Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Human PDAC tumors were obtained with written informed consent (Comunidad de Madrid, Spain (C.P. CNIO-CTC-11 – C.I. 11/103-E). Implantation of human pancreatic tumors into immunodeficient mice requires the approval of Institutional Review Board as well as Institutional Animal Care and Use Committee (IACUC). The procedures of xenograft pancreatic tumor mouse models must be conducted in accordance with the institutional and national regulations (here Ethics Committee of the Instituto de Salud Carlos III; Madrid, Spain; Protocol PA 34_2012).

1. Culture Media

1. Prepare Complete Medium.
   1. Supplement RPMI medium with 10% FBS, 100 units/ml penicillin/streptomycin. For 500 ml of RPMI add 55 ml of heat-inactivated FBS and 6 ml of penicillin/streptomycin (10,000 U/ml, 100X).
   2. Store the complete medium at 4 °C.
2. Prepare CSCs Medium.
   1. Supplement the serum-free DMEM/F12 medium with 100 units/ml penicillin/streptomycin and 2 mM glutamine.
   2. Store the CSC medium at 4 °C. B27 (1:50) and 20 ng/ml bFGF are freshly added to the medium before each experiment.
      NOTE: The addition of B27 has been shown to increase tumor sphere formation and to sustain several passages of sphere cultures ³². The medium composition is a crucial step and has to be tested for each cell type in culture. We recommend testing self-renewal and differentiation capacity of spheres and analyze expression of CSC markers by flow cytometry (i.e., CD133+ and CD44+).
3. Prepare Extracellular Flux Assay Medium.
   1. This specific medium is basal DMEM 5030 containing vitamins, aminoacids and other supplements, but lacking glutamine, glucose, pyruvate and bicarbonate.
   2. For the current assay, supplement the medium with 2 mM glutamine, 8 mM glucose and 2 mM sodium pyruvate to a full mitochondrial metabolic activity.
      NOTE: The absence of sodium bicarbonate is essential in order to accurately measure extracellular acidification of cells growing in culture, since regular medium containing bicarbonate will buffer pH variations during the assay. In addition, the use of a basal medium allows a tight control of the concentration of L-glutamine (as L-alanyl-glutamine) glucose, or sodium pyruvate necessary for different assay conditions and/or applications.

2. Sphere Forming Assay and Analysis

NOTE: All tissue culture protocols and manipulations must be performed using sterile techniques with great attention using clean, detergent-free, sterile glassware. Before use, pre-warm all medium and solution in a 37 °C water-bath (as an alternative, you can use medium and solutions pre-warmed at RT). Obtain Human PDAC tumors as previously detailed ²¹.

