Name: defining gene functions in tumorigenesis by ex vivo ablation of floxed alleles in malignant peripheral nerve sheath tumor cells
Uploaded: 2021-08-25T14:00:00
Description: Watch this Scientific Journal Video about Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells at JoVE.com

This work was supported by grants from the National Institute of Neurological Diseases and Stroke (R01 NS048353, R01 NS109655), the National Cancer Institute (R01 CA122804), the Department of Defense (X81XWH-09-1-0086, W81XWH-11-1-0498, W81XWH-12-1-0164, W81XWH-14-1-0073, and W81XWH-15-1-0193), and The Children&#39;s Tumor Foundation (2014-04-001 and 2015-05-007).

Prior to performing any procedures with mice, all procedures must be reviewed and approved by the Institutional Animal Care and Use Committee. The protocol described in this manuscript was approved by the Institutional Animal Care and Use Committee of the Medical University of South Carolina. This protocol was performed by properly trained personnel following MUSC&#39;s institutional animal care guidelines.
1. Generation of mice that develop MPNSTs homozygous for Erbb4 flox</su...

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The detailed methods presented here were developed using a GEM model of MPNSTs. However, if the tumor tissue of interest can be dispersed into individual cells, these methodologies are easily adaptable for various tumor types arising in GEMs. It is important to ensure that the floxed allele does not result in i) decreased survival that can make it difficult to obtain sufficient mice to screen for tumors, or ii) increased tumor latency that can make it difficult to obtain enough tumor-bearing mice. If the floxed allele do...

