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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Cancer Research

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 endothelia typically lack the fluid dynamics that invading cells are otherwise exposed to in vivo. However, in vivo systems such as mouse models, though more physiologically relevant, require longer experimental timescales and present unique challenges associated with monitoring and data analysis. Here we describe a zebrafish assay that seeks to bridge this technical gap by allowing for the rapid assessment of cancer cell vascular invasion and extravasation. The approach involves injecting fluorescent cancer cells into the precardiac sinus of transparent 2-day old zebrafish embryos whose vasculature is marked by a contrasting fluorescent reporter. Following injection, the cancer cells must survive in circulation and subsequently extravasate from vessels into tissues in the caudal region of the embryo. Extravasated cancer cells are efficiently identified and scored in live embryos via fluorescence imaging at a fixed timepoint. This technique can be modified to study intravasation and/or competition amongst a heterogeneous mixture of cancer cells by changing the injection site to the yolk sac. Together, these methods can evaluate a hallmark behavior of cancer cells and help uncover mechanisms indicative of malignant progression to the metastatic phenotype.

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

This method utilizes zebrafish embryos to efficiently test the vascular invasive ability of cancer cells. Fluorescent cancer cells are injected into the precardiac sinus or yolk sac of developing embryos. Cancer cell vascular invasion and extravasation is assessed via fluorescence microscopy of the tail region 24 to 96 hr later.

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

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.

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

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human central nervous system (CNS). However, both the cytological origin and the evolutionary process of HBs (including neovascularization) remain controversial, and anti-angiogenesis for VHL-HBs, based on classic HB angiogenesis, have produced disappointing results in clinical trials. One major obstacle to the successful clinical translation of anti-vascular treatment is the lack of a thorough understanding of neovascularization in this vascular tumor. In this article, we present a comprehensive procedure to evaluate in vitro whether classic tumor angiogenesis exists in HBs, as well as its role in HBs. With this procedure, researchers can accurately understand the complexity of HB neovascularization and identify the function of this common form of angiogenesis in HBs. These protocols can be used to evaluate the most promising anti-vascular therapy for tumors, which has high translational potential either for tumors treatment or for aiding in the optimization of the anti-angiogenic treatment for HBs in future translations. The results highlight the complexity of HB neovascularization and suggest that this common form angiogenesis is only a complementary mechanism in HB neovascularization.

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

This paper presents a comprehensive procedure to evaluate in vitro whether classic tumor angiogenesis exists in hemangioblastomas (HBs) and its role in HBs. The results highlight the complexity of HB-neovascularization and suggest that this common form of angiogenesis is only a complementary mechanism in the HB-neovascularization.

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

Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in Prostate Cancer

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction pathways and is tightly regulated by kinases and phosphatases. Therefore, characterizing the phosphoproteome may provide insights into identifying novel targets and biomarkers for oncologic therapy. Mass spectrometry provides a way to globally detect and quantify thousands of unique phosphorylation events. However, phosphopeptides are much less abundant than non-phosphopeptides, making biochemical analysis more challenging. To overcome this limitation, methods to enrich phosphopeptides prior to the mass spectrometry analysis are required. We describe a procedure to extract and digest proteins from tissue to yield peptides, followed by an enrichment for phosphotyrosine (pY) and phosphoserine/threonine (pST) peptides using an antibody-based and/or titanium dioxide (TiO2)-based enrichment method. After the sample preparation and mass spectrometry, we subsequently identify and quantify phosphopeptides using liquid chromatography-mass spectrometry and analysis software.

This protocol describes a procedure to extract and enrich phosphopeptides from prostate cancer cell lines or tissues for an analysis of the phosphoproteome via mass spectrometry-based proteomics.

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction ...

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.

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 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 malignant brain tumor with no cure, has up to a half the tumor mass TAMs. TAMs can be pro-tumoral or anti-tumoral, depending on the activation of specific genes in the cells. Genetic mutations in the tumors, through regulating cytokine expression, can affect recruitment of TAMs to the tumor microenvironment. Here, we describe a quantitative cell-based assay to assess macrophage recruitment by the conditioned medium from the tumor cells. This assay uses the human macrophage cell line MV-4-11 to study macrophage attraction by the conditioned medium from glioblastoma, allowing for high reproducibility and low variability. Data generated with this assay can contribute to a better understanding of the interaction between the tumor and the tumor microenvironment. Similar assay can be used to assess interaction between the tumor cells and other immune cells, including T cells and natural killer (NK) cells.

This article represents a useful in vitro assay to evaluate the capability of conditioned medium from tumor cells to attract macrophages.

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 growth and metastasis. Paracrine signals can involve cell-cell contact between different cell types, such as PD-L1 expressed on the surface of tumors interacting directly with PD-1 on the surface of T cells, or the secretion of ligands by a tumor cell to affect an immune cell. Here we describe a co-culture method to interrogate the effects of tumor-secreted ligands on immune cell (macrophage) activation. This straightforward procedure utilizes commercially available 0.4 &#181;m polycarbonate membrane permeable supports and standard tissue culture plates. In the process described, macrophages are cultured in the upper chamber and tumor cells in the lower chamber. The presence of the 0.4 &#181;m barrier allows for the study of intercellular signaling without the confounding variable of physical contact, because the two cell types share the same medium and exposure to paracrine ligands. This approach can be combined with others, such as genetic alterations of the macrophage (e.g., isolation from genetic knock-out mice) or tumor (e.g., CRISPR-mediated alterations) to study the role of specific secreted factors and receptors. The approach also lends itself to standard molecular biological analyses such as quantitative reverse transcription polymerase chain reaction (qRT-PCR) or Western blot analysis, without the need for flow sorting to separate the two cell populations. Enzyme-linked immunosorbent assays (ELISAs) can similarly be utilized to measure secreted ligands to better understand the dynamic interaction of cell signaling in the multiple cell type context. Duration of co-culture can also be varied for the study of temporally regulated events. This co-culture method is a robust tool that facilitates the study of tumor-secreted signals in the immune context.

