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Isolation and Separation of Epithelial CD34+ Cancer Stem Cells from Tgfbr2-deficient Squamous Cell Carcinoma
从Tgfbr2缺乏鳞状细胞癌分离上皮CD34+癌干细胞   

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Abstract

Most epithelial tumors have been shown to contain cancer stem cells that are potentially the driving force in tumor progression and metastasis (Kreso and Dick, 2014; Nassar and Blanpain, 2016). To study these cells in depth, cell isolation strategies relying on cell surface markers or fluorescent reporters are essential, and the isolation strategies must preserve their viability. The ability to isolate different populations of cells from the bulk of the tumor will continue to deepen our understanding of the biology of cancer stem cells. Here, we report the strategy combining mechanical tumor dissociation, enzymatic treatment and flow cytometry to isolate a pure population of epithelial cancer stem cells from their native microenvironment. This technique can be useful to further functionally profile the cancer stem cells (RNA sequencing and epigenetic analysis), grow them in culture or use them directly in transplantation assays.

Keywords: Cancer stem cells(癌症干细胞), Flow cytometry(流式细胞仪 ), Cell isolation(细胞分离), Squamous cell carcinoma (鳞状细胞癌)

Background

Tumor recurrence and metastasis is the leading cause of most deaths related to cancer. Malignant tumors may be initiated and maintained by a stem cell population (Nassar and Blanpain, 2016; Bonnet and Dick, 1997), and these cells represent an important therapeutic target to prevent relapse (Baumann et al., 2008). Studies suggest that squamous cell carcinomas are maintained by a subpopulation of tumor cancer stem cells that are resistant to therapy and can initiate tumor recurrence by undergoing self-renewal and differentiation, like normal stem cells, giving rise to proliferating progenitor cells that differentiate and form the bulk of the tumor (Locke et al., 2005; Prince et al., 2007; Malanchi et al., 2008; de Sousa e Melo et al., 2017). In this scenario, tumor cell fate and behavior are determined by the specific combination of changes in genes or their expression that have occurred during tumor development (Wang, 2010). Alternatively, progenitor cells can also acquire mutations that give them the potential to self-renew or can acquire some plasticity that give them cancer stem cell properties (Shimokawa et al., 2017). Regardless of the origin of cancer stem cells, efficient techniques to isolate these cells while maintaining their viability is essential. We have extensively characterized cancer stem cells from anorectal transition zone squamous cell carcinoma which arise spontaneously in the absence of epithelial TGFβ signaling (Keratin14Cre; Tgfbr2flox/flox mice) (Guasch et al., 2007; McCauley and Guasch, 2013; McCauley et al., 2017 ). In this protocol, we describe a method to isolate cancer stem cells from these anorectal transition zone squamous cell carcinomas.

Materials and Reagents

  1. Tissue culture dishes, 100 x 20 mm (Corning, Falcon®, catalog number: 353003 ) and 60 x 15 mm (Corning, Falcon®, catalog number: 353002 )
  2. Sterile disposable scalpel, #21 (Sklar)
  3. 50 ml conical tubes (BD, Falcon)
  4. Pipettes (25 ml, 10 ml and 5 ml serological and 1,000 µl, 200 µl, 20 µl and 2 µl pipette tips)
  5. Sterile nylon cell strainers, 70 μm (Fisher Scientific, catalog number: 22-363-548 ) and 40 µm (Fisher Scientific, catalog number: 22-363-547 )
  6. 5 ml polystyrene round-bottom tubes with cell strainer caps, 12 x 75 mm style (Corning, Falcon®, catalog number: 352235 )
  7. Sterile screw cap tubes and caps with ‘O’ rings, 1.5 ml (Corning, Axygen®, catalog number: SCT-150-C-S )
  8. Nalgene sterile disposable vacuum filters (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 167-0045 )
  9. Healthy and Tumor-bearing mice
    1. For healthy control mice, we used any mouse in the colony that did not express a transgene
    2. We used Keratin14Cre; Tgfbr2flox/flox; R26R-eYFPSTOP-flox-STOP mice, which spontaneously develop anorectal squamous cell carcinoma (Guasch et al., 2007; McCauley and Guasch, 2013; McCauley et al., 2017) in the development of this protocol. All three alleles are available from Jackson Labs (THE JACKSON LABORATORY, catalog number: 018964 , 012603 , and 006148 , respectively)
  10. 1x Hanks’ balanced salt solution (HBSS) (Thermo Fisher Scientific, GibcoTM, catalog number: 14170112 )
  11. Deoxyribonuclease (DNAse) I, from bovine pancreas, 10 mg/ml (Sigma-Aldrich, catalog number: D4263-5VL )
  12. 1x phosphate-buffered saline, sterile (1x PBS) (made in-house)
  13. Trypsin-EDTA, 0.25% (Thermo Fisher Scientific, GibcoTM, catalog number: 25200056 ) pre-warmed to 37 °C
  14. 7-AAD, 0.05 mg/ml (BD, BD Biosciences, catalog number: 559925 )
  15. Antibodies
    1. PE-Cy7 rat-anti-mouse CD11b, clone M1/70, 1/200 dilution (BD, BD Biosciences, catalog number: 561098 , RRID:AB_ 2033994)
    2. PE-Cy7 rat-anti-mouse CD31, clone 390, 1/100 dilution (BD, BD Biosciences, catalog number: 561410 , RRID:AB_10612003)
    3. PE-Cy7 rat-anti-mouse CD45, clone 30-F11, 1/200 dilution (Thermo Fisher Scientific, eBioscienceTM, catalog number: 25-0451-82 , RRID:AB_469625)
    4. PE rat-anti-human CD49f, 1/50 dilution (BD, BD Biosciences, catalog number: 555736 , RRID AB_396079)
    5. Pacific Blue anti-mouse/rat CD29, clone HMb1-1, 1/100 dilution (BioLegend, catalog number: 102224 , RRID:AB_2128079)
    6. Biotin anti-mouse CD34, clone RAM34, 1/50 dilution (Thermo Fisher Scientific, eBioscienceTM, catalog number: 13-0341-85 , RRID:AB_ 466426)
    7. APC Streptavidin, 1/200 dilution (BD, BD Bioscience, catalog number: 554067 , RRID:AB_10050396)
  16. β-Mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )
  17. QIAGEN RNeasy Micro kit (QIAGEN, catalog number: 74004 )
  18. Matrigel Matrix Basement Membrane, 5 ml vial (Corning, catalog number: 356234 )
  19. Sterile distilled, deionized water
  20. Collagenase (Sigma-Aldrich, catalog number: C2674-1G )
  21. 20% collagenase from Clostridium histolyticum (see Recipes)
  22. Chelated fetal bovine serum (FBS) (see Recipes) (Nowak and Fuchs, 2009)
    1. Chelex, 100 resin, sodium, 200-400 dry mesh, 75-150 µm wet bead (Bio-Rad Laboratories, catalog number: 1422842 )
  23. Epithelial cell culture medium (see Recipes) (Nowak and Fuchs, 2009)
    1. Dulbecco’s modified Eagle’s medium/Ham’s F-12 nutrient medium (3:1 Mix) without Calcium (Gibco Invitrogen, special order custom powdered media, catalog number: 90-5010EA)
    2. Ham’s F-12 nutrient mixture (Thermo Fisher Scientific, GibcoTM, catalog number: 11765054 )
    3. Penicillin-streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140148 )
    4. Sodium bicarbonate (Sigma-Aldrich, catalog number: S5761 )
    5. L-Glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 21051024 )
    6. Hydrocortisone (EMD Millipore, catalog number: 3867 )
    7. Cholera toxin (MP Biomedicals, catalog number: 02150005 )
    8. Insulin (Sigma-Aldrich, catalog number: I6634 )
    9. Transferrin (Roche Diagnostics, catalog number: 10652202001 )
    10. 3,3’,5-Triiodo-L-thyronine (T3) (Sigma-Aldrich, catalog number: T2877 )
    11. HCl and NaOH for pH adjustment and preparing dilutions of media additives
  24. Staining buffer (2% FBS) (see Recipes)