Before the last two decades, the treatment of human cancers relied heavily on radiotherapy and chemotherapeutic agents that broadly targeted rapidly proliferating cellular populations by damaging DNA or inhibiting DNA synthesis. Although these approaches did inhibit cancer cell growth, they also had deleterious side effects on normal rapidly proliferating cell types such as intestinal epithelial cells and hair follicle cells. More recently, cancer therapy has begun to utilize chemotherapeutic agents that precisely target proteins within signaling pathways that are critically important for the growth of an individual patient&#39;s neoplasm. This approach, commonly referred to as &#34;Precision Medicine,&#34; has led to the development of an ever-expanding repertoire of monoclonal antibodies and small molecular inhibitors. These agents effectively inhibit tumor cell proliferation and survival while avoiding the deleterious side effects on normal cell types seen with conventional chemotherapeutic agents and radiotherapy. Monoclonal antibodies used for the treatment of human cancers most commonly target cell surface molecules such as growth factor receptors1 (e.g., the large family of membrane-spanning receptor tyrosine kinases) and immune response modulators2 (e.g., programmed cell death protein 1, programmed death-ligand 1). Small molecular inhibitors can inhibit either cell surface proteins or signaling proteins that are located intracellularly3. However, to effectively employ these new therapeutic agents, it must be established that a particular cancer is dependent upon the molecule that is being targeted by a candidate therapeutic agent.
Although these new therapeutic agents have more focused effects, many of them still inhibit the action of more than one protein. In addition, multiple agents with varying effectiveness and specificity are often available to target a specific protein. Consequently, during preclinical investigations, it is wise to use additional approaches such as genetic ablation to validate a candidate protein as a therapeutic target. One especially useful approach to validating a protein as a therapeutic target is to ablate the gene encoding the candidate protein in a genetically engineered animal model that develops the specific cancer type of interest. This approach can be relatively straightforward if mice with a null mutation (either a natural mutation, a genetically engineered null mutation [a &#34;knockout&#34;], or a null mutation introduced by a gene trap)&#160;are available, and the mice are viable into adulthood. Unfortunately, mice with a null mutation that meet these criteria are often not available, typically because the null mutation results in death embryonically or in the first days of postnatal life. In this circumstance, mice prone to develop the tumor type of interest may instead be crossed to mice in which key segments of the gene of interest are flanked by loxP sites (&#34;floxed&#34;), which allows the gene to be ablated by introducing a transgene expressing Cre recombinase into the tumor cells (a conditional knockout). This approach provides several advantages. First, if a Cre driver is available that is expressed in the tumor but not in the cell type that led to death in conventional knockouts, this approach can potentially validate the candidate therapeutic target. Second, ablating the gene encoding the candidate protein in tumor cells but not in other intratumoral elements such as tumor-associated fibroblasts or immune cells allows the investigator to distinguish between cell-autonomous and non-cell-autonomous effects of the therapeutic target. Finally, a tamoxifen-inducible Cre driver (CreERT2) allows the investigator to delete the gene of interest at different stages in tumor development and define the window in which the candidate therapeutic agent is most likely to be effective.
Unfortunately, there are also technical issues that can limit the use of conditional knockouts in tumors arising in GEM models. For instance, a Cre driver that is expressed in tumor cells and avoids gene deletion in normal cells essential for life may not be available. Another issue, which may be underestimated, is that Cre and CreERT2 drivers often variably ablate floxed alleles in mice, resulting in mosaicism for the null mutation in a GEM cancer. When this occurs, tumor cells in which the targeted gene has not been ablated will continue to proliferate rapidly, overgrowing the tumor cells with ablated alleles. Mosaicism in Cre driver lines can occur due to non-ubiquitous Cre expression in the lineage targeted and by failed recombination in individual cells independent of Cre expression4. This is a known phenomenon of Cre drivers that is cell-type dependent and should be considered during experimental design and data interpretation. Mosaicism can mask the effect of the knockout and lead an investigator to erroneously conclude that the gene of interest is not essential for tumor cell proliferation and/or survival and thus is not a valid therapeutic target.
Several of these problems were encountered in a previous study that attempted to determine whether the receptor tyrosine kinase erbB4 was a potential therapeutic target in MPNST cells5. In these studies, mice were used that express a transgene encoding the neuregulin-1 (NRG1) isoform glial growth factor-&#946;3 (GGF&#946;3) under the control of the Schwann cell-specific myelin protein zero promoter (P0-GGF&#946;3 mice). P0-GGF&#946;3 mice develop multiple plexiform neurofibromas that progress to become MPNSTs via a process that recapitulates the processes of neurofibroma pathogenesis and plexiform neurofibroma-MPNST progression seen in patients with the autosomal dominant tumor susceptibility syndrome neurofibromatosis type 1 (NF1)6. When crossed to mice with a Trp53 null mutation, the resulting P0-GGF&#946;3;Trp53+/- mice develop MPNSTs de novo as is seen in cis-Nf1+/-;Trp53+/- mice.
These MPNSTs recapitulate the progression from World Health Organization (WHO) grade II to WHO grade IV lesions seen in humans7. In P0-GGF&#946;3 mice, MPNSTs arise within pre-existing plexiform neurofibromas in the trigeminal nerve (58%) and spinal dorsal nerve roots (68%)7; the MPNSTs arising in P0-GGF&#946;3;Trp53+/- mice have a highly similar distribution. In humans, MPNSTs most commonly arise in the sciatic nerve followed by the brachial plexus, spinal nerve roots, vagus, femoral, median, sacral plexus, popliteal obturator, and posterior tibial and ulnar nerves8. This tumor distribution in these GEM models is somewhat different from what is seen in humans. However, the MPNSTs that arise in P0-GGF&#946;3 and P0-GGF&#946;3;Trp53+/- mice are histologically identical to human MPNSTs, carry many of the same mutations seen in human MPNSTs, and recapitulate the process of neurofibroma-MPNST progression seen in NF1 patients. The generation of P0-GGF&#946;3 or P0-GGF&#946;3;Trp53+/- mice that were Erbb4-/- was not feasible as mice with two Erbb4 null alleles die in utero at embryonic day 10.5 secondary to cardiac defects9. Because rescuing Erbb4 expression in the heart by introducing a cardiac-specific Erbb4 transgene (&#945;-myosin heavy chain (MHC)-Erbb4) results in viable Erbb4-/- mice10, the generation of mice with a complicated P0-GGF&#946;3;Trp53+/-;&#945;-MHC-Erbb4;Erbb4-/- genotype was attempted.
