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 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.</li>
		<li>Grow the cells in Dulbecco&#180;s modified Eagle&#180;s medium (DMEM) supplemented with penicillin, streptomycin and 10% fetal bovine serum (FBS) at 37 &#176;C and 5% CO2.</li>
		<li>Suspend 1 x 106 cells in 50 &#956;L of phosphate buffered saline (PBS) at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Cell inoculation into the flanks of mice 
	<ol>
		<li>Mix the cells with the same amount of basement membrane matrix. For example, add 1 x 106 cells in 50 &#956;L of PBS to 50 &#956;L of basement membrane matrix (see Table of Materials) and mix gently. 
		NOTE:</li>
		<li>Inject 100 &#956;L 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&#39;&#39; (0.4x13 mm) needle.</li>
	</ol>
	</li>
	<li>Intratumoral antagomiR treatment. 
	<ol>
		<li>Suspend the antagomiR (see Table of Materials) or the negative control in 500 &#181;L of RNase-free distilled water.</li>
		<li>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.
		<ol>
			<li>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 &#956;L of miRNA-inhibitor solution (16 nmol) to 80 &#956;L of complexation buffer (included in the in vivo delivery reagent kit, see Table of Materials).</li>
			<li>Bring the in vivo delivery reagent to room temperature. Add 160 &#956;L to a 1.5-mL tube and immediately add 160 &#956;L of diluted antagomiR solution. Return remaining reagent to -20 &#176;C. If necessary, store the reagent at 4 &#176;C for up to one week after thawing.</li>
			<li>Vortex immediately (10 s) to ensure complexation of the in vivo delivery reagent-antagomiR.</li>
			<li>Incubate the in vivo delivery reagent-antagomiR mixture for 30 min at 50 &#176;C. Centrifuge the tube briefly to recover the sample.</li>
			<li>Dilute the complex 6-fold by adding 1360 &#181;L of PBS pH 7.4 and mix well.</li>
			<li>Proceed with in vivo delivery of the reagent-antagomiR complex (8 mice/condition), or store the complex at 4 &#176;C for up to one week prior to injection. Inject 200 &#956;L intratumorally into each tumor (2 nmol of antagomiR). 
			NOTE: The volume and quantity of the antagomiR is independent of the tumor volume.</li>
		</ol>
		</li>
		<li>Perform the treatment 3 times each week (Monday, Wednesday and Friday) for 2 weeks.</li>
	</ol>
	</li>
	<li>Analysis of tumor growth
	<ol>
		<li>Inject 50 &#956;L 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&#39;&#39; (0.4 mm x 13 mm) needle.
		<ol>
			<li>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.</li>
			<li>Apply ophthalmic ointment to both eyes to prevent desiccation.</li>
		</ol>
		</li>
		<li>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 the tumor growth.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Tumor excision
	<ol>
		<li>Seven days after the end of the antagomiR treatment, sacrifice the mice, excise the tumor and extract protein and/or RNA for future analysis. Alternatively, section the tumors and fix them for immunohistochemistry.</li>
	</ol>
	</li>
</ol>
2. Thyroid orthotopic inoculation of cells and systemic antagomiR treatment
<ol>
	<li>Cell preparation
	<ol>
		<li>Engineer a Cal62 human thyroid cancer cell line to overexpress a transgenic construct that constitutively expresses GFP and luciferase. Select the transgenic cells by antibiotic resistance or by sorting the GFP-positive cells with flow cytometry.</li>
		<li>Grow these cells in DMEM supplemented with antibiotics (penicillin and streptomycin) and 10% FBS at 37 &#176;C and 5% CO2.</li>
		<li>Suspend 1 x 105 cells in 5 &#956;L of PBS.</li>
	</ol>
	</li>
	<li>Cell inoculation into the thyroid gland of mice
	<ol>
		<li>Inject 100 &#956;L of analgesic (buprenorphine) and 100 &#956;L of antibiotic (cephalosporin) subcutaneously into 7-week-old BALB/c nu/nu mice.&#160;Disinfect the injection site with alcohol.</li>
		<li>Inject 5 &#956;L of the cell solution into the thyroid gland of the mice.
		<ol>