Equipment

  1. Rocking platform (VWR)
  2. Precision gravity convection incubator set to 37 °C and 5% CO2 (GCA corporation)
  3. Pipet aid (Drummond) and micro-pipettors (Rainin)
  4. Refrigerated centrifuge (Eppendorf, model: 5810 R )
  5. Laminar flow hood (Labgard)
  6. Autoclave (Steris)
  7. 4 L beaker (Pyrex)
  8. 6 L Erlenmeyer flask (Pyrex)
  9. Glass screw-top bottles, autoclaved (Pyrex)
  10. Graduated cylinders (Nalgene)
  11. Stir plate and stir bars (Fisher Scientific)
  12. pH meter (Fisher Scientific)
  13. Compressed CO2 source (Praxair)
  14. Sterile scissors, Iris Ribbon, 4” (Sklar)
  15. Sterile dissecting forceps, Half-curved 1 mm tip, 4” (George Tiemann & Co)
  16. FACS Aria II, 4 lasers 405/488/561/633 nm (Becton Dickinson) with 130 μm nozzle (BD, model: FACSAriaTM II )

Procedure

  1. Dissociation of squamous cell carcinoma into single-cells
    1. Sacrifice mice according to standard protocol. We use CO2 inhalation followed by cervical dislocation to ensure death has occurred.
    2. Our tumor-bearing mice contain the eYFP reporter allele (Figure 1A). Because of this, we also sacrifice a healthy, wild-type mouse which does not contain a fluorescent reporter and dissociate keratinocytes from the wild-type anorectal transition zone to obtain cells for unstained and single-stained controls. Dissociate cells from the anorectal transition zone of this healthy, wild-type mouse in parallel to dissociation of tumor cells from the tumor-bearing mouse.
    3. Carefully dissect the tumor, ensuring that it is separated from skin, fat, muscle and other contaminating tissue (Figure 1A). Remove any necrotic tissue.
    4. Place the tumor, approximately 0.5 cm2, in 8 ml sterile 1x HBSS in a 10 cm Petri dish and mince using a scalpel until all pieces are uniform and small (Figure 1B).
    5. Add 100 µl 20% collagenase (see Recipes) to dissociate the minced tumor. Incubate for 45 min while shaking at 37 °C. We place a rocking platform within a dry incubator set at speed 3 rpm.
    6. After 45 min, the tumor mixture should be homogeneous and may appear viscous (Figure 1C). Add 8.3 µl DNase I (10 mg/ml) to the minced tumor and incubate for an additional 10 min while shaking at 37 °C. After DNAse I treatment, the viscosity of the tumor mixture should be greatly reduced (Figure 1D).


      Figure 1. Dissociation of squamous cell carcinoma. A-A’. Keratin14Cre; Tgfbr2 flox/flox mice develop squamous cell carcinoma at the transition between the anal canal and the rectum. We have crossed these mice into mice containing the R26R-eYFPSTOP-flox-STOP allele such that the resulting Keratin14-positive cells, including the anorectal squamous cell carcinoma, express YFP. B. After removing the skin, fat and rectum from the tumor, mince the tumor into small pieces and incubate in HBSS containing 20% collagenase for 45 min (steps A4-A5). C. After 45 min of collagenase treatment, the tumor mixture will appear viscous (step A6). D. After 10 minutes of DNAse I treatment, the viscosity of the tumor mixture will be greatly reduced (step A6). Figures 1B-1D is adapted from McCauley and Guasch, 2013.

    7. Using a 25 ml pipette, add 10 ml cold 1x PBS to the minced tumor and pipette up and down vigorously 8 to 10 times to further dissociate the tumor mixture. Add the minced tumor to a 50 ml conical tube on ice.
    8. Wash the plate twice with 10-15 ml cold 1x PBS to obtain the maximum number of cells extracted from the tumor and add to the same 50 ml conical tube for each sample dissected.
    9. Centrifuge for 10 min at 2,000 x g at 4 °C.
    10. Carefully aspirate the supernatant, as the cell pellet may be very loose at this step. Using a 10 ml pipette, resuspend the cell pellet with 20 ml cold 1x PBS and add 200 µl chelexed FBS.
    11. Place a 70 µm filter set atop a new 50 ml conical tube. Pre-wet the filter with 1x PBS and filter the tumor cell suspension.
    12. Place the 70 µm filter in a 60 mm culture dish and add 2 ml of pre-warmed 0.25% trypsin-EDTA to the filter to maximize the number of cells extracted from the minced tumor. Incubate the filter for 10 min at 37 °C. This step is crucial as most of the epithelial CD34+ cells will be obtained at this dissociation step.
    13. After 5 min, block the activity of the trypsin by adding 5 ml epithelial cell culture media (see Recipes) containing serum to the filter. Mix well the cells with a 5 ml pipette to completely dissociate any clumps. Add the filtered trypsinized cells to the filtered cells in the same 50 ml conical tube from step 11. Wash the 60 mm plate twice with another 5 ml media to maximize the number of cells obtained.
    14. Place a 40 µm filter set atop a new 50 ml conical tube and pre-wet with epithelial cell culture media. Using a 5 ml pipette, pass the cells through the 40 µm filter.
    15. Centrifuge for 10 min at 200 x g at 4 °C.
    16. Aspirate the supernatant and resuspend the cell pellet in 1 ml staining buffer (see Recipes) in PBS to create a single-cell suspension.

