Tumorsphere Derivation and Treatment from Primary Tumor Cells Isolated from Mouse Rhabdomyosarcomas

The housing, treatment, and sacrifice of mice were performed following the approved IACUC protocol of the Sanford Burnham Prebys Medical Discovery Institute.

1. Reagent preparation

1. Prepare 100 mL of cell isolation media: F10 medium supplemented with 10% horse serum (HS).
2. Prepare 50 mL of collagenase type II solution: dissolve 1 g of collagenase type II powder in 50 mL of cell isolation media (note the units of the enzyme per 1 mL of media, since the number of units changes depending on the lot). Aliquot the solution and store in a -20 °C freezer until ready for use.
   NOTE: Every lot of collagenase should be tested before use, since the enzyme activity may change across different lots.
3. Prepare 10 mL per sample of second digestion solution: cells isolation media with 100 units/mL of collagenase type II solution and 2 units/mL of dispase II freshly weighted. Prepare immediately before use.
4. Prepare 500 mL of tumor cells media: DMEM high glucose supplemented with 20% FBS and 1% pen/strep.
5. Prepare 500 mL of FACS buffer: 1x PBS supplemented with 2.5% v/v normal goat serum and 1 mM EDTA.
6. Prepare 500 mL of tumorsphere media: DMEM/F12 supplemented with 1% pen/strep. Just before use, add the following growth factors: 1% N2 supplement, 10 ng/mL EGF, and 10 ng/mL β-FGF.

2. Cell isolation and culture

1. Prepare a 10 cm plate containing 5 mL of cells isolation media (one plate per tumor sample) and place it in an incubator at 37 °C until ready to harvest the tumor tissue.
2. RMS have been reported to spontaneously develop in both male and female mouse models for Duchenne muscular dystrophy, such as B10 mdx mice at around 18 months of age and in B6 mdx/mTR mice by 9 months^22,23. Anesthetize the mouse that develops the RMS tumor using isoflurane, and sacrifice the animal through cervical dislocation or according to the IACUC guidelines of the institution. With scissors, make an incision on the skin in the area where the tumor is localized, and (using tweezers) pull the skin away from the area of interest. Employing a razor blade, excise the tumor from the animal.
3. Weigh 500–1,000 mg of the tumor tissue and place it in the plate prepared in step 2.1 (Figure 1A).
   NOTE: Larger amounts of tissue negatively affect the digestion steps and decrease the overall yield. If the harvested tumor is larger than 1000 mg, divide it into parts and sample randomly until the desired weight is reached. Random sampling is necessary for evaluating tissue heterogeneity.
4. Place the plate containing tumor tissues in the sterile cell culture hood and mince it with a razor blade. Tumor tissue from RMS is heterogeneous; thus, different areas may present different resistance to the cuts. Make sure that the sizes of the resulting minced pieces are uniform to ensure optimal digestion.
   NOTE: RMS exists as mixtures of different tissue types (mainly fibrotic, vascularized, and fatty tissues) that can be identified in every tumor. To 1) isolate a heterogeneous cell population accurately recapitulating the composition of the original tumor and 2) not bias the isolation procedure, perform random sampling of the harvested tissue. Cells from different areas of the tumor should be digested and tested in parallel in vitro in the assay described in section 3.
5. Move the minced tissue and cell isolation media in a 15 mL centrifuge tube, wash the plate with 4 mL of cell isolation media and place it in the tube.
6. Add 700 units/mL of collagenase solution to digest the tissue and incubate in a shaking water bath at 37 °C for 1.5 h.
7. After incubation, spin down the tissue at 300 x g for 5 min at RT. Aspirate the supernatant without disturbing the pellet, resuspend the pellet in 10 mL of second digestion solution (dispase), and incubate in a shaking water bath at 37 °C for 30 min.
8. Once the second digestion is completed, pipet up and down and pass the cell suspension through a 70 μm nylon filter on a 50 mL centrifuge tube. Then add 10 mL of cell isolation media to wash the filter and dilute the digestion solution, and spin down the tissue at 300 x g and RT for 5 min.
9. Aspirate the supernatant and resuspend the pellet in 20 mL of tumor cells media. Then transfer the cell suspension in a 15 cm cell culture plate. Place the cells in the incubator at 37 °C overnight. This plate will be identified as P0.
10. The day after isolation, change the media. This step is necessary for ensuring removal of debris and dead cells that may negatively influence cell survival.
11. Assess cell confluency after media is changed, which ranges from 30%–60% depending on the amount of starting material and cell size. Leave the cells growing in the incubator until they reach 90% confluency. Monitor cells every day and change media every 2 days. The time necessary for tumor cells to become confluent varies depending on multiple parameters: tumor aggressiveness, genotype of the tumor, age of the mouse, heterogeneity of the tissue.
12. For cell passaging, do the following:
    1. Pre-warm the cell detachment solution and tumor cells media in a water bath at 37 °C.
    2. Wash the cells with 1x sterile PBS and incubate them at 37 °C in 10 mL of warm cell detachment solution for 5–10 min.
    3. When all the cells are detached from the plate, add 10 mL of warm tumor cells media, move the solution into a 50 mL centrifuge tube and spin cells down at 300 x g for 5 min at RT.
    4. Resuspend the cells in 5–10 mL of tumor cells media, depending on the pellet size, and count live cells using Trypan blue (1:5 dilution) to exclude dead cells.
    5. Plate 10⁵ cells in 10 cm plates or 3 x 10⁵ cells in 15 cm plates. Cell doubling time varies depending on the factors detailed in step 2.10.

