; Fisher healthcare,目录号:S47299)
  8. 人前列腺成纤维细胞和Ras V12 (Mishra et al。,2018)
    注意:与普通对等体相比,这些细胞类型具有活跃的Ras活动。
  9. 胎牛血清(亚特兰大生物制品,目录号:S11550) 
  10. DMEM / F12培养基(Fisher Scientific,目录号:SH300404)
  11. 四甲基罗丹明 - 共轭高分子量葡聚糖(TMR-葡聚糖,75kDa),赖氨酸可固定(Invitrogen Molecular Probes,目录号:D1818)
  12. DQ-BSA(Thermo Fisher Scientific,Invitrogen TM ,目录号:D12050)
  13. LysoTracker ®绿色DND-26(Life Technologies,目录号:L7526)
  14. 5-(N-乙基-N-异丙基)阿米洛利(EIPA)(Invitrogen Molecular Probes,目录号:e-3111) 
  15. 带DAPI的Vectashield Antifade安装介质(Vector Laboratories,目录号:H-1200)
  16. 甲醛溶液,ACS试剂级,37%(vol / vol)(Sigma,目录号:252549) 
  17. NuSerum(Fisher Scientific,目录号:355500)
  18. 胰岛素,人重组,锌溶液(Invitrogen,目录号:12585-014)
  19. 氯化钠(NaCl)(Fisher Biosciences,目录号:BP358-1)
  20. 二甲基亚砜(DMSO)(Fisher Biosciences,目录号:D128-1)
  21. 磷酸二氢钠(NaH 2 PO 4 )(Fisher Biosciences,目录号:ICN19550080) 
  22. 磷酸钠,二元,无水,Na 2 HPO 4 (Fisher Biosciences,目录号:AC424375000)
  23. 10 -8 M睾酮(Nacalai Tesque,目录号:32811-61)
  24. 聚L-赖氨酸(Sigma,目录号:P4707)
  25. EIPA(5-(N-乙基-N-异丙基)阿米洛利)(Sigma,目录号:A3085) 
  26. 无菌蒸馏水
  27. 基质完全培养基(见食谱)
  28. PBS 1x,pH 7.4(参见食谱)
  29. 葡聚糖原液(见食谱)
  30. EIPA库存解决方案(参见食谱)
  31. LysoTracker绿色DND-26(见食谱)
  32. DQ-Green BSA原液(见食谱)
  33. 葡聚糖细胞培养培养基(参见食谱)
  34. 固定缓冲液(见食谱)

设备

  1. 移液器援助
  2. 组织培养罩
  3. 用适配器离心15 ml锥形管
  4. 加湿的细胞培养箱设定为37℃和5%CO 2
  5. 共聚焦激光扫描显微镜(德国莱卡)
  6. 细尖镊子
  7. 4°C冰箱
  8. -20°C冰箱

程序

  1. 在盖玻片上生长成纤维细胞
    1. 将无菌盖玻片放入12孔板中;用磷酸盐缓冲盐水(PBS)冲洗盖玻片,然后用培养基快速冲洗。将盖板上的细胞以约3,000 /孔的密度在500μl培养基中铺板。
    2. 巨噬菌体的标记涉及血清饥饿条件下的细胞生长。因此,需要事先进行优化以找出获得50%-75%细胞融合的合适时间点。细胞的融合对于正确标记巨噬细胞并在盖玻片上扩散非常关键。

  2. 巨蛋白体标记
    1. 将细胞在5%CO 2 培养箱中于37℃培养,并且所有后续处理在37℃下使用预热的培养基进行。
    2. 将在12孔板中的盖玻片上生长的细胞血清饥饿18小时,然后与1mg / ml TMR-葡聚糖在CO 2 培养箱中的无血清DMEM / F12培养基中孵育30分钟。
      注意:葡聚糖有不同的颜色和分子量,可根据实验的要求和预期目标进行选择。
    3. 尽可能去除培养基,用500μl冰冷的PBS洗涤细胞5次。用PBS中的150μl3.7%甲醛固定细胞。在室温下将细胞用铝箔覆盖,在黑暗中孵育20分钟。
      注意:正确冲洗样品对于避免荧光背景信号至关重要。
    4. 使用镊子从12孔板上取下盖玻片。保持盖玻片垂直,底部边缘接触纸巾以吸收剩余的PBS并完全干燥盖玻片。
    5. 对于核染色和安装,在每个载玻片上涂一滴DAPI封固剂。取下盖玻片,使其与滑块成一定角度,使其一边接触DAPI安装介质,然后小心地将其放在墨滴上,使盖玻片覆盖介质而不会在下方夹住气泡。使用纸巾的一角将任何多余的DAPI安装介质吸干盖玻片边缘。 
    6. 将盖玻片放在DAPI安装介质上,使细胞朝下。确保,如果没有,细胞会变干,然后可以在室温下放置更长时间。然后用清晰的指甲油仔细密封。在室温下在黑暗中干燥密封2小时。