  2. Separation of cancer stem cells from squamous cell carcinoma
    1. Filter the single-cell suspension through pre-blocked FACS tubes with a cell strainer cap. Pre-wet the cell strainer with PBS before applying the cell suspension to maximize cell number obtained.
      Note: To pre-block FACS tubes, we fill each tube that will be used, including those for single-stained and unstained controls, with FBS, allowing the FBS to coat the walls of the tubes. We then decant the FBS and reserve for reuse, and fill each blocked tube with 1x PBS until needed.
    2. Aliquot approximately 100 µl of cell suspension to the appropriate number of pre-blocked FACS tubes for unstained and single-stained controls. For our experiments:
      1. Wild-type anorectal transition zone cells are split into:
        1. Unstained control
        2. 7AAD control (reserve until immediately before sort)
        3. PE-Cy7 control
        4. PE control
        5. Pacific Blue control
        6. APC control
      2. YFP+ tumor cells are split into:
        1. YFP control (100 µl)
        2. The remainder of the cell suspension (approximately 1 ml) contains all colors and is the sample which will be sorted
    3. Add the appropriate antibody to each tube.
    4. Incubate the cells on ice and in the dark for 30 min. Tap the tubes every 5-10 min to disturb the cells from settling at the bottom of the tube.
    5. Fill each tube with 2% FBS to wash.
    6. Centrifuge for 5 min at 200 x g.
    7. Dump the supernatant into a waste container. Approximately 200 µl of buffer, containing the cell pellet, should remain at the bottom of the tube.
    8. We found that using the biotin-CD34 with the SAV-APC optimized the signal for the rare CD34+ cells within our tumors. For the APC control tube and the sample to be sorted, incubate with secondary antibody SAV-APC for 20 min on ice, protected from light.
      Note: Reserve the remainder of the control tubes on ice and protected from light during this time.
    9. Fill these tubes with 2% FBS to wash.
    10. Centrifuge for 5 min at 200 x g.
    11. Dump the supernatant into a waste container. Approximately 200 µl of buffer, containing the cell pellet, should remain at the bottom of the tube.
    12. Resuspend the cell pellet of the tumor sample with 300-800 µl 2% FBS. The volume should be adjusted to account for the size of the cell pellet to optimize flow rate on the cytometer. Keep in mind that a concentrated sample can easily be diluted.
    13. Bring the 7AAD with the sample to the flow cytometer. We prefer to add the 7AAD to the sample immediately before sorting to minimize cell death due to toxicity. We found 20 µl per million cells to be sufficient to label dead cells.
    14. Record at least 10,000 events from the unstained and single-color controls to enable compensation.
    15. See Figure 2 for an example of our gating strategy to isolate epithelial CD34+ cancer stem cells. The population hierarchy is:
      1. Forward scatter (A) versus side scatter (A).
      2. Side Scatter (H) versus Side Scatter (W) doublet discrimination.
      3. Forward Scatter (H) versus Forward Scatter (W) doublet discrimination.
      4. 7AAD negative (live), PE-Cy7 negative (CD11b, CD31, CD45 staining).
      5. YFP positive.
      6. PE positive (a6-integrin), Pacific Blue positive (b1-integrin).
      7. APC negative and positive (CD34).


        Figure 2. Gating strategy to isolate epithelial CD34+ cancer stem cells from Tgfbr2-deficient tumors. After dissociation and filtering into a single cell suspension, cells are subjected to fluorescence-activated cell sorting to isolate the rare epithelial CD34+ cancer stem cells. Cells of the appropriate size and granularity are included in P1, and doublets are excluded by the SSC and FSC gates. Live cells are selected by 7AAD exclusion, and CD11b+, CD31+ and CD45+ cells are excluded in the DUMP channel by PE-Cy7 staining. LIVE cells which express YFP under control of the Keratin 14 promoter are then further purified by α6- and β1-integrin expression (PE and Pacific Blue, respectively). Of these YFP+α6+β1+ cells, a pure population of CD34 (APC+) cancer stem cells can then be isolated.

    16. We have successfully isolated live cells for RNA extraction, for returning to culture, and for direct transplantation into recipient mice. Depending on the size of the initial tumor, the efficiency of dissociation, and individual composition of the tumor, we typically obtain between 25,000 and 250,000 live, YFP+, a6-integrin+ b1-integrin+ tumor cells from a tumor approximately 0.5 cm3, and from that tumor population, we typically obtain between 2,000-10,000 CD34+ cancer stem cells.
      1. For RNA extraction, collect 300 to 10,000 cells directly into 350 µl lysis buffer containing 3.5 µl β-mercaptoethanol in screw-top tubes. Vortex the tubes immediately after sorting and store at -80 °C until RNA extraction. We prefer the QIAGEN RNeasy Micro kit and use the buffer RLT from the kit.
      2. For return to culture, collect as many cells as possible in 500 µl epithelial cell culture media in a screw-top tube. Centrifuge tubes at 200 x g for 5 min and very carefully aspirate supernatant. Resuspend in epithelial cell culture media and plate. We found that cells did not survive when plating directly on plastic. We prefer to plate cells on irradiated mouse fibroblasts to improve colony formation and survival. We found that plating less than 1,000 cells did not allow for cell survival.
      3. For direct transplantation, collect as many cells as possible in 500 µl epithelial cell culture media without serum in a screw-top tube. The serum generates bubbles which negatively affect the resuspension in 30% Matrigel. Centrifuge tubes and very carefully aspirate the supernatant. Resuspend in 30% Matrigel, and keep tubes on ice until transplantation (see McCauley and Guasch [2013] for the detailed protocol of the orthotopic transplantation). We have successfully generated tumors by transplanting as few as 100 CD34+ cells. Transplanting larger numbers of cells will accelerate tumor formation.

Data analysis

  1. Because the isolation of rare cells requires inclusion and exclusion of a number of markers, it is imperative to have unstained and single-stained controls for effective compensation of spectral overlap.
  2. The frequency of epithelial CD34+ cells ranged from 7% to 22%. This is due to the heterogeneity of the squamous cell carcinoma from mouse to mouse. It is therefore recommended to perform at least six biological repeats within each experimental group. 

