Name: a comprehensive procedure to evaluate the in vivo performance of cancer nanomedicines
Uploaded: 2017-03-04T14:00:00
Description: Watch this Scientific Journal Video about A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines at JoVE.com

The authors would like to thank Drs. Helene Salmon and Miriam Merad from Icahn School of Medicine at Mount Sinai for providing the B16-F10-YFP cells and for their expert advice on melanoma mouse models. The authors further thank the Animal Imaging Core Facility, the Radiochemistry and Molecular Imaging Probes Core Facility, and the Molecular Cytology Core Facility at Memorial Sloan Kettering Cancer Center (MSK) for their support. This work was supported by National Institutes of Health grants NIH 1 R01 HL125703 (W.J.M.M.), R01CA155432 (W.J.M.M.), K25 EB016673 (T.R.) and P30 CA008748 (MSK Center Grant). The authors also thank the Center for Molecular Imaging and Nanotechnology (CMINT) at MSK for their financial support (T.R.).

The procedure consists of the dual radioactive and fluorescent labeling of nanoparticles, in vivo PET-CT imaging, ex vivo biodistribution measurements, and ex vivo immunostaining and flow cytometry analyses. All animal experiments were approved by the Institutional Animal Care and Use Committee of Memorial Sloan Kettering Cancer Center.
1. Preparation of Dual-labeled Liposomes
NOTE: Syngeneic B16 melanoma tumors can be induced by injecting 3...

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Critical Steps within the Protocol:
The high quality of dual-labeled liposomes is the key to producing consistent results over a long period of time. Free fluorescent dyes or 89Zr ions can generate totally different targeting patterns and must be completely removed during the purification step. In addition, if the immune system significantly affects experimental cancer nanomedicine performance, the use of immunocompetent mouse models should be preferable, such as the B16-F10 melanoma m...

Nanomedicine is shifting the paradigm of cancer treatment development1. Inspired by the tremendous clinical impact of previous cancer nanomedicines, such as liposome- and albumin-based nanotherapies2,3, many novel formulations have been produced in the past decade. However, recent analyses of the clinical translation success of these cancer nanomedicines indicate that only a few of them have been approved for clinical use4,5. One major obstacle to the clinical translation of new cancer nanomedicines is their limited improvement of the therapeutic index compared with the direct administration of the free therapeutic compounds6. As such, accurate evaluation of the in vivo performance of nanomedicines at systemic, tissue, and cellular levels in preclinical animal models is essential to identify those with optimal therapeutic indices for future clinical translation.
Nanomaterials can be radiolabeled for quantitative characterization in living animals with positron emission tomography (PET) imaging, which has superb sensitivity and reproducibility among all clinical imaging modalities7. For example, 89Zr-labeled long-circulating nanomedicines have been characterized in mouse models for cancer8,9,10, as well as in other disease models11. In addition, the blood half-life and biodistribution of the nanomedicines can be extensively evaluated by using ex vivo radioactivity measurements in individual tissues8. Therefore, radiolabeling allows for the quantitative evaluation of nanomedicines at systemic and tissue levels.
Importantly, radiolabeled nanomedicines generally cannot be analyzed at the single-cell or subcellular levels due to the limited spatial resolution of the radioactive signal. Therefore, fluorescent labeling proves to be a complementary modality for the evaluation of nanoparticles with optical imaging techniques such as flow cytometry and fluorescence microscopy12. To this end, nanoparticles labeled with radioisotopes and fluorescent tags can be quantitatively evaluated in vivo by nuclear imaging and ex vivo by radioactivity counting, and they can also be extensively characterized at the cellular level by optical imaging.
Previously, we have developed modular procedures to incorporate radioactive and fluorescent labels into various nanoparticles, including high-density lipoprotein (HDL)11, liposomes9,10, polymeric nanoparticles, antibody fragments, and nanoemulsions10,13. These labeled nanoparticles have allowed for quantitative characterization in relevant animal models at different levels, which guided the optimization of these nanomaterials for their specific applications. In the current study, the aim is to use liposomal nanoparticles&#8212;the most established nanomedicine platform14&#8212;as an example to demonstrate comprehensive procedures to generate a dual-labeled nanoparticle and to thoroughly characterize it in a classic syngeneic melanoma B16-F10 mouse model15. From the results, we are confident this nanoparticle characterization approach can be adapted to evaluate other cancer nanomedicines in relevant mouse models.

