We are grateful to Raquel Arocha Riesco for her assistance with the treatment and care of mice. We thank Dr. J. Blanco (Catalonian Institute for Advance Chemistry-CSIC) and Dr E. Mato (Institut de Reserca de l&#8217;Hospital de la Santa Creu i Sant Pau) Barcelona (Spain) for gifting the CMV-Firefly luc- IRES-EGFP and Cal62-Luc cells, respectively.&#160;Funding:&#160; SAF2016-75531-R (MICIU), Fondo Europeo de Desarrollo Regional FEDER, B2017/BMD-3724 (Comunidad de Madrid), and GCB14142311CRES (Fundaci&#243;n Espa&#241;ola contra el C&#225;ncer, AECC).

Animal experimentation was performed in compliance with the European Community Law (86/609/EEC) and the Spanish law (R.D. 1201/2005), with approval of the Ethics Committee of the Consejo Superior de Investigaciones Cient&#237;ficas (CSIC, Spain).
1. Flank inoculation of cells and intratumoral antagomiR treatment
<ol>
	<li>Cell preparation
	<ol>
		<li>Engineer a Cal62 human thyroid cancer cell line (KRASG12R and p53A161D mutations) to overexpress a trans...

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The authors have nothing to disclose.

This paper describes a method for studying the in vivo function of a miRNA in order to better understand its role in tumor initiation and progression, and its potential as a therapeutic target in thyroid cancer. The tumor xenograft models here described are based on the use of cells that can be tracked by their bioluminescence signal, permitting the measurement of tumor growth in vivo under the influence of a treatment. In addition, we describe the use of a miRNA-based treatment for thyroid cancer, which is currently in ...

Thyroid cancer is an endocrine malignancy with an increasing incidence, although in general terms it has a good outcome1. Nevertheless, some patients develop aggressive forms of the disease that are untreatable and the molecular bases are poorly understood2.
miRNAs are 22-nucleotide-long non-coding RNAs that regulate gene expression in many tissues, typically by base-pair binding to the 3&#39; untranslational region (3&#8217;UTR) of target messenger RNAs (mRNAs), triggering mRNA degradation or translational repression3,4. There is increasing evidence demonstrating that the deregulation of microRNA expression is a hallmark of cancer, as these molecules modulate proliferative signaling, migration, invasion and metastasis, and can provide resistance to apoptosis5,6. In recent years, many studies have identified miRNAs as potential biomarkers for cancer diagnosis and prognosis as well as therapeutic targets7, providing a new dimension to cancer evaluation and treatment.
miRNAs have taken center stage in human molecular oncology as key drivers of human thyroid neoplasms8,9,10,11,12. Among the miRNAs up-regulated, miR-146b is highly overexpressed in Papillary Thyroid Carcinoma (PTC) tumors and was shown to significantly increase cell proliferation, and to be associated with aggressiveness and dismal prognosis6,12,13,14,15. Furthermore, miR-146b regulates several thyroid genes involved in differentiation12, and also important tumor suppressor genes such as PTEN16 and DICER117. Despite their importance in cancer biology, miRNA-based cancer therapy is still in its early stages, and very few studies have addressed thyroid cancer - the most frequent of the endocrine tumors18. Here we describe a protocol using two different mouse models with human-derived tumors, in which the administration of a synthetic miRNA-inhibitor (antagomiR) that specifically inhibits a cellular miRNA can block tumor growth. We first used a common xenograft model, and the local intratumor administration of an antagomiR decreased tumor growth measured as a reduction in tumor bioluminescence16. Because the establishment of robust mouse models mimicking human tumor progression is essential to develop unique therapeutic approaches, orthotopic implantation of primary human tumors is a more valuable platform for clinical validation of new drugs than subcutaneous implantation models. Thus, in order to better assess the therapeutic potential of the antagomiR, we used an orthotopic mouse model with systemic delivery in the blood stream, obtaining the same results.