Notes

  1. All experiments were approved by the Cincinnati Children’s Hospital Research Foundation Institutional Animal Care and Use Committee (protocol number 1D10087) and in agreement with European and national regulation (protocol number 4572), and carried out using standard procedures.
  2. Trypsinizing the filter is a crucial step that can drastically impact the efficiency of CD34+ cancer stem cells isolated, as the cells may remain in clumps on top of the filter and be unintentionally discarded. Be sure to use pre-warmed 0.25% trypsin-EDTA, to incubate for the full 10:00 at 37 °C, and to vigorously pipet the trypsinized cells to ensure proper dissociation and maximal number of cells obtained.
  3. It is possible to store the tumor samples in media containing serum overnight at 4 °C and perform the cell isolation the next day. Cell mortality will be increased but the overall percentage of cancer stem cells will not be altered.
  4. FBS lots need to be tested for optimal growth of the epithelial cells.
  5. Because epithelial cells are very sensitive to calcium, chelating the FBS is essential.
  6. While it is possible to collect sorted cells using a 100 μm nozzle, we recommend using a 130 μm nozzle to improve the survival of the large epithelial cells post-sort.

Recipes

  1. 20% collagenase from Clostridium histolyticum
    1. Prepare a 20% stock by dissolving 1 g of powdered collagenase in 5 ml 1x sterile phosphate-buffered saline
    2. Aliquot 250 μl into Eppendorf tubes and store at -20 °C to avoid repeated freezing and thawing that will decrease enzyme activity
  2. Chelated fetal bovine serum (FBS), prepared as described in (Nowak and Fuchs, 2009)
    Day 1
    1. Add 400 g of dry Chelex to a 4 L beaker with a stir bar. Add distilled water to a total volume of 4 L. Cover and stir continuously
    2. Adjust pH to 7.4 using 10 N HCl. Stir for 20 min, readjust pH with 10 N HCl, and repeat as needed until pH remains stable for more than 20 min
    3. Place the beaker at 4 °C overnight to allow the Chelex to form a compact pellet
    Day 2
    1. Carefully decant H2O. Add fresh H2O to 4 L
    2. Adjust the pH to 7.4 as in Day 1
    3. Place the beaker at 4 °C for 1 h to allow the Chelex to form a compact pellet
    4. Carefully decant H2O
    5. Slowly add two 500 ml bottles of characterized or defined fetal bovine serum to the Chelex
    6. Stir slowly at 4 °C for 1 h. Try to minimize bubbles
    7. Place the beaker at 4 °C for 1 h to allow the Chelex to form a compact pellet
    8. Decant the serum into a 1 L glass bottle and filter the serum through a Nalgene bottle top filter under sterile conditions
    9. Store unused FBS at 20 °C or use immediately to make E media without calcium
  3. Epithelial cell culture media, prepared as described in (Nowak and Fuchs, 2009)
    1. In a 6 L Erlenmeyer flask, combine six packets of Gibco Invitrogen customized DMEM:F12 (3:1) without calcium with distilled water to reach a final volume of 5.5 L
    2. Add 18.42 g of sodium bicarbonate, 2.85 g of L-glutamine, and 60 ml of 100x penicillin-streptomycin solution
    3. Adjust pH to 7.2 using 10 N HCl and adjust the volume to 6 L with H2O
    4. Apply compressed CO2 to the media for 15 min. The media should reach an amber color
    5. Prepare the following cocktail of media additives:
      1. 20 ml of 5 mg/ml insulin, dissolved in 0.1 N HCl
      2. 20 ml of 5 mg/ml transferrin, dissolved in sterile PBS
      3. 20 ml of 2 x 10-8 M T3, dissolved to 2 x 10-4 in 0.02 N NaOH then further diluted to final concentration in 1x PBS
      4. 140 ml 1x PBS
      5. Filter sterilize and store in 37.5 ml aliquots at -20 °C
    6. Add 75 ml of the above cocktail, 750 ml 10-6 M cholera toxin (dissolved in water), and 750 ml 4 mg/ml hydrocortisone to 1 L of chelated FBS
    7. Produce final 15% FBS media in 1 L batches by combining 850 ml of the DMEM:F12 media base from step 2 with 150 ml of the supplemented chelated FBS and sterilize using a Nalgene bottle top filter
    8. Media can be stored in 250 or 500 ml bottles at 20 °C
  4. Staining buffer (2% FBS)
    1 ml chelexed FBS
    49 ml 1x PBS
    Note: Make fresh for each use, and keep on ice.

Acknowledgments

The protocol to dissociate the tumor is based on our original protocol published in Methods Mol Biol (McCauley and Guasch, 2013), with minor modifications. The protocol to make chelexed FBS and E media are abbreviated here, but made exactly as described in (Nowak and Fuchs, 2009). An abbreviated description of the flow cytometry protocol appeared in eLife (McCauley et al., 2017). We would like to acknowledge the assistance of the Research Flow Cytometry Core in the Division of Rheumatology at Cincinnati Children’s Hospital Medical Center (supported in part by P30 DK07839) and the Flow Cytometry Core at the CRCM, FRANCE. The protocol related to this work was supported by grants from the V Foundation, the Sidney Kimmel Foundation and in part from the Foundation ARC pour la recherche sur le cancer (GG).