数据分析

我们的方案描述了前列腺成纤维细胞中巨噬细胞的可视化。这些巨噬细胞进一步融合到溶酶体中并被其酸性酶(溶菌酶)降解。为了排除标记巨噬细胞体的背景信号可能性,我们还通过用巨噬细胞增多抑制剂处理Ras过表达的小鼠成纤维细胞来包括阴性对照组。除了阴性对照之外,还建立了实验方法以观察溶酶体中的巨噬菌体融合及其通过溶菌酶的降解。为了追踪溶酶体,使用LysoTracker green和降解DQ TM -BSA。分析步骤总结如下:

  1. 我们遵循四个标准来识别巨噬细胞:
    1. 右旋糖酐阳性。
    2. 被排除在核心之外。
    3. 被GFP阳性细胞质包围。
    4. 巨噬菌体的荧光强度与细胞外空间的荧光强度相当或更高。 
  2. 我们使用共聚焦激光扫描显微镜(德国莱卡)对这些样品进行了最佳成像。 
  3. 使用60x1.4NA油浸镜头获取图像。使用不同的激光,其跨越蓝色(457,477和488纳米),绿色(514和543纳米)和红色(633和647纳米)光谱区域。在顺序模式下获得的各个通道中记录荧光,以避免使用高灵敏度的32通道砷化镓磷化物检测器进行串扰。针孔设置为1艾里单位。
  4. 作为该方案的体外实施的一个例子,我们评估了Ras过表达小鼠成纤维细胞中巨胞饮作用的程度。共焦图像的代表性示例如图1所示。


    图1.监测巨噬细胞。代表性图像显示Ras V12 前列腺小鼠成纤维细胞(表达GFP)中的TMR-葡聚糖阳性巨噬细胞体(箭头)。比例尺=7.5μm。

  5. 如下所述分析验证控制:
    1. 阴性对照:如方法A所述,在盖玻片上培养Ras V12 小鼠前列腺成纤维细胞。在加入无血清葡聚糖之前,用25μM的EIPA处理细胞30分钟。基质媒体。 EIPA是位于质膜中的Na + / H + 交换剂的临床抑制剂。如方法B中所述用TMR-葡聚糖标记细胞。固定细胞并通过共聚焦显微镜分析。共焦图像的代表性示例如图2所示。


      图2.通过EIPA减少巨胞饮作用。 Ras V12 前列腺小鼠成纤维细胞中的巨噬细胞体可视化用30分钟预处理5-(N-乙基-N-异丙基)阿米洛利(EIPA)。比例尺=24μm。

    2. 监测巨噬细胞溶酶体融合:如程序A所述,在盖玻片上培养Ras V12 小鼠前列腺成纤维细胞。将细胞与Lyso跟踪绿色DND-26的混合物一起孵育(75 nM)和TMR-葡聚糖(各1mg / ml)在37℃下保持30分钟。 30分钟后,将细胞置于冰上,在冰冷的无染料培养基中洗涤3分钟三次,并在37℃下用3.7%甲醛在冰上固定20分钟。具有TMR-葡聚糖阳性染色的Lysotracker发出的荧光信号表明巨噬菌体的溶酶体降解。使用显微镜获得图像(图3)。


      图3.监测溶酶体中巨噬菌体的内化。通过LysoTracker(绿色)与TMR-葡聚糖的共定位监测巨噬菌体的酸化,表明巨噬菌体与溶酶体融合(参见合并图像中的橙色斑点) 。图像显示代表性的显微图像。比例尺,30μm。

    3. 通过巨噬细胞体和溶酶体定位测试白蛋白摄取:大粒细胞增多症从细胞外液中显着提高蛋白质如牛血清白蛋白(BSA)的摄取量(Commisso et al。,2013) 。我们选择DQ TM -BSA来确认白蛋白摄取和溶酶体功能。 DQ TM -BSA是猝灭的荧光化合物,其需要在酸性细胞内区室(即,溶酶体)中进行酶促切割以获得强荧光。如方法A所述,在盖玻片上培养癌症相关的前列腺成纤维细胞。将细胞与TMR-葡聚糖和DQ TM -BSA(10μg/ ml,在无血清培养基中)在37℃温育1小时。确保巨噬细胞形成,并且试剂到达溶酶体区室。固定细胞并通过共聚焦显微镜分析。共焦图像的代表性示例如图4所示。


      图4.监测巨噬细胞体中细胞外白蛋白的内化。与荧光DQ-BSA(绿色)和TMR-葡聚糖(红色)共培养的CAF然后在1小时追踪后固定。具有TMR-葡聚糖阳性染色(箭头)的DQ-BSA发出的荧光信号表明巨噬菌体对白蛋白的摄取和随后的溶酶体分解。比例尺=7.5μm。