However, the matings did not produce mice in the expected Mendelian ratios, indicating that the desired genotype was deleterious. Therefore, the generation of P0-GGF&#946;3;Trp53+/- mice with floxed Erbb4 alleles11 and a CreERT2 driver was attempted to allow the deletion of Erbb4 in the MPNSTs arising in these mice. In these animals, numerous tumor cells with intact Erbb4 alleles were still present (mosaicism). The mosaicism observed could result from inefficient tamoxifen delivery, which resulted in differences in recombination efficiency within the tissue. The possibility of spontaneous compensatory mechanisms could further contribute to mosaicism in tamoxifen-mediated recombination by bypassing the requirement for Erbb4 expression. It is feasible that the loss of Trp53 makes tumor cells susceptible to additional spontaneous &#34;permissive&#34; mutations that could confuse the interpretation of the data. As it seemed likely that the Erbb4-intact MPNST cells would mask the consequences of ablating Erbb4 in other tumor cells, this approach was abandoned.
These obstacles led to the development of a methodology for ablating Erbb4 in very early passage MPNST cells using an adenovirus expressing Cre recombinase and eGFP. These cells can be separated from non-infected cells using FACS, which markedly reduces mosaicism for the ablated Erbb4 gene. Below, the methods used to achieve this, together with the methods used to assess the effects of gene ablation in vitro and in vivo, are described. The following protocol is an example of how to produce tumor-bearing mice that yield tumors carrying floxed alleles of embryonic lethal genes of interest for ex vivo excision prior to in vivo allograft tumor growth assessment. This includes a description of the approaches used to analyze the effect that Erbb4 ablation exerts on tumor cell proliferation, survival, and gene expression in vitro and proliferation, survival, and angiogenesis in orthotopic allografts.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ad5CMV-eGFP</td>
			<td>Gene Transfer Vector Core, Univ of Iowa</td>
			<td>VVC-U of Iowa-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Ad5CMVCre-eGFP</td>
			<td>Gene Transfer Vector Core, Univ of Iowa</td>
			<td>VVC-U of Iowa-1174</td>
			<td></td>
		</tr>
		<tr>
			<td>alexa 568 secondary antibody</td>
			<td>Thermo/Fisher</td>
			<td>GaR A11036</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioconductor Open Source Software for Bioninformatics</td>
			<td>Bioconductor</td>
			<td>http://www.bioconductor.org</td>
			<td>alternative statistical analysis tool used for step 4.4</td>
		</tr>
		<tr>
			<td>CD31</td>
			<td>Abcam</td>
			<td>ab28364</td>
			<td></td>
		</tr>
		<tr>
			<td>Celigo Image Cytometer</td>
			<td>Nexcelom Bioscience</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Stripper</td>
			<td>Corning</td>
			<td>25-056-Cl</td>
			<td>mixture of chelators</td>
		</tr>
		<tr>
			<td>DAB staining kit</td>
			<td>Vector Labs</td>
			<td>MP-7800</td>
			<td></td>
		</tr>
		<tr>
			<td>DAVID (Database for Annotation, Visualization, and Integrated Discovery)</td>
			<td>DAVID</td>
			<td>https://david.ncifcrf.gov</td>
			<td>functional enrichment analysis software used for step 4.5</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Corning</td>
			<td>15-013-Cl</td>
			<td></td>
		</tr>
		<tr>
			<td>DreamTaq and Buffer (Genotyping PCR)</td>
			<td>Thermo/Fisher</td>
			<td>EP0701 and K1072</td>
			<td></td>
		</tr>
		<tr>
			<td>erbB4 antibodies</td>
			<td>Santa Cruz</td>
			<td>sc-284</td>
			<td></td>
		</tr>
		<tr>
			<td>erbB4 antibodies</td>
			<td>Abcam</td>
			<td>ab35374</td>
			<td></td>
		</tr>
		<tr>
			<td>erbB4 antibodies</td>
			<td>Millipore</td>
			<td>HFR1: 05-1133</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS Sorter</td>
			<td>BD Biosciences</td>
			<td>Aria II</td>
			<td></td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>Sigma</td>
			<td>F6886</td>
			<td></td>
		</tr>
		<tr>
			<td>GenomeSpace Tools and Data Sources</td>
			<td>GenomeSpace</td>
			<td>https://genomespace.org/support/tools/</td>
			<td>general resource for several types of open source bioinformatic tools for step 4.5</td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Corning</td>
			<td>25-005-Cl</td>
			<td></td>
		</tr>
		<tr>
			<td>Gorilla Gene Ontology enRIchment anaLysis and visuaLizAtion tool</td>
			<td>Gorilla</td>
			<td>N/A, http://cbl-gorilla.cs.technion.ac.il</td>
			<td>functional enrichment analysis software used for step 4.5</td>
		</tr>
		<tr>
			<td>GSEA Gene Set Enrichment Analysis</td>
			<td>Broad Institute</td>
			<td>N/A, https://www.gsea-msigdb.org/gsea/index.jsp</td>
			<td>functional enrichment analysis software used for step 4.5</td>
		</tr>
		<tr>
			<td>HSD: Athymic Nude-FOxn1nu mice</td>
			<td>Envigo (Previously Harlan Labs)</td>
			<td>69</td>
			<td></td>
		</tr>
		<tr>
			<td>Illumina HiSeq2500 (next generation DNA sequencer)</td>
			<td>Illumina</td>
			<td>Hi Seq 2500</td>
			<td>DNA sequencer used for step 4.2</td>
		</tr>
		<tr>
			<td>Lasergene: ArrayStar Gene expression and variant analysis</td>
			<td>DNAStar LaserGene software</td>
			<td>N/A</td>
			<td>software statistical and normalization analysis used for step 4.4</td>
		</tr>
		<tr>
			<td>Lasergene: SeqMan NGen sequence alignment assembly</td>
			<td>software alignment used for step 4.3</td>
			<td>N/A</td>
			<td>software alignment used for step 4.3</td>
		</tr>
		<tr>
			<td>Matrigel, low growth factor basement membrane matrix</td>
			<td>Corning</td>
			<td>354230</td>
			<td></td>
		</tr>
		<tr>
			<td>NRG1-beta</td>
			<td>In house</td>
			<td>Generated by SLC, also commercially available from R &#38; D Systems(396-HB-050/CF).</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclear Stain Hoeschst 33342</td>
			<td>Thermo</td>
			<td>62249</td>
			<td></td>
		</tr>
		<tr>
			<td>Panther Gene Ontology Classification System</td>
			<td>Panther</td>
			<td>http://pantherdb.org</td>
			<td>functional enrichment analysis software used for step 4.5</td>
		</tr>
		<tr>
			<td>Partek (BWA aligner and analyzer)</td>
			<td>Partek, Ver 7</td>
			<td>N/A</td>
			<td>software alignment and statistical/normalization used for step 4.3</td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>Corning</td>
			<td>30-002-Cl</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer 1: 5&#8242;-CAAATGCTCTCTCTGTTCTTTGT 
			GTCTG- 3&#8242;</td>
			<td>Eurofins Genomics</td>
			<td>Primer 1 + 2: 250&#8201;bp ErbB4 null product and a 350&#8201;bp Floxed ErbB4 product;</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer 2: 5&#8242;-TTTTGCCAAGTTCTAATTCCATC 
			AGAAGC-3&#8242;</td>
			<td>Eurofins Genomics</td>
			<td>Primer 1 + 2: 250&#8201;bp ErbB4 null product and a 350&#8201;bp Floxed ErbB4 product;</td>
			<td></td>
		</tr>
		<tr>
			<td>
			Primer 3: 5&#8242;-TATTGTGTTCATCTATCATTGCA 
			ACCCAG-3&#8242;
			</td>
			<td>Eurofins Genomics</td>
			<td>Primer 1 + 3: 350&#8201;bp wild-type ErbB4 product.</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium Iodine</td>
			<td>Fisher</td>
			<td>51-351-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteom Profiler Phospho-Kinase Arrays</td>
			<td>R&#38;D Systems</td>
			<td>ARY003B</td>
			<td></td>
		</tr>
		<tr>
			<td>Real time glo</td>
			<td>Promega</td>
			<td>G9712</td>
			<td>bioluminescent cell viability assay</td>
		</tr>
		<tr>
			<td>ToppGene Suite</td>
			<td>ToppGene</td>
			<td>https://toppgene.cchmc.org</td>
			<td>functional enrichment analysis software used for step 4.5</td>
		</tr>
		<tr>
			<td>Trizol (acid-quanidinium-phenol and choloroform based reagent)</td>
			<td>Invitrogen</td>
			<td>15596026</td>
			<td></td>
		</tr>
		<tr>
			<td>Tyramide Signal Amplification Kit</td>
			<td>Perkin Elmer</td>
			<td>NEL721001EA</td>
			<td></td>
		</tr>
	</tbody>
</table>