3. Tumorsphere derivation

1. Use tumor cells at passage P1 or P2 to avoid cell selection through multiple passages (Figure 1B). To detach cells from the plate, first wash the dish with 1x PBS, without disturbing the cells, then cover them using cell detachment solution (5 mL for a 10 cm plate or 10 mL for a 15 cm plate) and place them in the incubator for 5–10 min.
2. Confirm that cells are detached by looking at the plate under a bright-field microscope, add 1:1 volume of tumor cells media (cell detachment solution:tumor cells media), place the cell suspension in a centrifuge tube and spin the cells down at 300 x g for 5 min at RT.
3. Resuspend cells in either FACS buffer (section 3.4) or tumorspheres media (section 3.5), according to the method used for plating.
4. Plating cells through flow cytometer
   1. Resuspend cells in FACS buffer (the amount depends on the pellet size) and manually count live cells using Trypan blue exclusion. Make sure that the final cell concentration is 10⁷ cells/mL (100 μL of FACS buffer per 10⁶ cells). Add 1 μL of Fx Cycle Violet stain per 10⁶ cells to discriminate live from dead cells during cell sorting. Prepare an unstained control in which Fx Cycle Violet stain is not added to the cells.
      NOTE: The concentration is optimized for efficient staining and speed during sorting. A lower cell concentration will result in a longer sorting time, while a higher concentration will affect staining.
   2. Employing the unstained control, set up a FACS gate segregating alive (Fx Cycle Violet^–) from dead (Fx Cycle Violet⁺) cells. Then, employ fluorescent-activated cell sorting (with 450/50 filter band pass) for separation and count of live/dead cells and for plating the desired number of live cells in each well of the 96 well low-attachment plates. Each well of the plate needs to be filled with 200 μL of tumorspheres media before starting the sorting.
      NOTE: Given that TPCs are a rare subpopulation within the whole tumor, optimize the protocol by plating 100 cells/well from mouse RMS to observe the formation of tumorspheres in suspension culture. The cell number per well should be adjusted for the specific tumor tested.
   3. Place cells in the incubator until the end of the experiment. Try not to disturb the plate unless necessary. For a 30 day experiment, each well should be replenished with media and an appropriate proportion of growth factors every week (media tend to evaporate and growth factors are not effective after 1 week).
   4. After completion of the experiment, manually screen plates under a bright-field microscope to identify tumorspheres (see step 3.6).
      NOTE: A timepoint of 30 days was optimized to easily detect mouse RMS tumorspheres of sizes ranging from 50–300 μm of diameter. The timepoint should be adjusted depending on the aggressiveness of the tumor tested and its proliferative rate.
5. Plating cells manually
   1. Resuspend cells in tumorsphere media (the amount depends on the pellet) and manually count live cells using trypan blue (1:10 dilution). Calculate the cell concentration in the tube and plate the proper number of cells in a 96 well low attachment plate. Place cells in the incubator until the end of the experiment. Try not to disturb the plate unless necessary for replenishing media and growth factors, as described in step 3.4.3.
   2. After completion of the experiment, screen plates manually under a bright-field microscope to identify tumorspheres or using the Celigo software, as previously described in Kessel et al.²⁴ See step 3.6 below.
6. Note that two separate readouts can be evaluated as result of this assay: number and size of formed tumorspheres.
   NOTE: When more than one cell is plated in a well, either tumorspheres or cell clusters can form (Figure 1C, third and fourth panels). Cell clusters are small cellular aggregates that form in suspension culture that enhance cell survival and are characterized by an irregular shape. Tumorspheres are large and have a more compact structure with a spheroid shape. They derive from a single cell that has the ability to survive in an anchorage-independent manner and proliferate at a high rate²⁵. Plating cells through flow cytometer or manually can be used interchangeably, depending on the capabilities available in the laboratory. Moreover, employing low-attachment plates of sized different than 96 well plates is possible and dependent on the required outcome. Indeed, assessment of tumorsphere frequency should be done employing 96 well low attachment plates, whereas initial screening for assessment of cells tumorigenic potential will yield faster and reliable results on 6 well low-attachment plates.