Here, we present a method using permeable membrane supports to facilitate the study of non-contact paracrine signaling used by tumor cells to suppress the immune response. The system is amenable to studying the role of tumor-secreted factors in dampening macrophage activation.

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

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 particularly devastating, difficult to treat, and confer a poor prognosis. While the precise incidence of brain metastases in the United States remains hard to estimate, it is likely to increase as extracranial therapies continue to become more efficacious in treating cancer. Thus, it is necessary to identify and develop novel therapeutic approaches to treat metastasis at this site. To this end, intracranial injection of cancer cells has become a well-established method in which to model brain metastasis. Previously, the inability to directly measure tumor growth has been a technical hindrance to this model; however, increasing availability and quality of small animal imaging modalities, such as magnetic resonance imaging (MRI), are vastly improving the ability to monitor tumor growth over time and infer changes within the brain during the experimental period. Herein, intracranial injection of murine mammary tumor cells into immunocompetent mice followed by MRI is demonstrated. The presented injection approach utilizes isoflurane anesthesia and a stereotactic setup with a digitally controlled, automated drill and needle injection to enhance precision, and reduce technical error. MRI is measured over time using a 9.4 Tesla instrument in The Ohio State University James Comprehensive Cancer Center Small Animal Imaging Shared Resource. Tumor volume measurements are demonstrated at each time point through use of ImageJ. Overall, this intracranial injection approach allows for precise injection, day-to-day monitoring, and accurate tumor volume measurements, which combined greatly enhance the utility of this model system to test novel hypotheses on the drivers of brain metastases.

Intracranial brain metastasis modeling is complicated by an inability to monitor tumor size and response to treatment with precise and timely methods. The presented methodology couples intracranial tumor injection with magnetic resonance imaging analysis, which when combined, cultivates precise and consistent injections, enhanced animal monitoring, and accurate tumor volume measurements.

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 a useful measure to provide an accurate analysis of the methylation status of gastrointestinal (GI) malignancies. Given the multiple potential translational uses of DNA methylation analysis, practicing clinicians and others new to DNA methylation studies need to be able to understand step by step how these genome-wide analyses are performed. The goal of this protocol is to provide a detailed description of how this method is used for the biomarker identification in GI malignancies. Importantly, we describe three critical steps that are needed to obtain accurate results during genome-wide analysis. Clearly and concisely written, these three methods are often lacking and not noticeable to those new to epigenetic studies. We used 48 samples of a GI malignancy (gastric cancer) to highlight practically how genome-wide DNA methylation analysis can be performed for GI malignancies.

Herein, we describe a procedure for genome-wide analysis of DNA methylation in gastrointestinal cancers. The procedure is of relevance to studies that investigate relationships between methylation patterns of genes and factors contributing to carcinogenesis in gastrointestinal cancers.

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

Cytokine therapy is a promising immunotherapeutic strategy that can produce robust antitumor immune responses in cancer patients. The proinflammatory cytokine interleukin-1 alpha (IL-1&#945;) has been evaluated as an anticancer agent in several preclinical and clinical studies. However, dose-limiting toxicities, including flu-like symptoms and hypotension, have dampened the enthusiasm for this therapeutic strategy. Polyanhydride nanoparticle (NP)-based delivery of IL-1&#945; would represent an effective approach in this context since this may allow for a slow and controlled release of IL-1&#945; systemically while reducing toxic side effects. Here an analysis of the antitumor activity of IL-1&#945;-loaded polyanhydride NPs in a head and neck squamous cell carcinoma (HNSCC) syngeneic mouse model is described. Murine oropharyngeal epithelial cells stably expressing HPV16 E6/E7 together with hRAS and luciferase (mEERL) cells were injected subcutaneously into the right flank of C57BL/6J mice. Once tumors reached 3-4 mm in any direction, a 1.5% IL-1a - loaded 20:80 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane:1,6-bis(p-carboxyphenoxy)hexane (CPTEG: CPH) nanoparticle (IL-1&#945;-NP) formulation was administered to mice intraperitoneally. Tumor size and body weight were continuously measured until tumor size or weight loss reached euthanasia criteria. Blood samples were taken to evaluate antitumor immune responses by submandibular venipuncture, and inflammatory cytokines were measured through cytokine multiplex assays. Tumor and inguinal lymph nodes were resected and homogenized into a single-cell suspension to analyze various immune cells through multicolor flow cytometry. These standard methods will allow investigators to study the antitumor immune response and potential mechanism of immunostimulatory NPs and other immunotherapy agents for cancer treatment.

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

A standard protocol is described to study the antitumor activity and associated toxicity of IL-1&#945; in a syngeneic mouse model of HNSCC.

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