References

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  2. Bonnet, D. and Dick, J. E. (1997). Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med 3(7): 730-737.
  3. de Sousa e Melo, F., Kurtova, A. V., Harnoss, J. M., Kljavin, N., Hoeck, J. D., Hung, J., Anderson, J. E., Storm, E. E., Modrusan, Z., Koeppen, H., Dijkgraaf, G. J., Piskol, R. and de Sauvage, F. J. (2017). A distinct role for Lgr5+ stem cells in primary and metastatic colon cancer. Nature 543(7647): 676-680.
  4. Guasch, G., Schober, M., Pasolli, H. A., Conn, E. B., Polak, L. and Fuchs, E. (2007). Loss of TGFβ signaling destabilizes homeostasis and promotes squamous cell carcinomas in stratified epithelia. Cancer Cell 12(4): 313-327.
  5. Kreso, A. and Dick, J. E. (2014). Evolution of the cancer stem cell model. Cell Stem Cell 14(3): 275-91.
  6. Locke, M., Heywood, M., Fawell, S. and Mackenzie, I. C. (2005). Retention of intrinsic stem cell hierarchies in carcinoma-derived cell lines. Cancer Res 65(19): 8944-8950.
  7. Malanchi, I., Peinado, H., Kassen, D., Hussenet, T., Metzger, D., Chambon, P., Huber, M., Hohl, D., Cano, A., Birchmeier, W. and Huelsken, J. (2008). Cutaneous cancer stem cell maintenance is dependent on β-catenin signalling. Nature 452(7187): 650-653.
  8. McCauley, H. A., Chevrier, V., Birnbaum, D. and Guasch, G. (2017). De-repression of the RAC activator ELMO1 in cancer stem cells drives progression of TGFβ-deficient squamous cell carcinoma from transition zones. Elife 6.
  9. McCauley, H. A. and Guasch, G. (2013). Serial orthotopic transplantation of epithelial tumors in single-cell suspension. Methods Mol Biol 1035: 231-245.
  10. Nassar, D. and Blanpain, C. (2016). Cancer stem cells: Basic concepts and therapeutic implications. Annu Rev Pathol 11: 47-76.
  11. Nowak, J. A. and Fuchs, E. (2009). Isolation and culture of epithelial stem cells. Methods Mol Biol 482: 215-232.
  12. Prince, M. E., Sivanandan, R., Kaczorowski, A., Wolf, G. T., Kaplan, M. J., Dalerba, P., Weissman, I. L., Clarke, M. F. and Ailles, L. E. (2007). Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma. Proc Natl Acad Sci U S A 104(3): 973-978.
  13. Shimokawa, M., Ohta, Y., Nishikori, S., Matano, M., Takano, A., Fujii, M., Date, S., Sugimoto, S., Kanai, T. and Sato, T. (2017). Visualization and targeting of LGR5+ human colon cancer stem cells. Nature 545(7653): 187-192. 
  14. Wang, J. C. (2010). Good cells gone bad: the cellular origins of cancer. Trends Mol Med 16(3): 145-151.

简介

大多数上皮肿瘤已经显示含有可能是肿瘤进展和转移的驱动力的癌症干细胞(Kreso和Dick,2014; Nassar和Blanpain,2016)。 为了深入研究这些细胞,依赖于细胞表面标志物或荧光报告基因的细胞分离策略是必不可少的,分离策略必须保持其活力。 从大部分肿瘤中分离不同细胞群的能力将继续加深我们对癌症干细胞生物学的认识。 在这里,我们报告了结合机械肿瘤解离,酶处理和流式细胞术的策略,从其天然微环境中分离出纯种群的上皮癌干细胞。 该技术可用于进一步功能性地分析癌症干细胞(RNA测序和表观遗传学分析),在培养物中培养它们或在移植测定中直接使用它们。
【背景】肿瘤复发和转移是大多数与癌症有关的死亡的主要原因。恶性肿瘤可能由干细胞群体启动和维持(Nassar和Blanpain,2016; Bonnet和Dick,1997),这些细胞是预防复发的重要治疗靶点(Baumann et al。,2008)。研究表明,鳞状细胞癌由肿瘤干细胞亚群维持,其抗药性,并通过进行自我更新和分化(如正常干细胞)引发肿瘤复发,产生增殖祖细胞,其分化形成肿瘤大部分(Locke et al。,2005; Prince et al。,2007; Malanchi et al。,2008; de Sousa e Melo et al。,2017)。在这种情况下,肿瘤细胞的命运和行为是通过肿瘤发生过程中发生的基因变化或其表达的特异性组合来确定的(Wang,2010)。或者,祖细胞也可以获得使它们具有自我更新潜力的突变,或者可以获得赋予它们癌症干细胞特性的一些可塑性(Shimokawa等,2017)。不管癌干细胞的来源如何,在保持其活力的同时分离这些细胞的有效技术是至关重要的。我们已经广泛地表征了来自肛门直肠过渡区鳞状细胞癌的癌症干细胞,其在不存在上皮TGFβ信号传导(Keratin14Cre; Tgfbr2flox / flox小鼠)时自发产生(Guasch等人,2007; McCauley和Guasch,2013; McCauley et al。 ,2017)。在这个方案中,我们描述了一种从这些肛门直肠过渡区鳞状细胞癌分离癌症干细胞的方法。