4. Tumorsphere treatment with recombinant proteins

1. Repeat steps 3.1 and 3.2.
2. If setting up a treatment with recombinant proteins, first determine the best concentration to use following section 4.3, or if the optimal concentration has been previously determined, skip to section 4.4.
3. Determine the recombinant protein treatment concentration.
   1. Resuspend the cells in tumorsphere media (the volume depends on pellet size) and manually count live cells using Trypan blue exclusion. Calculate cell concentration in the tube and plate 100,000 cells in a 6 well low attachment plate. Plate two wells per each concentration tested and two wells for untreated controls (Figure 2). Protein concentrations to be tested are based on literature search.
   2. Treat each well of suspension cells with a different protein concentration and place the cells in the incubator for 48 h (time necessary to be able to evaluate the effect of the treatment on both cell viability and on the expression of downstream target genes). Then, assess the following parameters:
      1. Cell survival: using a bright-field microscope as well as comparison with untreated controls, check cell morphology. Healthy cells appear bright and reflective under the microscope, while excessive cell death will induce the accumulation of debris in the media. For a quantifiable determination of cell death, Trypan blue exclusion, crystal violet staining, MTT, or TUNEL assay can be employed (in this case, add one well to the original plating).
      2. Effect of recombinant proteins on downstream pathways: perform a literature search using PubMed for identification of the downstream genes known to be affected by the tested protein. Design qRT-PCR primers for the target genes, and perform qRT-PCR analysis on the RNA isolated from the treated cells (Figure 2).
4. Treat with recombinant protein.
   1. Resuspend the cells in tumorsphere media (the volume depends on pellet size) and manually count live cells using Trypan blue exclusion. Determine the total number of cells needed for the desired experiment (100 cells per each well of a 96 well low attachment plate) and dilute them in the appropriate tumorsphere media volume. If performing more than one treatment, prepare separate cell tubes.
   2. Treat each tube with the appropriate concentration of recombinant protein and plate the cells in a separate well of 96 well low-attachment plate. The treatment will be repeated on each well depending on the half-life of the recombinant protein until the 30 day endpoint of the experiment.
   3. At the end of the experiment, follow steps 3.4.4 and 3.6 to analyze the data.

5. Tumorsphere treatment with overexpression plasmids

1. Repeat steps 3.1 and 3.2.
2. If setting up the treatment on a new tumor type, first determine the best concentration of plasmid to use following section 5.3, or if the optimal DNA concentration has been previously determined, skip to section 5.4.
3. Determine optimal plasmid concentration.
   1. Resuspend cells in tumor cells media (the volume depends on pellet size) and manually count live cells using Trypan blue exclusion. Plate the counted cells to achieve a confluency from 70%–90% (cell number is highly dependent in cell size and morphology). GFP-plasmid will be employed to test transfection efficiency following the manufacturer protocol of the transfection reagent for adherent cells. In parallel, also test an untreated control (Figure 3A). Perform an efficiency test in a 24 well plate.
      NOTE: Adherent cells are used to enhance the transfection efficiency, as transfection performed on suspension cells is not efficient and negatively affects cell viability.
   2. 48 h after transfection assess cells for the following parameters:
      1. Cell survival: using a bright-field microscope, compare the number of cells present in each-well and compare it to the untreated cells well.
      2. GFP expression: count the percentage of cells that are GFP positive over the total number of cells in each well (Figure 3B).
4. Overexpression plasmid treatment
   1. Resuspend the cells in tumor cell media (the volume depends on pellet size) and manually count live cells using Trypan blue exclusion. Plate 200,000 cells per well of a 6 well plate. Each well will be used for an independent transfection event. Each well will be transfected using the set-up developed for the specific tumor type (Figure 3A).
   2. 24 h after transfection, wash each well with 1x PBS and incubate the cells in warm cell detachment solution (enough to cover the well). Place the plate in the incubator at 37 °C for 5–10 min, depending on factors detailed in section 2.15.
   3. When cells are detached, count the cells derived from each single well independently using Trypan blue exclusion. Place 100,000 cells per well of a 6 well low attachment plate, making sure not to mix cells derived from different wells. Place the plate in the incubator at 37 °C and leave undisturbed for a week.
      NOTE: The duration of this assay is 7 days, a sufficient time to allow tumorsphere formation while preventing tumorsphere fusion. Tumorsphere fusion is a phenomenon that becomes evident when 100,000 or more cells are plated together in suspension for over 1 week, which can bias the assessment of tumorsphere formation ability. In the event that the experiment requires longer incubation time, cell density should be adjusted or polymeric scaffolds used to avoid tumorsphere fusion²⁶.
   4. At the end of the experiment, follow steps 3.5.2 and 3.6 to analyze the data.