			<li>Anesthetize the mouse using 3% isoflurane mixed with oxygen and place it under a stereomicroscope in a sterile flow cabinet.&#160;&#160;Assess the level of anesthesia by pedal reflex (firm toe pinch) and adjust anesthetic delivery as appropriate to maintain surgical plane.</li>
			<li>Apply ophthalmic ointment to both eyes to prevent desiccation.&#160;</li>
			<li>For each mouse, disinfect the neck with iodopovidone and make an incision of approximate 2 cm in the skin with scissors. Once open, expose the neck by displacing the salivary glands.</li>
			<li>Dissect the strap muscles using dissection forceps and/or scissors to expose the trachea and the thyroid gland. Using a 10 &#956;L microliter syringe inject 5 &#956;L of the cell solution into the right thyroid lobule, located at the side of the cricoid cartilage.</li>
			<li>Reposition the salivary glands and suture the incision with silk using braided, coated, non-absorbable sutures.</li>
			<li>Add iodopovidone to the wound area and place the mouse on a thermic blanket while it recovers from the anesthesia.</li>
		</ol>
		</li>
		<li>The following day post-surgery, inject subcutaneously analgesic (buprenorphine) and add 3 mL of liquid ibuprofen (40 mg/mL) into 250 mL of the drinking water for 1 week.</li>
	</ol>
	</li>
	<li>Systemic antagomiR treatment 
	NOTE: Perform this step 2-3 weeks after the cell injection, when the bioluminescent signal is detectable.
	<ol>
		<li>Suspend the antagomiR or the negative control in distilled RNase-free water.</li>
		<li>Prior to the injection, prepare 7 nmol of the antagomiR or the control together with the in vivo delivery reagent for each injection.
		<ol>
			<li>For the antagomiR and the in vivo delivery reagent solution preparation see step 1.3 but add 7 nmol of the miRNA-inhibitor per mouse.</li>
		</ol>
		</li>
		<li>Administer the solution intravenously by retro-orbital injection of the venous sinus of the mouse. Use isoflurane to induce anesthesia. 
		NOTE: Assess the level of anesthesia by pedal reflex (firm toe pinch).</li>
		<li>Treat the mice 3 times a week (Monday, Wednesday and Friday) for 2 weeks.</li>
	</ol>
	</li>
	<li>Analysis of tumor growth
	<ol>
		<li>Determine the tumor bioluminescent signal twice weekly to calculate tumor growth.
		<ol>
			<li>Inject 50 &#956;L of a 40 mg/mL solution of D-luciferin substrate at each time point via subcutaneous injection.</li>
			<li>At 8 min post-injection, anesthetize the mice using 3% isoflurane mixed with oxygen and image the bioluminescent signal with in vivo imaging software.</li>
		</ol>
		</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Tumor excision
	<ol>
		<li>Seven days after the end of the antagomiR treatment period, sacrifice the mice, excise the tumor and extract protein and/or RNA for future analysis. Alternatively, section the tumors and fix them for immunohistochemistry.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Lim, H., Devesa, S. S., Sosa, J. A., Check, D., Kitahara, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trends+in+Thyroid+Cancer+Incidence+and+Mortality+in+the+United+States.">Trends in Thyroid Cancer Incidence and Mortality in the United States.</a> Journal of the American Medical Association. 317 (13), 1338-1348 (2017).</li><li>Landa, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+and+transcriptomic+hallmarks+of+poorly+differentiated+and+anaplastic+thyroid+cancers.">Genomic and transcriptomic hallmarks of poorly differentiated and anaplastic thyroid cancers.</a> The Journal of Clinical Investigation. 126 (3), 1052-1066 (2016).</li><li>Gregory, R. I., Shiekhattar, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+biogenesis+and+cancer.">MicroRNA biogenesis and cancer.</a> Cancer Research. 65 (9), 3509-3512 (2005).</li><li>Lin, S., Gregory, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+biogenesis+pathways+in+cancer.">MicroRNA biogenesis pathways in cancer.</a> Nature Reviews Cancer. 15 (6), 321-333 (2015).</li><li>Hammond, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+as+oncogenes.">MicroRNAs as oncogenes.</a> Current Opinion In Genetics &amp; Development. 16 (1), 4-9 (2006).</li><li>Cancer Genome Atlas Reserch Network. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+genomic+characterization+of+papillary+thyroid+carcinoma.">Integrated genomic characterization of papillary thyroid carcinoma.</a> Cell. 159 (3), 676-690 (2014).</li><li>Li, Z., Rana, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+targeting+of+microRNAs:+current+status+and+future+challenges.">Therapeutic targeting of microRNAs: current status and future challenges.</a> Nature Reviews. Drug Discovery. 13 (8), 622-638 (2014).</li><li>Pallante, P., Battista, S., Pierantoni, G. M., Fusco, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deregulation+of+microRNA+expression+in+thyroid+neoplasias.">Deregulation of microRNA expression in thyroid neoplasias.</a> Nature Reviews. Endocrinology. 10 (2), 88-101 (2014).</li><li>Fuziwara, C. S., Kimura, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+in+thyroid+development,+function+and+tumorigenesis.">MicroRNAs in thyroid development, function and tumorigenesis.</a> Molecular and Cellular Endocrinology. 456, 44-50 (2017).</li><li>Fuziwara, C. S., Kimura, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+Deregulation+in+Anaplastic+Thyroid+Cancer+Biology.">MicroRNA Deregulation in Anaplastic Thyroid Cancer Biology.</a> International Journal of Endocrinology. 2014, 743450 (2014).</li><li>Riesco-Eizaguirre, G., Santisteban, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocrine+Tumours:+Advances+in+the+molecular+pathogenesis+of+thyroid+cancer:+lessons+from+the+cancer+genome.">Endocrine Tumours: Advances in the molecular pathogenesis of thyroid cancer: lessons from the cancer genome.</a> European Journal of Endocrinology. 175 (5), R203-R217 (2016).</li><li>Riesco-Eizaguirre, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+miR-146b-3p/PAX8/NIS+Regulatory+Circuit+Modulates+the+Differentiation+Phenotype+and+Function+of+Thyroid+Cells+during+Carcinogenesis.">The miR-146b-3p/PAX8/NIS Regulatory Circuit Modulates the Differentiation Phenotype and Function of Thyroid Cells during Carcinogenesis.</a> Cancer Research. 75 (19), 4119-4130 (2015).</li><li>Lima, C. R., Geraldo, M. V., Fuziwara, C. S., Kimura, E. T., Santos, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MiRNA-146b-5p+upregulates+migration+and+invasion+of+different+Papillary+Thyroid+Carcinoma+cells.">MiRNA-146b-5p upregulates migration and invasion of different Papillary Thyroid Carcinoma cells.</a> BMC Cancer. 16, 108 (2016).</li><li>Lee, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-222+and+microRNA-146b+are+tissue+and+circulating+biomarkers+of+recurrent+papillary+thyroid+cancer.">MicroRNA-222 and microRNA-146b are tissue and circulating biomarkers of recurrent papillary thyroid cancer.</a> Cancer. 119 (24), 4358-4365 (2013).</li><li>Deng, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MiR-146b-5p+promotes+metastasis+and+induces+epithelial-mesenchymal+transition+in+thyroid+cancer+by+targeting+ZNRF3.+Cellular+Physiology+and+Biochemistry.">MiR-146b-5p promotes metastasis and induces epithelial-mesenchymal transition in thyroid cancer by targeting ZNRF3. Cellular Physiology and Biochemistry.</a> International Journal of Experimental Cellular Physiology, Biochemistry, and Pharmacology. 35 (1), 71-82 (2015).</li><li>Ramirez-Moya, J., Wert-Lamas, L., Santisteban, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-146b+promotes+PI3K/AKT+pathway+hyperactivation+and+thyroid+cancer+progression+by+targeting+PTEN.">MicroRNA-146b promotes PI3K/AKT pathway hyperactivation and thyroid cancer progression by targeting PTEN.</a> Oncogene. , (2018).</li><li>Ramirez-Moya, J., Wert-Lamas, L., Riesco Eizaguirre, G., Santisteban, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+microRNA+processing+by+DICER1+downregulation+endows+thyroid+cancer+with+increased+aggressiveness.">Impaired microRNA processing by DICER1 downregulation endows thyroid cancer with increased aggressiveness.</a> Oncogene. , (2019).</li><li>Xing, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+pathogenesis+and+mechanisms+of+thyroid+cancer.">Molecular pathogenesis and mechanisms of thyroid cancer.</a> Nature Reviews. Cancer. 13 (3), 184-199 (2013).</li></ol>