defining gene functions in tumorigenesis by ex vivo ablation of floxed alleles in malignant peripheral nerve sheath tumor cells

The development of new drugs that precisely target key proteins in human cancers is fundamentally altering cancer therapeutics. However, before these drugs can be used, their target proteins must be validated as therapeutic targets in specific cancer types. This validation is often performed by knocking out the gene encoding the candidate therapeutic target in a genetically engineered mouse (GEM) model of cancer and determining what effect this has on tumor growth. Unfortunately, technical issues such as embryonic lethality in conventional knockouts and mosaicism in conditional knockouts often limit this approach. To overcome these limitations, an approach to ablating a floxed embryonic lethal gene of interest in short-term cultures of malignant peripheral nerve sheath tumors (MPNSTs) generated in a GEM model was developed.
This paper describes how to establish a mouse model with the appropriate genotype, derive short-term tumor cultures from these animals, and then ablate the floxed embryonic lethal gene using an adenoviral vector that expresses Cre recombinase and enhanced green fluorescent protein (eGFP). Purification of cells transduced with adenovirus using fluorescence-activated cell sorting (FACS) and the quantification of the effects that gene ablation exerts on cellular proliferation, viability, the transcriptome, and orthotopic allograft growth is then detailed. These methodologies provide an effective and generalizable approach to identifying and validating therapeutic targets in vitro and in vivo. These approaches also provide a renewable source of low-passage tumor-derived cells with reduced in vitro growth artifacts. This allows the biological role of the targeted gene to be studied in diverse biologic processes such as migration, invasion, metastasis, and intercellular communication mediated by the secretome.

Figure 4&#160;illustrates a typical result obtained when transducing P0-GGF&#946;3;Trp53-/+;Erbb4fl/fl MPNST cells with either the Ad5CMV-eGFP adenovirus or Ad5CMV-Cre/eGFP adenovirus (Figure 4A). Cultures are viewed with fluorescence microscopy to identify eGFP-expressing cells and by phase-contrast microscopy to determine the total number of cells present in the same field at 10x (top) and 40x (bottom). The pe...

Watch this Scientific Journal Video about Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells at JoVE.com

Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells

Here, we present a protocol for performing gene knockouts that are embryonic lethal in vivo in genetically engineered mouse model-derived tumors and then assessing the effect that the knockout has on tumor growth, proliferation, survival, migration, invasion, and the transcriptome in vitro and in vivo.

Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells

The development of new drugs that precisely target key proteins in human cancers is fundamentally altering cancer therapeutics. However, before these ...

This protocol provides an alternative in vivo method to study the tumor growth role of an embryonic lethal gene knockout. The advantage of this method is that gene knockouts that are normally embryonic lethal can be performed in tumors derived from a genetically engineered mouse model to study their loss of function effects in vivo. This technique can be applied to diseases that require studying the in vivo role of embryonic essential genes unattainable from traditional gene knockout mouse models.Generating the animal model takes time and empirical testing is required to determine the optimal MOI for adenoviral infection to ensure that appropriate recombination is achieved. To begin with, identify the mouse with the desired genotype. When a tumor-bearing mouse reaches its humane endpoint, euthanize the mouse and use a sterile scalpel knife to remove the tumor.Dissect the tumor into three sections in bread loaf-style cuts using a scalpel knife to generate the tissue segments for formalin fixation paraffin embedding early passage culture generation and flash freezing for analytical analyses. Stain a five-micrometer thick formalin-fixed, paraffin-embedded tissue section with hematoxylin and eosin. Once a pathologist confirms the tumor, perform the immunohistochemical staining to confirm the tumor diagnosis.Place the freshly-collected malignant peripheral nerve sheath tumor tissue in 10 milliliters of ice-cold sterile PBS on ice, and then transfer it to a sterile work area to establish early passage cultures. Mince the tumor tissue into two to four-millimeter pieces and triturate eight to 10 times in a 10-square centimeter treated tissue culture dish containing 10 milliliters of growth medium. Culture these tissues in high glucose DMEM-10 growth medium supplemented with 10 nanomolar neuregulin-1 beta and two micromolar forskolin.Seed the early passaged tumor cells at a density of 1.5 times 10 to the sixth cells per one 10-square centimeter treated tissue culture dish containing DMEM-10 growth medium. After 12 to 16 hours, wash the adherent cultures with two to four milliliters of DPBS, and infect the cells with adenovirus at approximately 400 plaque-forming units per cell in 10 milliliters of serum-free DMEM. After 48 hours of the infection, briefly check the cells for EGFP signal on a fluorescence microscope to ensure efficient infection with approximately 50 to 100%positive cells, and then FACS sort the EGFP-positive infected cells.After FACS sorting, let the cells recover in a tissue culture incubator for at least 24 to 48 hours, and then prepare the cells for in vitro cell-based analyses or in vivo grafting. Confirm Erbb4 deletion by performing PCR using genomic DNA isolated from a portion of the sorted EGFP-positive cells. Perform the proliferation assays over the next seven days on sorted tumor cells plated in a 96-well plate using an image-based automated cytometer.Stain the cells with hooks and propidium iodide dyes and capture the images of the cells using an automated plate reader to count the number of live and dead cells in each well. Isolate total RNA from sorted tumor cells using standard acid guanidinium phenol and chloroform-based method. Open the software to perform the RNA sequence alignment following the program-specific steps using the default settings.Select the analysis method, RNA seek, and then select the mouse reference genome. Upload the BED file if provided one by the sequencing core. Upload the FASTQ sequencing files and assign unique replicate names to the files.Group replicate the FASTQ files, and designate the files to a replicate set. For the statistical and normalization analysis to identify the differential gene expression signals with robust statistical power, open the analysis software and select the GFP FASTQ files as the control data set. Next, select DESeq2 as the statistical and normalization method and start the assembly and analysis.Use the gene ontology data sets integrated into the open access functional enrichment analysis tools to perform the functional enrichment analysis on the identified statistically significant Erbb4-mediated DEG to determine the biological and pathway significance of Erbb4 gene loss. Inject the tumor cells into the sciatic nerve of the anesthetized mouse to assess the in vivo allograft growth potential in post-infected cells. Once the tumor reaches the required size, dissect the tumor from a euthanized mouse in two-three sections as demonstrated previously.Fix one tissue section in 4%Paraformaldehyde overnight, and then embed the fixed section in the paraffin. After making five-micrometer thick sections from the FFPE tissue, mount the section on the slides and confirm the presence of tumor cells in the grafted tissue using H&E and immunostaining as demonstrated previously. To confirm in vivo Erbb4 expression differences between the two experimental conditions, stain the formalin-fixed, paraffin-embedded tissue with Erbb4-specific antibodies.The transduction of the malignant peripheral nerve sheath tumor tissue cells with adenovirus was analyzed. The EGFP expressing cells were detected using fluorescence microscopy, and the total number of cells was determined using phase-contrast microscopy. After the Cre-mediated gene ablation, depending on the transfection efficiency, the PCR genotyping showed a complete gene knockout and a heterogeneous population of induced and uninduced cells compared to the control transduction.The characteristic histopathology found in the orthotopic allografts of malignant peripheral nerve sheath tumor tissues compared to the tumors derived from the original genetically-engineered animal models demonstrated a difference in the cell morphology. The Erbb4 ablation decreased cellular density and cellular proliferation in adeno5-CMV-Cre-ablated cells control adeno5-CMV-GFP-transduced cells. The expression of Erbb4 in the tumor cells transduced with adeno5-CMV-Cre, or EGFP virus, was decreased in the result in adeno5-CMV-Cre transduced allografts compared to the control adeno5-CMV-EGFP-treated allograft tumors.The vascular density was assessed via immunoreactivity detection for CD31, and the representative results showed markedly reduced vascular density in allografts of adeno5-CMV-Cre-transduced cells compared to the control adeno5-CMV-EGFP-transduced cells. After gene ablation, the cells can be used for many different types of cell-based assays such as proliferation, migration, 3D growth, or microenvironment alterations, as well as for in vivo orthotopic injections.