1. Isolation of CSCs from Human PDAC (Figure 1)
   1. In a sterile biosafety cabinet transfer the human PDAC tissue to a 60 x 15 mm culture dish containing 1 ml of sterile 1X phosphate buffered saline (PBS). Mince the tumor into small pieces (1 – 5 mm) using a sterile scalpel and forceps.
   2. Add 1 ml of sterile 1X PBS to the culture dish and repeat the trituration step until the tissue is completely dissociated, regularly this requires 3-4 rounds. Transfer the tissue suspension (top up to a final volume of 5 ml with 1X PBS) into a sterile tube and mechanically homogenize it with a dissociator such as gentleMACS.
   3. Incubate the homogenized tissue with collagenase (use 2.5 mg/ml of collagenase in 1X PBS) for 60 min at 37  °C and then centrifuge for 5 min at 900 x g. Decant the supernatant and resuspend the cell pellets in 10 ml of complete medium. Filter the cell suspension through a 40 µm strainer and centrifuge for 5 min at 900 x g.
   4. Decant the supernatant and resuspend the pellet with 5 ml of red blood cell lysis buffer (ammonium-chloride-potassium, ACK), and incubate the
   5. Resuspend the pellet in CSCs medium, plate on a gelatin-coated dish, and incubate at 37 °C for 1 h. This step will remove most of the fibroblast cells that quickly attach to the plate.
   6. Remove the plate from the incubator and carefully recover cell suspension and quantify the number of viable (trypan blue-negative) cells using a hemocytometer. For this purpose, gently mix the suspension and pipette 20 µl of the suspension into an Eppendorf tube. Add 20 µl (1:1 ratio) of trypan blue to the cells in the microcentrifuge tube and mix well. Pipette approximately 10 µl of the mixture onto the hemocytometer.
   7. Count all clear cells within the four corner quadrants of the counting chamber for viable cell count. Note: The viability and yield of cells may vary considerably between tumor specimens. For PDAC samples viability regularly ranges between 45-70%, and tissue pieces from a macroscopic tumor sample with a diameter of 3 – 5 mm should yield to approximately 5×10⁶ viable cells.
2. Sphere Formation Assay and Metformin Treatment
   1. Take the required number of cells and add the appropriate volume of CSCs medium to prepare a cell concentration of 2,000 cells/ml. Do not keep the cell suspension on ice for no longer than 1h and mixed well prior to plating.
   2. Add 500 µl of 1X PBS to the first and last row of a 24-well plate for humidification in order to help minimize medium evaporation. Seed the cells into ultra-low attachment cells plates at a density of 2,000 cells per well in 1 ml of tumor sphere medium (2,000 cells/ml).
      ​NOTE: The number of cells used for tumor sphere formation assays may vary between tumor types.
   3. Treat at least 4 wells with vehicle (negative control) and 4 wells with each allocated treatment, e.g., 3 mM of metformin. Place the cells in an incubator set to 37 °C and supply the cells with 5% CO₂ for one week.
      NOTE: The medium should not be changed in order to allow the undisturbed formation of tumor spheres, but can be topped up with growth factors on a daily basis as they are not stable in culture medium.
   4. Every other day add treatment, e.g., 3 mM of metformin or vehicle to each of the wells. Assess the number of formed tumor spheres after 5 and/or 7 days by the use of an automated cell counter that allows for counting of larger structures (Figure 2).
      NOTE: Only tumor spheres with at least 40 µm should be considered and can be categorized as small (40 – 80 µm) or large (80 – 120 µm), respectively. The results can be illustrated as the percentage of tumor spheres divided by the initial number of cells seeded (e.g., 2,000 cells).
3. Serial Passaging of First Generation Tumor Spheres
   1. After 7 days of incubation harvest the tumor spheres using a 40 µm cell strainer and centrifuge them for 5 min at 900 x g at RT.
   2. Dissociate the pellet of tumor spheres to single cells using trypsin, and then expand the obtained single cell cells suspension again for another 7 days as described above (see 2.2).