食谱

  1. 基质完全培养基
    DMEM / F12媒体
    5%FBS
    5%NuSerum(HyClone)
    0.1%胰岛素
  2. PBS 1x,pH 7.4(1L)
    NaCl 9克
    NaH 2 PO 4 0.23 g
    Na 2 HPO 4 1.15 g
    无菌蒸馏水,最高1升
  3. Dextran库存解决方案
    将25毫克荧光标记的70-kDa葡聚糖溶于1.25毫升PBS中,得到最终浓度为20毫克/毫升的葡聚糖原液
    在-20°C黑暗中以100μl等分试样储存储备溶液
    注意:确保右旋糖酐溶液变得清澈透明。通过上下移液,葡聚糖需要10-15分钟才能完全溶解。
  4. EIPA股票解决方案
    在二甲基亚砜(DMSO)中制备EIPA储备溶液(10mM),并以25mM的工作浓度使用
  5. LysoTracker绿色DND-26绿色
    1. 将一小瓶1mM LysoTracker DND-26溶液(50μl,在DMSO中)(Invitrogen L7526)温热至室温
    2. 在打开之前,简单地离心以将溶液沉积在小瓶的底部
    3. 从1mM溶液中,使用分子级H 2 O制备500nM储备溶液
    4. 用铝箔覆盖,以尽量减少光线照射
    5. 储存溶液低于-20°C
    6. 在即将使用之前,通过将1.5μl的500nM储备溶液与8μl分子级H 2 O组合,制备大约75nM LysoTracker Green的染色溶液。
  6. DQ-Green BSA库存解决方案
    1. 将1 mg DQ-BSA冻干粉末溶于500μlPBS中,制成2 mg / ml DQ-Green BSA原液
    2. 通过涡旋和移液彻底混合
    3. 重新配制后,用铝箔包裹以保护其免受光照,并在4°C下储存。
  7. 固定缓冲区
    向PBS中加入37%(体积/体积)ACS试剂级甲醛溶液至终浓度为3.7%(体积/体积)
    注意:使用新制备的解决方案。

致谢

这项工作得到了国家癌症研究所(CA108646至NAB)和退伍军人事务部(BX001040至NAB)的资助。感谢印度拉贾斯坦邦斋浦尔马尼帕尔大学的增强种子资助EF / 2018-19 / QE04-11(至R.M.)。

利益争夺

作者声明他们没有利益冲突。

伦理

根据机构动物护理和使用委员会批准,收获原代小鼠成纤维细胞并使用批准的程序生长。

参考

  1. Bar-Sagi,D。和Feramisco,J.R。(1986)。 通过ras蛋白诱导静息成纤维细胞膜起皱和液相胞饮作用。 Science 233(4768):1061-1068。
  2. Commisso,C.,Davidson,SM,Soydaner-Azeloglu,RG,Parker,SJ,Kamphorst,JJ,Hackett,S.,Grabocka,E.,Nofal,M.,Drebin,JA,Thompson,CB,Rabinowitz,JD, Metallo,CM,Vander Heiden,MG和Bar-Sagi,D。(2013)。 蛋白质的细胞增多症是Ras转化细胞中的一种氨基酸供应途径。 自然 497(7451):633-637。
  3. Davidson,SM,Jonas,O.,Keibler,MA,Hou,HW,Luengo,A.,Mayers,JR,Wyckoff,J.,Del Rosario,AM,Whitman,M.,Chin,CR,Condon,KJ,Lammers ,A.,Kellersberger,KA,Stall,BK,Stephanopoulos,G.,Bar-Sagi,D.,Han,J.,Rabinowitz,JD,Cima,MJ,Langer,R。和Vander Heiden,MG(2017)。 胰腺肿瘤中癌细胞自主细胞外蛋白分解代谢的直接证据。 Nat Med 23(2):235-241。
  4. Lim,J。P.和Gleeson,P。A.(2011)。 大粒细胞增多症:内吞大口的内吞途径。 Immunol Cell Biol 89(8):836-843。
  5. Mishra,R.,Haldar,S.,Placencio,V.,Madhav,A.,Rohena-Rivera,K.,Agarwal,P.,Duong,F.,Angara,B.,Tripathi,M.,Liu,Z 。,Gottlieb,RA,Wagner,S.,Posadas,EM和Bhowmick,NA(2018)。 基质表观遗传改变驱动代谢和神经内分泌前列腺癌重编程。 J Clin Invest 128(10):4472-4484。
  6. Swanson,J。A.和Watts,C。(1995)。 大粒细胞增多症。 趋势细胞生物学 5(11): 424-428。
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引用:Mishra, R. and Bhowmick, N. A. (2019). Visualization of Macropinocytosis in Prostate Fibroblasts. Bio-protocol 9(10): e3235. DOI: 10.21769/BioProtoc.3235.
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