Modelling Intratumoral Treatment with miRNA Inhibitors: A Method to Analyze the Effect of miRNA Inhibitors on Tumor Growth in Murine Model

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Flank Inoculation of Cells and Intratumoral antagomiR Treatment

1. Cell preparation
   1. Engineer a Cal62 human thyroid cancer cell line (KRASG12R and p53A161D mutations) to overexpress a transgenic construct that constitutively expresses GFP and luciferase (CMV-Firefly Luc-IRES-eGFP). Select the transgenic cells by antibiotic resistance or by sorting the GFP-positive cells with flow cytometry.
   2. Grow the cells in Dulbecco's modified Eagle's medium (DMEM) supplemented with penicillin, streptomycin, and 10% fetal bovine serum (FBS) at 37 °C and 5% CO₂.
   3. Suspend 1 x 10⁶ cells in 50 μL of phosphate-buffered saline (PBS) at 4 °C.
2. Cell inoculation into the flanks of mice
   1. Mix the cells with the same amount of basement membrane matrix. For example, add 1 x 10⁶ cells in 50 μL of PBS to 50 μL of basement membrane matrix (see Table of Materials) and mix gently.
   2. Inject 100 μL of the sample (cells in PBS + basement membrane matrix) subcutaneously into the left flank of 6-week-old immunodeficient BALB/c nu/nu mice using a 1 mL insulin syringe with a 27G 1/2'' (0.4×13 mm) needle.
3. Intratumoral antagomiR treatment
   1. Suspend the antagomiR (see Table of Materials) or the negative control in 500 μL of RNase-free distilled water.
   2. Prior to the injection, prepare 2 nmol of the antagomiR or the control together with an in vivo delivery reagent (see Table of Materials) for each injection.
      1. First mix the antagomiR solution (see Table of Materials) and the complexation buffer (see Table of Materials) in a 1:1 ratio. For example, add 80 μL of miRNA-inhibitor solution (16 nmol) to 80 μL of complexation buffer (included in the in vivo delivery reagent kit, see Table of Materials).
      2. Bring the in vivo delivery reagent to room temperature. Add 160 μL to a 1.5 mL tube and immediately add 160 μL of diluted antagomiR solution. Return the remaining reagent to -20 °C. If necessary, store the reagent at 4 °C for up to one week after thawing.
      3. Vortex immediately (10 s) to ensure complexation of the in vivo delivery reagent-antagomiR.
      4. Incubate the in vivo delivery reagent-antagomiR mixture for 30 min at 50 °C. Centrifuge the tube briefly to recover the sample.
      5. Dilute the complex 6-fold by adding 1360 μL of PBS pH 7.4 and mix well.
      6. Proceed with in vivo delivery of the reagent-antagomiR complex (8 mice/condition) or store the complex at 4 °C for up to one week prior to injection. Inject 200 μL intratumorally into each tumor (2 nmol of antagomiR).
         NOTE: The volume and quantity of the antagomiR is independent of the tumor volume.
   3. Perform the treatment 3 times each week (Monday, Wednesday, and Friday) for 2 weeks.
4. Analysis of tumor growth
   1. Inject 50 μL of a 40 mg/mL solution of D-luciferin substrate (see Table of Materials) subcutaneously at each time point with a 1 mL syringe with a 27G 1/2'' (0.4 mm x 13 mm) needle.
      1. At 8 min post injection, anesthetize the mice using 3% isoflurane mixed with oxygen. Assess the level of anesthesia by pedal reflex (firm toe pinch) and adjust anesthetic delivery as appropriate to maintain surgical plane.
      2. Apply ophthalmic ointment to both eyes to prevent desiccation.
5. Image the bioluminescent signal with in vivo imaging software (see Table of Materials).
   NOTE: Calipers can also be used to measure the tumor volume and tumor growth.
6. Once the bioluminescence signals are obtained, analyze the tumoral growth comparing both treatments and determine the significance by using a t-test. To analyze the within-group variance use the SEM.