关键字:癌症干细胞, 流式细胞仪 , 细胞分离, 鳞状细胞癌

材料和试剂

  1. 组织培养皿100×20mm(Corning,Falcon ,目录号:353003)和60×15mm(Corning,Falcon,目录号:353002) br />
  2. 无菌一次性手术刀#21(Sklar)
  3. 50ml锥形管(BD,Falcon)
  4. 移液管(25 ml,10ml,5ml血清学和1000μl,200μl,20μl和2μl移液器吸头)
  5. 无菌尼龙细胞过滤器,70μm(Fisher Scientific,目录号:22-363-548)和40μm(Fisher Scientific,目录号:22-363-547)
  6. 5 ml聚苯乙烯圆底管,带有12 x 75 mm样式的细胞过滤器盖(Corning,Falcon ®,目录号:352235)
  7. 无菌螺旋盖管和带有"O"形圈的帽,1.5毫升(康宁,Axygen,目录号:SCT-150-C-S)
  8. Nalgene无菌一次性真空过滤器(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:167-0045)
  9. 健康和肿瘤小鼠
    1. 对于健康对照小鼠,我们在殖民地使用任何不表达转基因的小鼠
    2. 我们使用了 Keratin14Cre R26R-eYFP 小鼠,自发发展肛门直肠鳞状细胞癌(Guasch等人,2007; McCauley和Guasch,2013; McCauley等人,2017)在本协议的开发中。所有三个等位基因可从Jackson Labs(THE JACKSON LABORATORY,目录号:018964,012603和006148)获得
  10. 1x Hanks平衡盐溶液(HBSS)(Thermo Fisher Scientific,Gibco TM,目录号:14170112)
  11. 来自牛胰腺的脱氧核糖核酸酶(DNAse)I,10mg / ml(Sigma-Aldrich,目录号:D4263-5VL)
  12. 1x磷酸盐缓冲盐水,无菌(1x PBS)(内部制成)
  13. 胰蛋白酶-EDTA,0.25%(Thermo Fisher Scientific,Gibco TM,目录号:25200056)预温至37℃
  14. 7-AAD,0.05mg / ml(BD,BD Biosciences,目录号:559925)
  15. 抗体
    1. PE-Cy7大鼠抗小鼠CD11b,克隆M1 / 70,1 / 200稀释(BD,BD Biosciences,目录号:561098,RRID:AB_2033994)
    2. PE-Cy7大鼠 - 抗小鼠CD31,克隆390,1/100稀释(BD,BD Biosciences,目录号:561410,RRID:AB_10612003)
    3. PE-Cy7大鼠 - 抗 - 小鼠CD45,克隆30-F11,1 / 200稀释(Thermo Fisher Scientific,eBioscience,目录号:25-0451-82,RRID:AB_469625) >
    4. PE大鼠抗人CD49f,1/50稀释(BD,BD Biosciences,目录号:555736,RRID AB_396079)
    5. 太平洋蓝抗小鼠/大鼠CD29,克隆HMb1-1,1/100稀释(BioLegend,目录号:102224,RRID:AB_2128079)
    6. 生物素抗小鼠CD34,克隆RAM34,1/50稀释(Thermo Fisher Scientific,eBioscience TM,目录号:13-0341-85,RRID:AB_466426)
    7. APC链霉亲和素,1/200稀释(BD,BD Bioscience,目录号:554067,RRID:AB_10050396)
  16. β-巯基乙醇(Sigma-Aldrich,目录号:M3148)
  17. QIAGEN RNeasy Micro kit(QIAGEN,目录号:74004)
  18. Matrigel Matrix基底膜,5ml小瓶(Corning,目录号:356234)
  19. 无菌蒸馏水,去离子水
  20. 胶原酶(Sigma-Aldrich,目录号:C2674-1G)
  21. 20%胶原酶从溶组织梭菌(见配方)
  22. 螯合胎牛血清(FBS)(见食谱)(Nowak和Fuchs,2009)
    1. Chelex 100树脂,钠200-400干网,75-150微米湿珠(Bio-Rad Laboratories,目录号:1422842)
  23. 上皮细胞培养基(见Recipes)(Nowak和Fuchs,2009)
    1. Dulbecco改良Eagle's Medium / Ham's F-12营养培养基(3:1 Mix),不含钙(Gibco Invitrogen,特殊订购粉末介质,目录号:90-5010EA)
    2. Ham's F-12营养混合物(Thermo Fisher Scientific,Gibco TM,目录号:11765054)
    3. 青霉素 - 链霉素(Thermo Fisher Scientific,Gibco TM,目录号:15140148)
    4. 碳酸氢钠(Sigma-Aldrich,目录号:S5761)
    5. L-谷氨酰胺(Thermo Fisher Scientific,Gibco TM,目录号:21051024)
    6. 氢化可的松(EMD Millipore,目录号:3867)
    7. 霍乱毒素(MP Biomedicals,目录号:02150005)
    8. 胰岛素(Sigma-Aldrich,目录号:I6634)
    9. 转铁蛋白(Roche Diagnostics,目录号:10652202001)
    10. 3,3',5-三碘-L-甲状腺素(T3)(Sigma-Aldrich,目录号:T2877)
    11. HCl和NaOH用于pH调节和制备介质添加剂的稀释剂
  24. 染色缓冲液(2%FBS)(参见食谱)

设备

  1. 摇摆平台(VWR)
  2. 精密重力对流培养箱设置为37℃和5%CO 2(GCA公司)
  3. 吸管辅助(Drummond)和微量移液器(Rainin)
  4. 冷冻离心机(Eppendorf,型号:5810 R)
  5. 层流罩(Labgard)
  6. 高压灭菌器(Steris)
  7. 4升烧杯(Pyrex)
  8. 6升三角烧瓶(Pyrex)
  9. 玻璃螺旋盖,高压灭菌(Pyrex)
  10. 量筒(Nalgene)
  11. 搅拌棒(Fisher Scientific)
  12. pH计(Fisher Scientific)
  13. 压缩的CO 2 源(Praxair)
  14. 无菌剪刀,Iris Ribbon,4"(Sklar)
  15. 无脊椎解剖镊子,半弯1毫米尖端,4"(乔治·蒂曼公司)
  16. 具有130μm喷嘴的FACS Aria II,4激光器405/488/561/633nm(Becton Dickinson)(BD,型号:FACSAria II)

程序

  1. 鳞状细胞癌分裂成单细胞
    1. 根据标准方案牺牲小鼠。我们使用CO 2 吸入,然后是颈椎脱位,以确保发生死亡。
    2. 我们的荷瘤小鼠含有eYFP报道等位基因(图1A)。因此,我们还牺牲不含荧光报告基因的健康的野生型小鼠,并将角质形成细胞从野生型肛门直肠过渡区解离以获得未染色和单染色对照的细胞。从这种健康的野生型小鼠的肛门直肠过渡区分离细胞,平行于肿瘤细胞与荷瘤小鼠的解离。
    3. 仔细切开肿瘤,确保与皮肤,脂肪,肌肉和其他污染组织分离(图1A)。去除任何坏死组织。
    4. 将肿瘤,大约0.5厘米<2>在10毫升培养皿中放入8毫升无菌1x HBSS中,并使用手术刀切片,直至所有部分均匀均匀(图1B)。
    5. 加入100μl20%的胶原酶(参见食谱)以解离切碎的肿瘤。在37℃下振荡孵育45分钟。我们将摆放平台放在干燥的孵化器中,速度为3 rpm。
    6. 45分钟后,肿瘤混合物应该是均匀的,并且可能看起来粘稠(图1C)。向切碎的肿瘤中加入8.3μlDNAse I(10mg / ml),并在37℃下振荡孵育另外10分钟。在DNAse I处理后,肿瘤混合物的粘度应大大降低(图1D)

      图1.鳞状细胞癌的分离。 A-A"。 Keratin14Cre;在肛管和直肠之间的过渡处,小鼠发生鳞状细胞癌,Tgfbr2 。我们将这些小鼠穿过含有R26R-eYFP 等位基因的小鼠,使得得到的角蛋白14阳性细胞,包括肛门直肠鳞状细胞癌,表达YFP。 B.从肿瘤中取出皮肤,脂肪和直肠后,将肿瘤切碎成小块,并在含有20%胶原酶的HBSS中孵育45分钟(步骤A4-A5)。 C.胶原酶处理45分钟后,肿瘤混合物显得粘稠(步骤A6)。 D.在DNA酶I处理10分钟后,肿瘤混合物的粘度将大大降低(步骤A6)。图1B-1D是从McCauley和Guasch,2013年改编而成的。