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Research

JoVE Journal

Cancer Research

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.

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

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the tumor microenvironment and respond adequately to tumor antigens. The recent development of improved imaging approaches, such as 2-photon microscopy, and the use of powerful mouse tumor models have shed light on some of the mechanisms that regulate anti-tumor T cell activities. Whereas such systems have provided valuable insights, they do not always predict human responses. In human, our knowledge in the field mainly comes from a description of fixed tumor samples from human patients, as well as in vitro studies. However, in vitro models lack the complex three-dimensional tumor milieu and, therefore, are incomplete approximations of in vivo T cell activities. Fresh slices made from explanted tissue represent a complex multi-cellular tumor environment that can act as an important link between co-cultured studies and animal models. Originally set up in murine lymph nodes1 and previously described in a JoVE article2, this approach has now been transposed to human tumors to examine the dynamics of both plated3&#160;as well as resident&#160;T cells4.
Here, a protocol for the preparation of human lung tumor slices, immunostaining of resident CD8 T and tumor cells, and tracking of CD8 T lymphocytes within the tumor microenvironment using confocal microscopy is described. This system is uniquely placed to screen for novel immunotherapy agents favoring T cell migration in tumors.

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

This protocol describes a method to image resident tumor-infiltrating CD8 T cells labeled with fluorescently coupled antibodies within human lung tumor slices. This technique permits real-time analyses of CD8 T cell migration using confocal microscopy.

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within 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.

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

Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for safe and effective therapies tailored to individual tumor immune profiles. Oncolytic viruses enable the induction of anti-tumor immune responses as well as tumor-restricted gene expression. This protocol describes the generation and ex vivo analysis of immunomodulatory oncolytic vectors. Focusing on measles vaccine viruses encoding bispecific T cell engagers as an example, the general methodology can be adapted to other virus species and transgenes. The presented workflow includes the design, cloning, rescue, and propagation of recombinant viruses. Assays to analyze replication kinetics and lytic activity of the vector as well as functionality of the isolated immunomodulator ex vivo are included, thus facilitating the generation of novel agents for further development in preclinical models and ultimately clinical translation.

This protocol describes a detailed workflow for the generation and ex vivo characterization of oncolytic viruses for expression of immunomodulators, using measles viruses encoding bispecific T cell engagers as an example. Application and adaptation to other vector platforms and transgenes will accelerate the development of novel immunovirotherapeutics for clinical translation.

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated molecular patterns and inducing the production of type I interferon. Immunotherapies, including checkpoint inhibition, primarily rely on preexisting tumor-specific T cells to unfold a therapeutic effect. Thus, synergistic therapeutic approaches that exploit immunogenic cell death as an intrinsic anti-cancer vaccine may improve their responsiveness. However, the spectrum of immunogenic factors released by cells under therapy-induced stress remains incompletely characterized, especially regarding extracellular vesicles (EVs). EVs, nano-scale membranous particles emitted from virtually all cells, are considered to facilitate intercellular communication and, in cancer, have been shown to mediate cross-priming against tumor antigens. To assess the immunogenic effect of EVs derived from tumors under various conditions, adaptable, scalable, and valid methods are sought-for. Therefore, herein a relatively easy and robust approach is presented to assess EVs' in vivo immunogenicity. The protocol is based on flow cytometry analysis of splenic T cells after in vivo immunization of mice with EVs, isolated by precipitation-based assays from tumor cell cultures under therapy or steady-state conditions. For example, this work shows that oxaliplatin exposure of B16-OVA murine melanoma cells resulted in the release of immunogenic EVs that can mediate the activation of tumor-reactive cytotoxic T cells. Hence, screening of EVs via in vivo immunization and flow cytometry identifies conditions under which immunogenic EVs can emerge. Identifying conditions of immunogenic EV release provides an essential prerequisite to testing EVs' therapeutic efficacy against cancer and exploring the underlying molecular mechanisms to ultimately unveil new insights into EVs' role in cancer immunology.