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<...

Watch this Scientific Journal Video about In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice at JoVE.com

In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice

In Vivo Hemmung von MicroRNA zur Verringerung des Tumorwachstums bei Mäusen

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 ...

in-vivo-inhibition-of-microrna-to-decrease-tumor-growth-in-mice

Research

JoVE Journal

Cancer Research

7.2K Aufrufe.  Instituto de Investigaciones Biomédicas, CSIC-UAM. Wir führen die erste Therapie durch, basierend auf einem microRNA-Hemmer in einem Schilddrüsenkrebsmodell. Diese sich entwickelnden Therapien versprechen die Behandlung dieser Krankheit. Diese Art von orthotopischem Modell, das einen systemischen Behandlungsweg nutzt, erleichtert die Validierung eines neuen mikroRNA-basierten Arzneimittels.Obwohl wir ein orthotopisches Modell verwenden, um menschliche Schilddrüsenkrebszellen in Mausschilddrüsen zu implantieren, könnten diese Model...

Serie: In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic effect is lacking and the exact mechanisms of its action are unclear. Here, we report an animal model that allows for direct testing of the effects of tumor hypoxia on ES dissemination and investigation into the underlying pathways involved. This approach combines two well-established experimental strategies, orthotopic xenografting of ES cells and femoral artery ligation (FAL), which induces hindlimb ischemia. Human ES cells were injected into the gastrocnemius muscles of SCID/beige mice and the primary tumors were allowed to grow to a size of 250 mm3. At this stage either the tumors were excised (control group) or the animals were subjected to FAL to create tumor hypoxia, followed by tumor excision 3 days later. The efficiency of FAL was confirmed by a significant increase in binding of hypoxyprobe-1 in the tumor tissue, severe tumor necrosis and complete inhibition of primary tumor growth. Importantly, despite these direct effects of ischemia, an enhanced dissemination of tumor cells from the hypoxic tumors was observed. This experimental strategy enables comparative analysis of the metastatic properties of primary tumors of the same size, yet significantly different levels of hypoxia. It also provides a new platform to further assess the mechanistic basis for the hypoxia-induced alterations that occur during metastatic tumor progression in vivo. In addition, while this model was established using ES cells, we anticipate that this experimental strategy can be used to test the effect of hypoxia in other sarcomas, as well as tumors orthotopically implanted in sites with a well-defined blood supply route.

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

This manuscript describes the development of an animal model that allows for the direct testing of the effects of tumor hypoxia on metastasis and the deciphering the mechanisms of its action. Although the experiments described here focus on Ewing sarcoma, a similar approach can be applied to other tumor types.

In - vivo - Modell für die Prüfung von Wirkung von Hypoxie auf die Tumormetastasierung

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic ...

Forschung

Krebsforschung

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

Das<em&gt; Drosophila</em&gt; Imaginalscheibe Tumormodell: Visualisierung und Quantifizierung von Genexpression und Tumorinvasivität mit Genetischen Mosaiken

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic probes to provide quantitative information on the chemical tumor microenvironment (TME), including pO2, pH, redox status, concentrations of interstitial inorganic phosphate (Pi), and intracellular glutathione (GSH). In particular, an application of a recently developed soluble multifunctional trityl probe provides unsurpassed opportunity for in vivo concurrent measurements of pH, pO2 and Pi in Extracellular space (HOPE probe). The measurements of three parameters using a single probe allow for their correlation analyses independent of probe distribution and time of the measurements.