3. Metabolism Analysis (ROS Production and Oxygen Consumption)

1. ROS Production
   1. In this protocol, we have used carboxy-DCFDA as an example to measure ROS production since it is an affordable, fast and widely used probe for ROS detection. However, there are several other probes and methodologies that can be used for this assay.
   2. Treat tumor spheres with metformin as described in 2.2 for the allocated amount of time. At the end of treatment, harvest tumor spheres using a 40 µm cell strainer, centrifuge the cells at 900 x g for 5 min and resuspend the pellet in 1 ml of trypsin. Incubate for 20 min at 37 °C.
   3. Once cells are singularized, centrifuge at 900 x g and resuspend the cells in HBSS containing 2.5 μM carboxy-DCFDA. Incubate the cells for 20 min at 37 °C in the dark.
   4. Prior to flow cytometry analysis protect stained cells from light as carboxy-DCFDA can be photo-oxidized giving false positive results. Place tubes on ice until analysis.
      NOTE: carboxy-DCFDA is fluorescent after reaction with a variety of reactive oxygen and nitrogen species, being detected in FL1 (ex. 488nm, em. 520nm).
2. Oxygen Consumption Measurement
   1. For this protocol, we will use the Extracellular Flux Analyzer system from Seahorse Biosciences, as an example. Coat the desired cell culture plate with a solution of 22.4 μg/ml of cell and tissue adhesive for 20 min at RT. Rinse the wells with water 2 – 3 times prior to seeding the cells.
   2. At the end of treatment, harvest tumor spheres using a 40 µm cell strainer, centrifuge the cells at 900 x g for 5 min and resuspend the pellet in 1 ml of trypsin. Incubate for 20 min at 37 °C.
   3. Plate singularized cells in the cell culture plate at a density of 30,000 cells/well for a 96 well plate (final volume: 150 µl). Resuspend the cells in Oxygen consumption assay medium containing 8 mM glucose and 2 mM sodium pyruvate.
   4. Centrifuge the cells to the bottom of the well by spin centrifugation of the wells until 100 x g has been reached and then let the centrifuge stop with brake off. Reverse the orientation of the plate and repeat the process. Transfer the plate to a 37 °C incubator without addition of CO₂ for 30 min.
   5. In the meantime, prepare the cartridge for the assay. Each well has 4 different injectors to load (25 µl/port):
      1. Port A: metformin. To obtain a final concentration of 3 mM in the well, prepare a 8x solution (24 mM) as the final volume will be 175 µl after injection of port A.
      2. Port B: metformin. To add 3 mM and obtain a final concentration of 6 mM in the well, prepare a 9x solution (27 mM) as the final volume will be 200 µl after injection of port B.
      3. Port C: metformin. To add 3 mM and obtain a final concentration of 9 mM in the well, prepare a 10x solution (30 mM) as the final volume will be 225 µl after injection of port C.
      4. Port D: rotenone 11 µM, to obtain 1µM as final concentration in the well (11x). Note: rotenone completely inhibits any residual mitochondrial activity.
   6. Calibrate the cartridge and load the cell culture plate. Perform the assay with the standard protocol of mixing and measurement.
   7. Calculate the percentage of inhibition of the total mitochondrial respiration achieved by metformin as the percentage of inhibition obtained with rotenone (set as 100%).

4. Xenograft Model for Human Pancreatic Cancer in Immunocompromised Mice

NOTE: Order sufficient numbers of female athymic nude or other, more immunocompromised mouse models such as NOD-SCID, SCID-Beige, or NSG for the experiments ³³. Autoclave all surgical instruments prior to the experiment and allow them to cool down to RT before use. For subcutaneous tumors we used a minimum of 4 mice per condition with one tumor per flank for a total of 8 tumors.

1. Xenograft Transplantation
   1. Prepare tumor pieces from human pancreatic cancer tissue specimens. Transfer the tumor into a Petri dish and dissect the tissue into small pieces (2 mm in diameter, ~8 mm³) using a scalpel and forceps to hold the tissue. Dip the obtained pieces into gelatinous protein mixture solution kept on ice.
   2. Anesthetize mice using 1 – 3% isofluorane or other inhalative or injectable anesthetics.
      NOTE: Before starting any surgical procedure, ensure that the mice have completely lost consciousness by stimulating the abdominal skin or tows with a pair of splinter forceps. Apply eye ointment to prevent dryness.
   3. Once anesthesia has taken effect, wipe the back of the mice with ethanol-containing skin antiseptic. Administer buprenorphine (0.05 mg/kg BW) prior to surgery to ensure post-operative analgesia.
   4. Lift the back skin with forceps, make a 0.7 – 1.0 cm long incision with sterile micro scissors and then bluntly prepare a small subcutaneous pocket, in which a piece of patient-derived pancreatic cancer tissue is inserted (~8 mm³).
   5. Close the wound using 1 – 2 skin staples. Remove them after 7 days. Return the mice to their cages and keep them on a heating pad or under a heating lamp until they are fully active again.
   6. Following tumor implantation, monitor the mice at least twice weekly for tumor growth and general signs of arising morbidity such as ruffled fur, hunched posture, and immobility.
   7. Once tumors have reached an average size of 200 mm³, mice are randomized to respective treatments. Administer vehicle or metformin daily via i.p. injections (150 mg/kg BW) or via the drinking water (150 mg/kg BW) with comparable treatment effects.
   8. Monitor tumor burden using a caliper and calculate tumor volume once a week (measurement of tumor volume = 1/2 length × breath × width).
   9. Sacrifice the mice once tumors have reached 1 cm in diameter or mice start showing any signs of severe pain or illness as specified above.