<table border="0" cellpadding="0" cellspacing="0" width="638">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DPPC</td>
			<td style="width:136px;">Avantilipids</td>
			<td align="right" style="width:119px;">850355</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cholesterol</td>
			<td style="width:136px;">Sigma-Aldrich</td>
			<td style="width:119px;">C8667</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DSPE-PEG2000</td>
			<td style="width:136px;">Avantilipids</td>
			<td style="width:119px;">880120P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DSPE-DFO</td>
			<td style="width:136px;">Home made</td>
			<td align="right" style="width:119px;">110634</td>
			<td>Perez-Medina et al, JNM, 2014</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DiIC12[5]-DS</td>
			<td style="width:136px;">AAT Bioquest</td>
			<td align="right" style="width:119px;">22051</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifugal filter</td>
			<td style="width:136px;">Vivaproducts</td>
			<td style="width:119px;">VS2061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rotary evaporator</td>
			<td style="width:136px;">Buchi</td>
			<td style="width:119px;">R-100</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Radio-HPLC</td>
			<td style="width:136px;">Shimadzu HPLC with 2 LC-10AT pumps</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">89Zr-oxalate</td>
			<td style="width:136px;">MSKCC</td>
			<td style="width:119px;">Synthesized in house</td>
			<td>TR19/9 variable beam cyclotron (Ebco Industries Inc)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Micro PET-CT</td>
			<td style="width:136px;">Siemens</td>
			<td style="width:119px;">Inveon Micro-PET/CT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gamma counter</td>
			<td style="width:136px;">PerkinElmer</td>
			<td style="width:119px;">2470-0150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Flow cytometry</td>
			<td style="width:136px;">BD Biosciences</td>
			<td style="width:119px;">Fortessa</td>
			<td>Any multi-parametric flow cytometry analyzers would suffice</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">C57BL/6 mice</td>
			<td style="width:136px;">Jackson Laboratories</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">B16-YFP melanoma cells</td>
			<td style="width:136px;">Home made</td>
			<td style="width:119px;">N/A</td>
			<td>Salmon et al, Immunity, 2016</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ly6C (clone HK1.4)--APC-Cy7</td>
			<td align="right" style="width:136px;">128025</td>
			<td style="width:119px;">Biolegend</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MHCII (M5/114/152)--APC</td>
			<td align="right" style="width:136px;">107613</td>
			<td style="width:119px;">Biolegend</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CD45 (30-F11)--BV510</td>
			<td align="right" style="width:136px;">103137</td>
			<td style="width:119px;">Biolegend</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CD64 (X54-5/7.1)--PE-Cy7</td>
			<td align="right" style="width:136px;">139313</td>
			<td style="width:119px;">Biolegend</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CD11b (M1/70)--BV605</td>
			<td align="right" style="width:136px;">101237</td>
			<td style="width:119px;">Biolegend</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CD3 (17A2)--BV711</td>
			<td align="right" style="width:136px;">100241</td>
			<td style="width:119px;">Biolegend</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CD31 (13.3)--PE</td>
			<td align="right" style="width:136px;">561073</td>
			<td style="width:119px;">Biolegend</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CD11c (M418)--PerCP-Cy5.5</td>
			<td align="right" style="width:136px;">117327</td>
			<td style="width:119px;">BD Biosciences</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CD31 (13.3) no fluorophore</td>
			<td align="right" style="width:136px;">550274</td>
			<td style="width:119px;">BD Biosciences</td>
			<td></td>
		</tr>
	</tbody>
</table>

a comprehensive procedure to evaluate the in vivo performance of cancer nanomedicines