    7. 使用25毫升移液管,将10毫升冷的1×PBS加入到切碎的肿瘤中,并将其高达8至10倍的上下移液,以进一步解离肿瘤混合物。将切碎的肿瘤加入到冰上的50ml锥形管中
    8. 用10-15ml冷的1x PBS洗板两次,以获得从肿瘤提取的最大数量的细胞,并将每个样品分开加入相同的50ml锥形管中。
    9. 在4℃下以2,000×g离心10分钟。
    10. 仔细吸取上清液,因为细胞沉淀物在此步骤可能非常松散。使用10ml移液管,用20ml冷的1x PBS重悬细胞沉淀,并加入200μl的chelexed FBS。
    11. 将一个70微米的过滤器置于新的50ml锥形管上。用1x PBS预过滤器过滤肿瘤细胞悬浮液。
    12. 将70μm过滤器置于60 mm培养皿中,加入2 ml预热的0.25%胰蛋白酶-EDTA至过滤器,以最大化从切碎的肿瘤提取的细胞数。在37℃下过滤10分钟。这个步骤是至关重要的,因为大多数上皮CD34 + 细胞将在此解离步骤获得。
    13. 5分钟后,通过向滤器中加入含有血清的5ml上清细胞培养基(参见食谱)来阻断胰蛋白酶的活性。用5ml移液管充分混合细胞以完全解离任何团块。将滤过的Trypisin化细胞加入到步骤11的相同50ml锥形管中的经过滤的细胞中。用另外的5ml培养基洗涤60mm板两次以使获得的细胞数量最大化。
    14. 将一个40μm的过滤器置于新的50ml锥形管顶上,并用上皮细胞培养基预湿。使用5ml移液器,将细胞通过40μm过滤器。
    15. 在4℃以200×g离心10分钟。
    16. 吸出上清液,并将细胞沉淀重悬于PBS的1ml染色缓冲液(参见Recipes)中以产生单细胞悬浮液。

  2. 从鳞状细胞癌分离癌干细胞
    1. 通过带有细胞过滤器盖的预先封堵的FACS管将单细胞悬浮液过滤。在使用细胞悬浮液以使获得的细胞数量最大化之前,用PBS将细胞过滤器预湿。
      注意:为了预先封堵FACS管,我们将每个将使用的管,包括用于单染色和未染色的对照的管与FBS填充,使FBS涂覆管的壁。然后我们滗出FBS并保留重新使用,并填充每个封闭的管与1x PBS直到需要。
    2. 将大约100μl的细胞悬浮液等分至适当数量的预先封闭的FACS管用于未染色和单染色对照。对于我们的实验:
      1. 野生型肛门直肠过渡区细胞分为:
        1. 无污染控制
        2. 7AAD控制(保留直到排序前)
        3. PE-Cy7控制
        4. PE控制
        5. 太平洋蓝调控制
        6. APC控制
      2. 将肿瘤细胞分为:YFP +
        1. YFP控制(100μl)
        2. 细胞悬浮液的剩余部分(约1毫升)包含所有颜色,并且将被分类的样品
    3. 向每个管中加入适当的抗体。
    4. 在冰上和黑暗中孵育细胞30分钟。每5-10分钟敲击管子,以扰乱细胞沉淀在管底部。
    5. 用2%FBS填充每个管子进行洗涤。
    6. 在200 x g离心5分钟。
    7. 将上清液倒入废物容器中。大约200μl含有细胞沉淀的缓冲液应保留在管的底部。
    8. 我们发现使用SAV-APC的生物素-CD34优化了我们肿瘤中稀有CD34 + 细胞的信号。对于APC对照管和要分选的样品,在冰上用二级抗体SAV-APC孵育20分钟,防止光照。
      注意:在此期间,将剩余的控制管保留在冰上,防止受光照射。
    9. 用2%FBS填充这些管子进行洗涤。
    10. 在200 x g离心5分钟。
    11. 将上清液倒入废物容器中。大约200μl含有细胞沉淀的缓冲液应保留在管的底部。
    12. 用300-800μl2%FBS重悬细胞沉淀。应调整体积以考虑细胞沉淀物的大小以优化流式细胞仪上的流速。请记住,浓缩样品可以轻松稀释。
    13. 将带有样品的7AAD带到流式细胞仪。我们更喜欢在分拣之前立即将7AAD加入到样品中,以尽量减少由于毒性引起的细胞死亡。我们发现每百万个细胞20微克足以标记死细胞。
    14. 从未染色和单色控件记录至少10,000个事件以实现补偿。
    15. 关于分离上皮CD34 + 癌症干细胞的门控策略的例子见图2。人口层级是:
      1. 前向散射(A)与侧向散射(A)。
      2. 侧面散射(H)与侧面散射(W)双峰鉴别。
      3. 正向散射(H)与向前散射(W)双峰鉴别。
      4. 7AAD阴性(活),PE-Cy7阴性(CD11b,CD31,CD45染色)
      5. YFP阳性。
      6. PE阳性(a6整合素),太平洋蓝阳性(b1-整联蛋白)。
      7. APC阴性和阳性(CD34)。


        图2.从分离上皮CD34 + 癌干细胞的门控策略 Tgfbr2 < / strong> - 缺乏肿瘤。 解离和过滤成单细胞悬液后,细胞进行荧光激活细胞分选以分离罕见的上皮CD34 +癌细胞。 P1中包含适当大小和粒度的单元,SSC和FSC门都排除了双引号。通过7AAD排除选择活细胞,并通过PE-Cy7染色在DUMP通道中排除CD11b + ,CD31 + 和CD45 + 细胞。在角蛋白14启动子控制下表达YFP的LIVE细胞然后通过α6和β1整联蛋白表达(分别为PE和Pacific Blue)进一步纯化。在这些YFP + α6 + β1 + 细胞中,纯种CD34(APC + )癌干细胞可以隔离。

    16. 我们已经成功地分离出活细胞进行RNA提取,恢复培养,并直接移植到受体小鼠中。根据初始肿瘤的大小,解离的效率和肿瘤的个体组成,我们通常获得25,000和250,000之间的活体,YFP +,/ sup,a6-整联蛋白来自肿瘤大约0.5cm 3的肿瘤细胞> b1-integrin + ,从该肿瘤群体中,我们通常获得2000-10,000个CD34 + / sup癌症干细胞。
      1. 对于RNA提取,将300至10,000个细胞直接收集到螺旋顶管中含有3.5μlβ-巯基乙醇的350μl裂解缓冲液中。分选后立即旋转管,并储存于-80°C直到RNA提取。我们更喜欢QIAGEN RNeasy Micro Kit,并使用试剂盒中的缓冲液RLT。
      2. 为了返回培养,在螺旋顶管中的500μl上皮细胞培养基中收集尽可能多的细胞。将离心管以200×g离心5分钟,并非常仔细地吸出上清液。重悬于上皮细胞培养基和培养板中。我们发现细胞直接在塑料上电镀时不能存活。我们更喜欢在照射的小鼠成纤维细胞上平板培养细胞以改善菌落形成和存活。我们发现,小于1000个细胞的电镀不允许细胞存活。
      3. 对于直接移植,在500μl上清细胞培养基中收集尽可能多的细胞,无螺旋顶管中没有血清。血清产生气泡,对30%Matrigel中的再悬浮产生负面影响。离心管,非常仔细地吸出上清液。重悬于30%的基质胶中,并将管保持在冰上直到移植(参见McCauley和Guasch [2013]了解原位移植的详细方案)。我们通过移植少至100个CD34 + 细胞成功地产生肿瘤。移植更多的细胞将加速肿瘤的形成