<table>
	<tbody>
		<tr>
			<td>AntagomiR: mirVana miRNA inhibitor</td>
			<td>Thermo Fisher</td>
			<td>4464088</td>
			<td>In Vivo Ready</td>
		</tr>
		<tr>
			<td>Basement Membrane Matrix: Matrigel Basement Membrane Matrix High Concentration</td>
			<td>Corning</td>
			<td>#354248</td>
			<td></td>
		</tr>
		<tr>
			<td>DICER antibody</td>
			<td>Abcam</td>
			<td>ab14601</td>
			<td>IHQ: 1/100</td>
		</tr>
		<tr>
			<td>In vivo delivery reagent: Invivofectamine 3.0 Reagent</td>
			<td>Thermo Fisher</td>
			<td>IVF3005</td>
			<td></td>
		</tr>
		<tr>
			<td>In vivo imaging software: IVIS-Lumina II Imaging System</td>
			<td>Caliper Life Sciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Negative control: mirVana miRNA Inhibitor, Negative Control #1</td>
			<td>Thermo Fisher</td>
			<td>4464077</td>
			<td>In Vivo Ready</td>
		</tr>
		<tr>
			<td>PCNA antibody</td>
			<td>Abcam</td>
			<td>ab92552</td>
			<td>WB: 1/2,000</td>
		</tr>
		<tr>
			<td>PTEN antibody</td>
			<td>Santa Cruz</td>
			<td>sc-7974</td>
			<td>WB: 1/1,000</td>
		</tr>
		<tr>
			<td>XenoLight D-Luciferin - K+ Salt Bioluminescent Substrate</td>
			<td>PerkinElmer</td>
			<td>122799</td>
			<td>Diluted in PBS</td>
		</tr>
	</tbody>
</table>

in vivo inhibition of microrna to decrease tumor growth in mice

MicroRNAs (miRNAs) are important regulators of gene expression through their ability to destabilize mRNA and inhibit translation of target mRNAs. An ever-increasing number of studies have identified miRNAs as potential biomarkers for cancer diagnosis and prognosis, and also as therapeutic targets, adding an extra dimension to cancer evaluation and treatment. In the context of thyroid cancer, tumorigenesis results not only from mutations in important genes, but also from the overexpression of many miRNAs. Accordingly, the role of miRNAs in the control of thyroid gene expression is evolving as an important mechanism in cancer. Herein, we present a protocol to examine the effects of miRNA-inhibitor delivery as a therapeutic modality in thyroid cancer using human tumor xenograft and orthotopic mouse models. After engineering stable thyroid tumoral cells expressing GFP and luciferase, cells are injected into nude mice to develop tumors, which can be followed by bioluminescence. The in vivo inhibition of a miRNA can reduce tumor growth and upregulate miRNA gene targets. This method can be used to assess the importance of a determined miRNA in vivo, in addition to identifying new therapeutic targets.

We used two different mice models to determine whether the neutralization of a miRNA could suppress tumor growth. Accordingly, human tumor thyroid Cal62-luc cells were subcutaneously injected into the flanks of nude mice to generate a xenograph model. After two weeks, tumors were established and could be measured with calipers. At that time point, mice were injected intratumorally with the miR-146b-inhibitor, or an appropriate control, and tumor volume was followed for a further two weeks (Figure 1A<...

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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.

MicroRNAs (miRNAs) are important regulators of gene expression through their ability to destabilize mRNA and inhibit translation of target mRNAs. An ...

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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7.1K visualizações.  Instituto de Investigaciones Biomédicas, CSIC-UAM. Realizamos a primeira terapia, baseada em um inibidor de microRNA em um modelo de câncer de tireoide. Essas terapias em desenvolvimento mostram-se promissoras para o tratamento desta doença. Esse tipo de modelo ortotópico, utilizando uma rota sistêmica de entrega de tratamento, facilita a validação de um novo sistema de microRNA.Embora usemos um modelo ortotópico para implantar células cancerígenas da tireoide humana em tireoides de camundongos, esses modelos podem ser todos s...