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

Low-field (L-band, 1.2 GHz) electron paramagnetic resonance using soluble nitroxyl and trityl probes is demonstrated for assessment of physiologically important parameters in the tumor microenvironment in mouse models of breast cancer.

In Vivo EPR-Bewertung der pH, pO2, Redox-Status, und Konzentrationen von Phosphat und Glutathion in der Tumor-Mikroumgebung

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

Utilizing High Resolution Ultrasound to Monitor Tumor Onset and Growth in Genetically Engineered Pancreatic Cancer Models

The LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mouse model represents an established and frequently used transgenic model to evaluate novel therapies in pancreatic cancer. Tumor onset is variable in the KPC model between 8 weeks and several months. Therefore, non-invasive imaging tools are required to screen for tumor onset and monitor for response to treatment. To address this issue, different approaches have emerged over the last years. High resolution ultrasound has major advantages such as non-invasiveness, fast session times and a high image resolution without radiation exposure. However, ultrasound in mice is not trivial and sufficient anatomical knowledge and practical skills are required to successfully perform high resolution ultrasound in preclinical pancreatic cancer models. With the following article, a detailed hands-on guide for abdominal ultrasound in murine models with a particular focus on endogenous pancreatic cancer models is presented. Furthermore, a summary of common mistakes and how to avoid them is provided.

This article describes the utilization of high-resolution ultrasound in genetically engineered pancreatic cancer mice. The primary aim is to provide a detailed instruction for detection and evaluation of endogenous pancreatic tumors.

Verwendung von hochauflösendem Ultraschall zu Monitor Tumor auftreten und Wachstum in gentechnisch Bauchspeicheldrüsenkrebs Modelle

The LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mouse model represents an established and frequently used transgenic model to evaluate novel ...

Transfer of Manipulated Tumor-associated Neutrophils into Tumor-Bearing Mice to Study their Angiogenic Potential In Vivo

The contribution of neutrophils to the regulation of tumorigenesis is getting increased attention. These cells are heterogeneous, and depending on the tumor milieu can possess pro- or anti-tumor capacity. One of the important cytokines regulating neutrophil functions in a tumor context are type I interferons. In the presence of interferons, neutrophils gain anti-tumor properties, including cytotoxicity or stimulation of the immune system. Conversely, the absence of an interferon signaling results in prominent pro-tumor activity, characterized with strong stimulation of tumor angiogenesis. Recently, we could demonstrate that pro-angiogenic properties of neutrophils depend on the activation of nicotinamide phosphoribosyltransferase (NAMPT) signaling pathway in these cells. Inhibition of this pathway in tumor-associated neutrophils leads to their potent anti-angiogenic phenotype. Here, we demonstrate our newly established model allowing in vivo evaluation of tumorigenic potential of manipulated tumor-associated neutrophils (TANs). Shortly, pro-angiogenic tumor-associated neutrophils can be isolated from tumor-bearing interferon-deficient mice and repolarized into anti-angiogenic phenotype by blocking of NAMPT signaling. The angiogenic activity of these cells can be subsequently evaluated using an aortic ring assay. Anti-angiogenic TANs can be transferred into tumor-bearing wild type recipients and tumor growth should be monitored for 14 days. At day 14 mice are sacrificed, tumors removed and cut with their vascularization assessed. Overall, our protocol provides a novel tool to in vivo evaluate angiogenic capacity of primary cells, such as tumor-associated neutrophils, without a need to use artificial neutrophil cell line models. vc

Here, we show therapeutic potential of anti-angiogenic tumor-associated neutrophils after their transfer into tumor-bearing mice. This protocol can be used to manipulate neutrophil activity ex vivo and to subsequently evaluate their functionality in vivo&#160;in developing tumors. It is an appropriate model for studying potential neutrophil-based immunotherapies.

Transfer von manipulierten Tumor-assoziierten Neutrophilen in tumortragende Mäuse zur Untersuchung ihres angiogenen Potenzials in Vivo

The contribution of neutrophils to the regulation of tumorigenesis is getting increased attention. These cells are heterogeneous, and depending on the ...