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

This manuscript describes how to assess in vivo immunogenicity of tumor cell-derived extracellular vesicles (EVs) using flow cytometry. EVs derived from tumors undergoing treatment-induced immunogenic cell death seem particularly relevant in tumor immunosurveillance. This protocol exemplifies the assessment of oxaliplatin-induced immunostimulatory tumor EVs but can be adapted to various settings.

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated ...

Ex Vivo Organoid Model of Adenovirus-Cre Mediated Gene Deletions in Mouse Urothelial Cells

Bladder cancer is an understudied area, particularly in genetically engineered mouse models (GEMMs). Inbred GEMMs with tissue-specific Cre and loxP sites have been the gold standards for conditional or inducible gene targeting. To provide faster and more efficient experimental models, an ex vivo organoid culture system is developed using adenovirus Cre and normal urothelial cells carrying multiple loxP alleles of the tumor suppressors Trp53, Pten, and Rb1. Normal urothelial cells are enzymatically disassociated from four bladders of triple floxed mice (Trp53f/f: Ptenf/f: Rb1f/f). The urothelial cells are transduced ex vivo with adenovirus-Cre driven by a CMV promoter (Ad5CMVCre). The transduced bladder organoids are cultured, propagated, and characterized in vitro and in vivo. PCR is used to confirm gene deletions in Trp53, Pten, and Rb1. Immunofluorescence (IF) staining of organoids demonstrates positive expression of urothelial lineage markers (CK5 and p63). The organoids are injected subcutaneously into host mice for tumor expansion and serial passages. The immunohistochemistry (IHC) of xenografts exhibits positive expression of CK7, CK5, and p63 and negative expression of CK8 and Uroplakin 3. In summary, adenovirus-mediated gene deletion from mouse urothelial cells engineered with loxP sites is an efficient method to rapidly test the tumorigenic potential of defined genetic alterations.

Ex Vivo Organoid Model of Adenovirus-Cre Mediated Gene Deletions in Mouse Urothelial Cells

This protocol describes the process of the generation and characterization of mouse urothelial organoids harboring deletions in genes of interest. The methods include harvesting mouse urothelial cells, ex vivo transduction with adenovirus driving Cre expression with a CMV promoter, and in vitro as well as in vivo characterization.

Bladder cancer is an understudied area, particularly in genetically engineered mouse models (GEMMs). Inbred GEMMs with tissue-specific Cre and loxP sites ...

Genome-Scale Screening to Identify and Compare Therapeutic Targets in Tumors

Genetic Profiling and Genome-Scale Dropout Screening to Identify Therapeutic Targets in Mouse Models of Malignant Peripheral Nerve Sheath Tumor

Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are derived from Schwann cells or their precursors. In patients with the tumor susceptibility syndrome neurofibromatosis type 1 (NF1), MPNSTs are the most common malignancy and the leading cause of death. These rare and aggressive soft-tissue sarcomas offer a stark future, with 5-year disease-free survival rates of 34-60%. Treatment options for individuals with MPNSTs are disappointingly limited, with disfiguring surgery being the foremost treatment option. Many once-promising therapies such as tipifarnib, an inhibitor of Ras signaling, have failed clinically. Likewise, phase II clinical trials with erlotinib, which targets the epidermal growth factor (EFGR), and sorafenib, which targets the vascular endothelial growth factor receptor (VEGF), platelet-derived growth factor receptor (PDGF), and Raf, in combination with standard chemotherapy, have also failed to produce a response in patients. 
In recent years, functional genomic screening methods combined with genetic profiling of cancer cell lines have proven useful for identifying essential cytoplasmic signaling pathways and the development of target-specific therapies. In the case of rare tumor types, a variation of this approach known as cross-species comparative oncogenomics is increasingly being used to identify novel therapeutic targets. In cross-species comparative oncogenomics, genetic profiling and functional genomics are performed in genetically engineered mouse (GEM) models and the results are then validated in the rare human specimens and cell lines that are available. 
This paper describes how to identify candidate driver gene mutations in human and mouse MPNST cells using whole exome sequencing (WES). We then describe how to perform genome-scale shRNA screens to identify and compare critical signaling pathways in mouse and human MPNST cells and identify druggable targets in these pathways. These methodologies provide an effective approach to identifying new therapeutic targets in a variety of human cancer types.

Author Spotlight: Finding New Therapeutic Targets for Malignant Peripheral Nerve Sheath Tumor Through Genome-Scale shRNA Screens 

We have developed a cross-species comparative oncogenomics approach utilizing genomic analyses and functional genomic screens to identify and compare therapeutic targets in tumors arising in genetically engineered mouse models and the corresponding human tumor type.

Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are derived from Schwann cells or their precursors. In patients with the tumor susceptibility syndrome ...