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past decade. However, only a small number of nanomedicines have been approved for clinical use, whereas the majority of nanomedicines under clinical development have produced disappointing results. One major obstacle to the successful clinical translation of new cancer nanomedicines is the lack of an accurate understanding of their in vivo performance. This article features a rigorous procedure to characterize the in vivo behavior of nanomedicines in tumor-bearing mice at systemic, tissue, single-cell, and subcellular levels via the integration of positron emission tomography-computed tomography (PET-CT), radioactivity quantification methods, flow cytometry, and fluorescence microscopy. Using this approach, researchers can accurately evaluate novel nanoscale formulations in relevant mouse models of cancer. These protocols may have the ability to identify the most promising cancer nanomedicines with high translational potential or to aid in the optimization of cancer nanomedicines for future translation.

Figure 1 shows an overview of the procedure. Figure 2 presents the schematic synthesis procedure of the dual-labeled liposomes described in step 110. Figure 3 displays a representative PET-CT image (Figure 3a), radioactivity quantification from PET imaging (Figure 3b), blood half-life (Figure 3c), and biodistribution (Figure 3d) of radioactive nano...

Watch this Scientific Journal Video about A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines at JoVE.com

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

The poor understanding of the in vivo performance of nanomedicines stymies their clinical translation. Procedures to evaluate the in vivo behavior of cancer nanomedicines at systemic, tissue, single-cell, and subcellular levels in tumor-bearing immunocompetent mice are described here. This approach may help researchers to identify promising cancer nanomedicines for clinical translation.

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past ...