Detection of Lung Tumor Progression in Mice by Ultrasound Imaging

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic alterations in oncogenes such as the KRAS oncogene, which constitutes ~25% of lung cancer cases. The difficulty in therapeutically targeting KRAS-driven lung cancer partly stems from having poor models that can mimic the progression of the disease in the lab. We describe a method that permits the relative quantification of primary KRAS lung tumors in a Cre-inducible LSL-KRAS G12D mouse model via ultrasound imaging. This method relies on brightness (B)-mode acquisition of the lung parenchyma. Tumors that are initially formed in this model are visualized as B-lines and can be quantified by counting the number of B-lines present in the acquired images. These would represent the relative tumor number formed on the surface of the mouse lung. As the formed tumors develop with time, they are perceived as deep clefts within the lung parenchyma. Since the circumference of the formed tumor is well-defined, calculating the relative tumor volume is achieved by measuring the length and width of the tumor and applying them in the formula used for tumor caliper measurements. Ultrasound imaging is a non-invasive, fast and user-friendly technique that is often used for tumor quantifications in mice. Although artifacts may appear when obtaining ultrasound images, it has been shown that this imaging technique is more advantageous for tumor quantifications in mice compared to other imaging techniques such as computed tomography (CT) imaging and bioluminescence imaging (BLI). Researchers can investigate novel therapeutic targets using this technique by comparing lung tumor initiation and progression between different groups of mice.

This protocol describes the steps taken to induce KRAS lung tumors in mice as well as the quantification of formed tumors by ultrasound imaging. Small tumors are visualized in early timepoints as B-lines. At later timepoints, relative tumor volume measurements are achieved by the measurement tool in the ultrasound software.

Nachweis der Lungentumorprogression bei Mäusen durch Ultraschall-Bildgebung

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic ...

A Mouse Model to Investigate the Role of Cancer-Associated Fibroblasts in Tumor Growth

Cancer-associated fibroblasts (CAFs) can play an important role in tumor growth by creating a tumor-promoting microenvironment. Models to study the role of CAFs in the tumor microenvironment can be helpful for understanding the functional importance of fibroblasts, fibroblasts from different tissues, and specific genetic factors in fibroblasts. Mouse models are essential for understanding the contributors to tumor growth and progression in an in vivo context. Here, a protocol in which cancer cells are mixed with fibroblasts and introduced into mice to develop tumors is provided. Tumor sizes over time and final tumor weights are determined and compared among groups. The protocol described can provide more insight into the functional role of CAFs in tumor growth and progression.

A protocol to co-inject cancer cells and fibroblasts and monitor tumor growth over time is provided. This protocol can be used to understand the molecular basis for the role of fibroblasts as regulators of tumor growth.

Ein Mausmodell zur Untersuchung der Rolle krebsassoziierter Fibroblasten beim Tumorwachstum

Cancer-associated fibroblasts (CAFs) can play an important role in tumor growth by creating a tumor-promoting microenvironment. Models to study the role ...

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in eradicating cancerous cells. However, the origins and replenishment of tumor antigen-specific CD8+ T cells within the tumor microenvironment (TME) remain obscure. This protocol employs the B16F10-OVA melanoma cell line, which stably expresses the surrogate neoantigen, ovalbumin (OVA), and TCR transgenic OT-I mice, in which over 90% of CD8+ T cells specifically recognize the OVA-derived peptide OVA257-264 (SIINFEKL) bound to the class I major histocompatibility complex (MHC) molecule H2-Kb. These features enable the study of antigen-specific T cell responses during tumorigenesis.
Combining this model with tumor transplantation surgery, tumor tissues from donors were transplanted into tumor-matched syngeneic recipient mice to precisely trace the influx of recipient-derived immune cells into transplanted donor tissues, allowing the analysis of the immune responses of tumor-inherent and periphery-originated antigen-specific CD8+ T cells. A dynamic transition was found to occur between these two populations. Collectively, this experimental design has provided another approach to precisely investigate the immune responses of CD8+ T cells in TME, which will shed new light on tumor immunology.

