In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice

This protocol describes xenograft and orthotopic mouse models of human thyroid tumorigenesis as a platform to test microRNA-based inhibitor treatments. This approach is ideal to study the function of non-coding RNAs and their potential as new therapeutic targets.

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We perform the first therapy, based in a microRNA inhibitor in a thyroid cancer model. These developing therapies show promise for the treatment of this disease. This type of orthotopic model, using a systemic route of treatment delivery, facilitates the validation of a new microRNA-based drugs.

Although we use an orthotopic model for implanting human thyroid cancer cells into mouse thyroids, these models could be all systemic for other cancer types. Demonstrating the procedure with Julia Ramirez-Moya and Adrian Acuna will be Raquel Arocha Riesco. She's a technician from our institute.

After establishing a CAL-62 human thyroid cancer cell line incomplete medium, suspend a 1 times 10 to the 6th cell aliquot in 50 microliters of PBS at 4 degrees celsius, and mix the cells with an equal volume of basement membrane matrix. Load the cells into a 1 milliliter syringe, equipped with a 27 gage half-inch needle, and subcutaneously inject 100 microliters of the sample into the left flank of a 6 week old immunodeficient valve C nude mouse. Two weeks after the injection, add the antagomiR or controlled treatment buffer solution to 160 microliters of room temperature in vivo delivery reagent in a 1 point 5 milliliter tube.

And immediately vortex the solution for 10 seconds to ensure complexation of the mixture. Incubate the treatment solution for 30 minutes at 50 degrees celsius, followed by a brief centrifugation in a micro centrifuge. Then, dilute the sedimented treatment complex sixfold with fresh PBS and thorough mixing.

And inject the entire 200 microliter volume of the treatment directly into the tumor. Inject 50 microliters of a 40 milligrams per liter D-Luciferin substrate solution subcutaneously 2 times a week, into each experimental animal. Making sure to confirm a lack of response to toe pinch after isoflurane anesthesia.

Place the animal in the chamber of an in vivo bioluminescence imaging system, and image the bioluminescence signal with the in vivo imaging software according to standard protocols. Then analyze the tumor growth. Comparing both treatments and determining the significance and growth differences using a T-test.

For orthotopic thyroid tumor cell inoculation suspend an aliquot of CAL-62 cells in 5 microliters of PBS, and subcutaneously inject 100 microliters of analgesic and 100 microliters of antibiotic into a 7 week old valve C nude mouse. After confirming a lack response to toe pinch, place the animal under a dissecting microscope and disinfect the neck of the animal with iodopovidone. Next, make an approximately 2 centimeter incision in the skin, and displace the salivary glans to expose the neck.

Use dissection forceps, and/or scissors, to dissect the strap muscles to expose the trachea and thyroid gland, and use a 10 microliter syringe, to inject the 5 microliter volume of tumor cells into the right thyroid lobule, located at the side of the cricoid cartilage. When all of the cells have been delivered, reposition the salivary glans, and use silk braided, coated, non absorbable sutures to close the incision. Then apply iodopovidone to the wound area, and place the mouse on a thermic blanket with monitoring until full recovery.

two to three weeks after the intrathyroid cell injection, prepare the treatment solution as demonstrated, and deliver the solution intravenously by retro-orbital injection of the venous sinus of the anesthetized thyroid tumor-bearing animal. In this representative experiment, the growth of tumors intratumorally injected without microRNA 146B inhibitor, was significantly suppressed with respect to the negative control. Intratumoral expression levels of some proliferation markers were also observed in low levels, in antagomiR treated tumors, compared to in control tumors.

In addition, the recovery of the micro-RNA targets can be studied through the analysis of tumor extracted RNA, or protein. Collectively revealing that the in vivo inhibition of individual micro RNA's is effective and may be exploited therapeutically for thyroid cancer treatments. Histological analysis of thyroid tumors established in mice as demonstrated, allows staining for the epithelial cells surrounding the tumor, revealing the thyroid follicle architecture of the tumor tissue.

Notably, the intravenous injection of microRNA 146B inhibitor into mice, with an established tumor, results in a significant decrease in the tumor volume compared to control-treated animals. Further, the expression of the newly described microRNA 146B target DICER1 in the primary tumor, increases after anti micro-RNA 146B treatment, further underscoring the potential of the inhibition of indogenous micro-RNA expression and therefore the restoration of it's target genes as a therapy in thyroid cancer. This technique, and the subsequent lipin results, open the possibility of exploring new therapies based on non-curing RNA's for the treatment of thyroid cancer.

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Journal (Cancer Research)

Journal (Cancer Research)

In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice

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