The overall goal of this procedure is to evaluate the in vivo behavior of cancer nanomedicines at systemic, tissue, single cell, and subcellular levels in tumor-bearing, immunocompetent mice. This method can help answer the key question in the cancer nanomedicine field. How do nanomedicines accumulate quantitatively in different tissues of living animals over time?The main advantage of this technique is that this dual-labeling approach allows for in vivo nanomedicine characterization from the macroscopic to the microscopic level. We choose liposomes as example to demostrate our procedure because liposomes are currently in clinical use, and they're also a common platform for experimental nanomedicines. To begin the procedure, dissolve 20 milligrams of a lipid mixture, which include 0.1%by mole of a lipophilic cationic fluorescent dye and two milliliters of chloroform.Then, dry the lipids with a rotary evaporator. Add 20 milliliters of phosphate-buffered saline to the dried lipids. Sonicate the mixture on ice with a 3.8 millimeter probe sonicator at 30 watts for 30 minutes, keeping the mixture between four and 20 degrees Celsius throughout.Centrifuge the resulting liposomal nanoparticle suspension at 4, 000 G for 10 minutes. Discard the pellet and wash the liposomes with PBS, using a 100 kilodalton MWCO centrifugal filter unit at 4000 G.Transfer the concentrated liposomes from the top chamber to a new centrifuge tube. The liposomes may be stored in PBS in a fridge for up to one week.Perform dynamic light scattering on a mixture of 50 microliters of the purified liposome suspension and 950 microliters of PBS. Measure the particle sizes and determine the size distribution. To begin the radiolabeling procedure, transfer to a 1.5 milliliter tube, the needed volume of purified liposomes and PBS, to obtain two milligrams of lipids.Add to this one millicurie of zurconium-89 oxalate for a final volume of 100 microliters and a pH of 6.9 to 7.1. Stir the mixture at 37 degrees Celsius for two hours. Then, remove free zirconium-89 by centrifugal filtration with a 100 kilodalton filter unit at 4, 000 G.Wash the retentate with sterile PBS three times at 4, 000 G.Then, dilute the retentate to about three microcurie per microliter for injection.Use size-exclusion HPLC paired with a radiation detector to determine the radiochemical purity of the radiolabeled liposomes. If the radiochemical purity is below 95%reformulate the dose. Use dynamic light scattering to confirm that the particle size and distribution values are both similar to those of the non-radiolabeled liposomes.To begin the imaging procedure, inject about 300 microcurie of radiolabeled liposomes into the tail vein of a restrained melanoma mouse. Two minutes after injection, use a 28 gauge insulin syringe to collect 10 to 20 microliters of blood from the tail vein of the mouse. Transfer the blood sample to a pre-weighed counting tube.Weigh the blood sample and measure the activity with a gamma counter. Calculate the radioactivity concentration as percentage of injected dose per gram. Fifteen minutes after injection, collect and measure another blood sample in the same way.Then, anesthetize the mouse by administration of 2%isoflurane and oxygen via a nosecone. Collect and measure a third blood sample one hour after injection. Then, allow the mouse to recover in a recovery cage until normal mobility has been regained.Collect additional samples four, eight, 24, and 48 hours after injection. Allow the mouse to recover fully between injections. House the mouse in a room designated for animals to which radioactive materials have been administered.At 24 or 48 hours after liposome injection, anesthetize the mouse with 2%isoflurane and oxygen. Place the mouse on its belly on a small animal microPET-CT scanner bed. Continue delivering 2%isoflurane and oxygen via a nose cone.Apply vet ointment to the mouse's eyes to prevent them from drying. Then, move the scanner bed into the instrument. Perform a 15 minute whole-body PET static scan with at least 50 million coincident events, with an energy of 250 to 700 kiloelectron volts, and a coincidence-timing window of six nanoseconds.Next, perform a five minute CT scan with 120 rotational steps over 220 degrees, with approximately 145 milliseconds per frame. After scan completion, immediately sacrifice the mouse by carbon dioxide asphyxiation. Confirm death by pinching the paw of the animal, and then perform cervical dislocation.Perfuse the tissues with PBS to remove blood, and harvest the relevant tissue samples. Weigh the collected tissues and measure their activity. Calculate the radiolabeled liposome accumulation in tissue as percentage of injected dose per gram.Store the tumor tissue in cold PBS. Perform ex vivo flow cytometry and immunoflurescence imaging on the harvested tumor tissue to measure accumulation with respect to cell populations. PET/CT imaging of tumor-bearing mice injected with nanoparticles labeled with zirconium-89 showed significant nanaparticle accumulation in the tumor tissue 24 hours after injection.The blood half-life of the nanoparticles was determined to be approximately 10 hours. Ex vivo biodistribution measurements at 24 hours post-injection, were consistent with the in vivo PET/CT measurements. Nanoparticle accumulation was then investigated on a single-cell level with immunostaining techniques.Flow cytometry indicated that nanoparticles were found to preferentially accumulate in tumor-assisted macrophages. Immunoflurescence imaging of tumor samples also indicated specific targeting of endothelial cells by the nanoparticles. This technique can help researchers identify promising nanomedicines for clinical translation.Once mastered, this technique can be done in three days if it is performed properly. When radiolabeling nanomedicines, it's important to apply high standards of quality control. Following this procedure, other nanomedicines, such as nanoemulsions, polymeric nanoparticles, micelles, antibodies, and antibody fragments, can be labeled in order to measure their in vivo performance in tumor-bearing mice.

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Testing the Vascular Invasive Ability of Cancer Cells in Zebrafish (Danio Rerio)

Testing the Vascular Invasive Ability of Cancer Cells in Zebrafish Danio Rerio

Cancer cell vascular invasion and extravasation is a hallmark of metastatic progression.  Traditional in vitro models of cancer cell invasion of ...

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

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 ...

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human ...

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 ...

In Vitro Assay to Study Tumor-macrophage Interaction

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

Studying the Effects of Tumor-Secreted Paracrine Ligands on Macrophage Activation using Co-Culture with Permeable Membrane Supports

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer ...

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous ...

Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are ...

Genome-Wide Analysis of DNA Methylation in Gastrointestinal Cancer

DNA methylation is an important epigenetic change that is biologically meaningful and a frequent focus of cancer research. Genome-wide DNA methylation is ...

Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1&#945; Nanoparticles

Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1&#945; Nanoparticles

Cytokine therapy is a promising immunotherapeutic strategy that can produce robust antitumor immune responses in cancer patients. The proinflammatory ...