Murine Model for Studying Leptomeningeal Disease Using Circulating Tumor Cells

Ex Vivo Culture of Circulating Tumor Cells in the Cerebral Spinal Fluid from Melanoma Patients to Study Melanoma&#45;Associated Leptomeningeal Disease

Melanoma-associated leptomeningeal disease (M-LMD) occurs when circulating tumor cells (CTCs) enter into the cerebral spinal fluid (CSF) and colonize the meninges, the membrane layers that protect the brain and the spinal cord. Once established, the prognosis for M-LMD patients is dismal, with overall survival ranging from weeks to months. This is primarily due to a paucity in our understanding of the disease and, as a consequence, the availability of effective treatment options. Defining the underlying biology of M-LMD will significantly improve the ability to adapt available therapies for M-LMD treatment or design novel inhibitors for this universally fatal disease. A major barrier, however, lies in obtaining sufficient quantities of CTCs from the patient-derived CSF (CSF-CTCs) to conduct preclinical experiments, such as molecular characterization, functional analysis, and in vivo efficacy studies. Culturing CSF-CTCs ex vivo has also proven to be challenging. To address this, a novel protocol for the culture of patient-derived M-LMD CSF-CTCs ex vivo and in vivo is developed. The incorporation of conditioned media produced by human meningeal cells (HMCs) is found to be critical to the procedure. Cytokine array analysis reveals that factors produced by HMCs, such as insulin-like growth factor-binding proteins (IGFBPs) and vascular endothelial growth factor-A (VEGF-A), are important in supporting CSF-CTC survival ex vivo. Here, the usefulness of the isolated patient-derived CSF-CTC lines is demonstrated in determining the efficacy of inhibitors that target the insulin-like growth factor (IGF) and mitogen-activated protein kinase (MAPK) signaling pathways. In addition, the ability to intrathecally inoculate these cells in vivo to establish murine models of M-LMD that can be employed for preclinical testing of approved or novel therapies is shown. These tools can help unravel the underlying biology driving CSF-CTC establishment in the meninges and identify novel therapies to reduce the morbidity and mortality associated with M-LMD.

Author Spotlight: Assessing the Potential of Circulating Tumor Cells in Leptomeningeal Disease Research

This article describes a protocol for the propagation of cerebral spinal fluid-circulating tumor cells (CSF-CTCs) collected from patients with melanoma-associated leptomeningeal disease (M-LMD) to develop preclinical models to study M-LMD.

Melanoma-associated leptomeningeal disease (M-LMD) occurs when circulating tumor cells (CTCs) enter into the cerebral spinal fluid (CSF) and colonize the ...

Prognostic Significance of Immune Cell Infiltration in Hepatocellular Carcinoma

Mast Cells in the Microenvironment of Hepatocellular Carcinoma Confer Favorable Prognosis: A Retrospective Study using QuPath Image Analysis Software

The insights provided by in-situ detection of immune cells within hepatocellular carcinoma (HCC) might present information on patient outcomes. Studies investigating the expression and localization of immune cells within tumor tissues are associated with several challenges, including a lack of precise annotation for tumor regions and random selection of microscopic fields of view. QuPath is an open-source, user-friendly software that could meet the growing need for digital pathology in whole-slide image (WSI) analysis.
The infiltration of HCC and adjacent tissues by CD1a+ immature dendritic cells (iDCs), CD117+ mast cells, and NKp46+ natural killer cells (NKs) cells was assessed immunohistochemically in representative specimens of 67 patients with HCC who underwent curative resection. The area fraction (AF) of positively stained cells was assessed automatically in WSIs using QuPath in the tumor center (TC), inner margin (IM), outer margin (OM), and peritumor (PT) area. The prognostic significance of immune cells was evaluated for time to recurrence (TTR), disease-free survival (DFS), and overall survival (OS).
The AF of mast cells was significantly greater than the AF of NKs, and the AF of iDCs was significantly lower compared to NKs in each region of interest. High AFs of mast cells in the IM and PT areas were associated with longer DFS. In addition, high AF of mast cells in IM was associated with longer OS.
Computer-assisted analysis using this software is a suitable tool for obtaining prognostic information for tumor-infiltrating immune cells (iDCs, mast cells, and NKs) in different regions of HCC after resection. Mast cells displayed the greatest AF in all regions of interest (ROIs). Mast cells in the peritumor region and IM showed a positive prognostic significance.

Author Spotlight: Investigating Immune Cell Dynamics in the Tumor Microenvironment &amp;#8212; Challenges and Innovations in Cancer Prognosis

The presence of mast cells in the inner margin and peritumor areas of hepatocellular carcinoma after resection confers a favorable prognosis. This study endorses QuPath image analysis software as a promising platform that could meet the need for reproducibility, consistency, and accuracy in digital pathology.

The insights provided by in-situ detection of immune cells within hepatocellular carcinoma (HCC) might present information on patient outcomes. Studies ...