6. Tumorsphere preparation for allograft transplantation

1. Place extracellular matrix (ECM) solution (50 μL per allograft) and tumor cells media (50 μL per allograft) on ice.
2. Tumorspheres can be used for allograft transplantations. Pull together all the tumorspheres obtained from a specific cell type or treatment into a 15 mL or 50 mL tube (according to the total volume of media) (Figure 1D). Spin the tumorspheres down at 300 x g for 5 min at RT. Remove the supernatant over the tumorspheres and wash them with 10 mL of sterile 1x PBS.
3. Spin the tumorspheres down again at 300 x g for 5 min at RT, aspirate the 1x PBS, and add 500 μL to 1 mL of cell detachment solution on top of the cell pellet, which starts the dissociation process. Incubate tumorspheres in digestive solution into a shaking water bath at 37 °C and check the progression of the digestion every 10 min. To help tumorspheres dissociation into a single cell solution, pipette the cells up and down for mechanical disruption. The digestion process might take up to 30 min.
   NOTE: Despite the fact that incubation time varies considerably when tumorspheres are derived from different primary RMS cells, a significant decrease in cell viability in relationship to digestion time was never observed.
4. When a single cell solution is obtained, add a volume of tumor cells media (1:1, cell detachment solution: tumor cells media) and spin it down at 300 x g for 5 min at 4 °C.
   NOTE: From this time on, all the steps need to be performed on ice. After spinning, tumorspheres do not form a stable pellet as the single cells solutions do. To make sure not to dislodge or aspirate the tumorspheres, use a 1 mL pipette and gently aspirate the liquid. Move to a 200 μL pipette when only 1 mL remains.
5. Resuspend cells in cold tumor cells media (the volume will depend on pellet size), place the cells on ice and count live cells using Trypan blue exclusion. After determining the proper amount of cells to use for the allograft, resuspend them in a total volume of 50 μL of cold tumor cells media. Cool down a pipette tip in cold tumor cells media. When the tip is cold, use it to take 50 μL of ECM solution and add it to the tube containing the cells. Do not remove the tubes from the ice during this process.
   NOTE: The number of cells to use for the transplant should be determined according to the tumor tested and experimental goal: a larger number of transplanted cells will decrease the time of tumor development (20,000 cells from mouse RMS tumorspheres have been shown to develop into a tumor 6 weeks after injection). To be able to compare different cell lines or different treatments, it is important to start from the same number of cells.
6. As cells are now ready for transplantation, maintain on ice until injection. Place a capped 0.5 mL insulin syringe with a 29 G needle on ice, to prevent the cell solution from becoming solid upon aspiration.
7. Turn on the flowmeter to 200 mL/min oxygen and the isoflurane vaporizer to 2.5%. Anesthetize a 2 month-old male NOD/SCID mouse by placing it inside the induction chamber. Wait 2–3 min until the mouse appears asleep and the breeding has slowed down. Before starting the procedure, first confirm through a foot pinch that the mouse is asleep, then apply vet ointment on the eyes. Shave the right side of the animal, aspirate the cell solution in the pre-cooled syringe, and inject them subcutaneously into the shaved area. A visible bump under the skin will form if the injection is performed correctly.
   NOTE: Cell allografts can be performed in the same mouse strain as that of the transplanted cells. For example, if the RMS cells were originally isolated from a C57BL/6 mouse, the allograft can be performed in C57BL/6 mice. If the strain is different, then immunodeficient recipient mice should be utilized to avoid rejection. The ages of recipient mice can also be adjusted depending on the experimental goal.
8. Monitor the mice for tumor formation once per week.
   NOTE: To validate identity of the tumor derived from allograft transplantation, it should be compared to the original tumor from which the cells were isolated. To this aim, histological analysis for morphological features and expression of myogenic markers as well as more comprehensive RNAseq can be performed.