数据分析

  1. 因为罕见细胞的分离需要包含和排除许多标记物,所以必须具有未染色和单染色的对照以有效补偿光谱重叠。
  2. 上皮CD34 + 细胞的频率范围为7%〜22%。这是由于鳞状细胞癌从小鼠到小鼠的异质性。因此,建议在每个实验组内至少进行六次生物重复。

笔记

  1. 所有实验均由辛辛那提儿童医院研究基金会机构动物保护和使用委员会(协议号1D10087)批准,并符合欧盟和国家规定(协议号4572),并使用标准程序进行。
  2. 过滤器的胰蛋白酶化是一个重要的步骤,可以大大影响分离的CD34 + 癌症干细胞的效率,因为细胞可能保留在过滤器顶部的团块中,并被无意丢弃。确保使用预热的0.25%胰蛋白酶-EDTA,在37℃下孵育完整的10℃,并强力吸移胰蛋白酶处理的细胞,以确保适当的解离和最大量的细胞获得。
  3. 可以在4℃下将肿瘤样品储存在含有血清的培养基中过夜,并在第二天进行细胞分离。细胞死亡率将会增加,但是干细胞的总体百分比不会改变
  4. 需要测试FBS批次以获得上皮细胞的最佳生长。
  5. 因为上皮细胞对钙非常敏感,螯合FBS是至关重要的。
  6. 虽然可以使用100μm喷嘴收集分选的细胞,但我们建议使用130μm喷嘴来提高分类后大型上皮细胞的存活率。

食谱

  1. 20%来自溶组织梭菌的胶原酶
    1. 通过将1克粉末胶原酶溶解在5ml 1x无菌磷酸盐缓冲盐水中来制备20%的原料
    2. 将等分试样250μl放入Eppendorf管中,储存于-20°C,以避免反复冷冻和解冻,从而降低酶活性。
  2. 如(Nowak和Fuchs,2009)中所述制备的螯合胎牛血清(FBS)
    第1天
    1. 将400克干燥Chelex加入到带有搅拌棒的4L烧杯中。加入蒸馏水至总体积为4L。覆盖并连续搅拌
    2. 使用10N HCl将pH调节至7.4。搅拌20分钟,用10 N HCl调节pH,并根据需要重复,直至pH保持稳定20分钟以上
    3. 将烧杯放置在4℃过夜,以使Chelex形成紧凑的颗粒
    第2天
    1. 仔细倾倒H 2 O。将新鲜H 2亚O添加到4L
    2. 将pH调节至7.4,如第1天
    3. 将烧杯放置在4℃下1小时,以使Chelex形成紧凑的颗粒
    4. 仔细倾倒H 2 2 O
    5. 慢慢地将两个500毫升的特征或定义的胎牛血清瓶装入Chelex
    6. 在4℃缓慢搅拌1小时。尽量减少气泡
    7. 将烧杯放置在4℃下1小时,以使Chelex形成紧凑的颗粒
    8. 将血清倒入1升玻璃瓶中,并在无菌条件下通过Nalgene瓶顶过滤器过滤血清
    9. 将未使用的FBS储存在20°C或立即使用,使E介质不含钙
  3. 如(Nowak和Fuchs,2009)所述制备的上皮细胞培养基,
    1. 在6升锥形瓶中,将不含钙的六份Gibco Invitrogen定制的DMEM:F12(3:1)与蒸馏水混合,达到5.5L的最终体积。
    2. 加入18.42g碳酸氢钠,2.85g L-谷氨酰胺和60ml 100x青霉素 - 链霉素溶液
    3. 使用10N HCl将pH调节至7.2,并用H 2 O→/
      调节体积至6L
    4. 将压缩的CO 2 应用于介质15分钟。媒体应达到琥珀色
    5. 准备以下混合媒体添加剂:
      1. 20毫升5毫克/毫升胰岛素,溶解在0.1 N HCl中
      2. 20毫升5毫克/毫升转铁蛋白,溶于无菌PBS
      3. 将20ml 2×10 -8 -8 M T3溶于0.02N NaOH溶解至2×10 -4,然后进一步稀释至1×PBS中的最终浓度。
      4. 140 ml 1x PBS
      5. 过滤消毒并在-20°C下储存37.5 ml等分试样
    6. 加入上述混合物75毫升,750毫升10毫升霍奇金杆菌毒素(溶解于水中)和750毫升4毫克/毫升氢化可的松至1升螯合FBS
    7. 通过将850ml来自步骤2的DMEM:F12培养基与150ml补充的螯合FBS组合并使用Nalgene瓶顶过滤器进行消毒,生成最终15%的FBS培养基,1批次。
    8. 介质可以储存在250或500毫升的瓶子中,在20°C
  4. 染色缓冲液(2%FBS)
    1毫升chelexed FBS
    49 ml 1x PBS
    注意:为每次使用新鲜,并保持冰上。

致谢

解离肿瘤的方案是基于我们原来的方案,在"Molmol"(McCauley和Guasch,2013)中公开的方案进行了微小修改。制作低通FBS和E媒体的协议在这里缩写,但完全按照(Nowak和Fuchs,2009)中所述进行。流式细胞术方案的简要描述出现在"生物学"(McCauley等人,2017)中。我们要感谢辛辛那提儿童医院医疗中心风湿病研究流程细胞计数核心(由P30 DK07839部分支持)和法国CRCM流式细胞计数核心的协助。与这项工作相关的议定书得到了V基金会Sidney Kimmel基金会的资助,部分来自"基金委员会"。

参考

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Copyright McCauley and Guasch. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. McCauley, H. A. and Guasch, G. (2017). Isolation and Separation of Epithelial CD34+ Cancer Stem Cells from Tgfbr2-deficient Squamous Cell Carcinoma. Bio-protocol 7(17): e2524. DOI: 10.21769/BioProtoc.2524.
  2. McCauley, H. A., Chevrier, V., Birnbaum, D. and Guasch, G. (2017). De-repression of the RAC activator ELMO1 in cancer stem cells drives progression of TGFβ-deficient squamous cell carcinoma from transition zones. Elife 6.
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