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

Here, we present a tumor transplantation protocol for the characterization of tumor-inherent and periphery-derived tumor-infiltrated lymphocytes in a mouse tumor model. Specific tracing of the influx of recipient-derived immune cells with flow cytometry reveals the dynamics of the phenotypic and functional changes of these cells during antitumor immune responses.

Tumortransplantation zur Beurteilung der Dynamik tumorinfiltrierender CD8+ T-Zellen in Mäusen

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in ...

An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth

Brain metastasis is a serious consequence of breast cancer for women as these tumors are difficult to treat and are associated with poor clinical outcomes. Preclinical mouse models of breast cancer brain metastatic (BCBM) growth are useful but are expensive, and it is difficult to track live cells and tumor cell invasion within the brain parenchyma. Presented here is a protocol for ex vivo brain slice cultures from xenografted mice containing intracranially injected breast cancer brain-seeking clonal sublines. MDA-MB-231BR luciferase tagged cells were injected intracranially into the brains of Nu/Nu female mice, and following tumor formation, the brains were isolated, sliced, and cultured ex vivo. The tumor slices were imaged to identify tumor cells expressing luciferase and monitor their proliferation and invasion in the brain parenchyma for up to 10 days. Further, the protocol describes the use of time-lapse microscopy to image the growth and invasive behavior of the tumor cells following treatment with ionizing radiation or chemotherapy. The response of tumor cells to treatments can be visualized by live-imaging microscopy, measuring bioluminescence intensity, and performing histology on the brain slice containing BCBM cells. Thus, this ex vivo slice model may be a useful platform for rapid testing of novel therapeutic agents alone or in combination with radiation to identify drugs personalized to target an individual patient&#39;s breast cancer brain metastatic growth within the brain microenvironment.

An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth

We introduce a protocol for measuring real-time drug and radiation response of breast cancer brain metastatic cells in an organotypic brain slice model. The methods provide a quantitative assay to investigate the therapeutic effects of various treatments on brain metastases from breast cancer in an ex vivo manner within the brain microenvironment interface.

Ein Ex-vivo-Gehirnschnittmodell zur Untersuchung und Bekämpfung des metastasierenden Tumorwachstums von Brustkrebs im Gehirn

Brain metastasis is a serious consequence of breast cancer for women as these tumors are difficult to treat and are associated with poor clinical ...

In Vivo Imaging to Measure Spontaneous Lung Metastasis of Orthotopically-injected Breast Tumor Cells

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple opportunities for therapeutic intervention, and the ability to accurately model them in mice is critical to evaluate their effects. Here, a step-by-step protocol is presented for the establishment of orthotopic primary breast tumors and the subsequent monitoring of the establishment and growth of metastatic lesions in the lung using in vivo bioluminescence imaging. This methodology allows for the evaluation of treatment or its biological effects along the entire range of metastatic development, from primary tumor escape to outgrowth in the lungs. Breast orthotopic tumors are generated in mice via injection of a luciferase-labeled cell suspension in the 4th mammary gland. Tumors are allowed to grow and disseminate for a specific amount of time and are then surgically resected. Upon resection, spontaneous lung metastasis is detected, and the growth over time is monitored using in vivo bioluminescence imaging. At the desired experimental endpoint, lung tissue can be collected for downstream analysis. The treatment of established, clinically evident metastasis is critical to improve outcomes for stage IV cancer patients, and it can be evaluated through tail vein models of experimental lung metastasis. However, metastatic dissemination occurs early in breast cancer, and many patients have latent, subclinical disseminated disease after surgery. Utilization of spontaneous models such as this one provides the opportunity to study the whole spectrum of the disease, especially the systemic effects driven by treatment of the primary tumor such as pre-metastatic niche priming, and evaluate treatments on dormant and subclinical disease after surgery.

<meta charset="utf8"></head><body>In-vivo-Bildgebung zur Messung spontaner Lungenmetastasen von orthotopisch injizierten Brusttumorzellen

The manuscript describes a methodology for the establishment as well as longitudinal growth monitoring of spontaneous lung metastasis from orthotopically-injected breast tumors, amenable to intervention at all stages of the metastatic cascade.