The authors would like to thank Carmina Espiritu and Robin E. Taylor for their support in automated dissection development as well as the Genentech Pathology Core Laboratory staff that supported this work.

Prior to initiation, obtain appropriate tissue specimens according to Institutional Review Board (IRB) protocols. All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Genentech, Inc.
1. Tissue and slide preparation
<ol>
	<li>Select FFPE or fresh frozen tissue blocks and utilize the corresponding processing method below.</li>
	<li>Cut tissue block sections onto positively charged glass slides at the desired thickness. Serially section the FFPE tissue in ribbons with the first reference section cut at a thickness appropriate for H&#38;E staining (i.e., 4 &#181;m) followed by 1-4 sections at a thickness ranging from 4-20 &#181;m based on the need and tissue availability. Collect the tissue sections onto positively charged glass microscope slides. 
	NOTE: Fresh frozen reference tissue sections should be stained immediately with Hematoxylin and Eosin (H&#38;E) using routine protocols for frozen sections and the unstained frozen sections held at -20 &#176;C until they are needed for harvest.</li>
	<li>Allow all the FFPE sections to dry at room temperature overnight.</li>
	<li>Bake the FFPE reference slides at 60 &#176;C for 30 min and then stain with H&#38;E using routine protocols.</li>
	<li>Scan the H&#38;E stained slides on a whole slide imager at 20x magnification or greater.</li>
	<li>Annotate the scanned slide images for tumor regions of interest using a vendor provided viewing platform or open-source viewer. Export these annotations as either a low-magnification screenshot or save them as a metadata file containing X-Y pixel coordinates corresponding to polygon vertices. 
	NOTE: The former is less technically challenging to work with, but the latter offers advantages in process automation.</li>
	<li>Create digital masks of the annotated regions of interest in line with the approach used and export the manual annotations. 
	&#8203;NOTE: If a screenshot/image of the annotations is used, simple image processing software can be used to select a region and to fill in the entire selection. Using X-Y coordinates for each ROI requires the use of a programming language to read both the image data and polygon coordinates to create a low-mag image with filled in regions of interest. The user should work with the automated dissection instrument vendor to establish a process based on their individual software availability and needs. If scanning, digital slide annotation and/or digital mask creation is unavailable, careful on-slide annotation using a marker can be performed and used in place of a digital mask as a reference image. Pseudocode for digital mask creation has been provided in Supplementary File 1.</li>
</ol>
2. Automated tissue dissection
<ol>
	<li>Place the unstained sample tissue slides onto the stage in the first through fourth slide positions when using digital slide reference. When using on-slide annotation rather than a digital option, place the unstained sample tissue slides onto the stage in the second through fourth slide positions with a reference slide in the first position.</li>
	<li>Create a milling job using the automated tissue dissection software: Job Selection &#62; Create New Job &#62; Case ID&#160;&#62; Name the milling job; go to Thickness &#62; Section Thickness using the up or down arrow tab; then go to Tissue Preparation &#62; Paraffinized or Deparaffinized, Reference Image &#62; From File &#62; Import Image &#62; File to import from the dropdown as the digital reference, if applicable. Select From Stage for on stage slide reference. When fields are complete, scan the stage by selecting the Scan Stage button in the bottom-right corner to capture each sample tissue slide in the first through fourth position.</li>
	<li>Select the tissue area for image capture.
	<ol>
		<li>If using an on-stage reference, drag the box from one corner to the opposite to create a rectangular area over tissue. Select the circular bubble under the rectangular area to capture the stage reference image. If using a digital reference image, overlay the image on the rectangular area selected. Resize and align the digital reference grossly in course zoom to best match the size and position over the sample tissue.</li>
		<li>Copy this rectangle field onto remaining sample tissue slides in the second through fourth slide positions by selecting the copy option in the upper-right corner of the reference image. Align and resize grossly as necessary. 
		NOTE: When using an on-slide reference image rather than a digital mask, select which slide on the stage should be used as the reference.</li>
	</ol>
	</li>
	<li>Align the reference and sample slides
	<ol>
		<li>When the reference image is grossly aligned on tissue sample slides in all slide positions, select the Scan Stage button in the lower-right corner of the screen to move into the fine adjustment step. Select the first stage position and Transform tool icon (the third icon down in the right-hand toolbar) to make fine alignment and zoom adjustments of the reference to best match the sample slide overlay. Use the Reference to Sample sliding bar at the bottom of the screen toggling between reference image and sample image along with the Zoom In and Zoom Out feature to adjust and achieve alignment of each slide position. Replicate this process in the second through fourth sample slide positions.</li>
	</ol>
	</li>
	<li>Select the milling area of region of interest
	<ol>
		<li>Once optimal sample overlay of each of the four slide positions is achieved, draw milling path designations using the Color Picker tool icon (the tenth icon down in the right-hand toolbar) on the colored portion of masked reference image. Select the Extend to Similar&#160;box if multiple slides or areas are annotated for dissection and then select the Get Annotation(s) button in lower right to draw milling paths onto sample slides.&#160;</li>
		<li>Select the milling path in the first slide position. 
		NOTE: When the milling path is selected in the first slide position, it will be copied onto the remaining slide positions and milling tip usage will be calculated. The milling tip usage in the upper-left corner is calculated based on the area covered and the tip size selected. If more than four tips are calculated, a larger tip size can be selected to capture the annotated ROI. Tip size can be selected or changed on the left side of the screen under the Milling Tip arrow and tip usage will be recalculated.</li>
		<li>When the milling path is calculated, collect the annotated ROI with four or less milling tips. Select the Setup Stage button in the lower right of the screen to prompt loading of milling tips from placed collection tubes in their proper designation on stage.</li>
	</ol>
	</li>
	<li>Fill the reservoir with 3.0 mL of the dissection buffer most appropriate for the tissue type (FFPE or fresh frozen) and downstream nucleic acid extraction kit needs and select the Dissect button in the lower-right corner of the screen. Use molecular grade mineral oil or an appropriate buffer from commercially available nucleic acid extraction kits. 
	NOTE: Automated dissection of slides and selected regions of interest then begins and samples are collected by the instrument. The unit head will pick up milling tips from the back of the stage and fill with dissection fluid from the reservoir. Tips then spin along the milling path aspirating sample tissue from slides until complete or full. The collected sample with dissection fluid is then dispensed into collection tubes located at the back of the stage.</li>
	<li>When automated dissection is complete, remove the collection tubes and the dissected sample slides from the stage and place them in a tube rack and slide rack, respectively. 
	NOTE: Fresh frozen harvests should be taken directly into nucleic acid extraction as per the manufacturer&#39;s instructions and post-dissection fresh frozen sections should be H&#38;E stained immediately using routine protocols for frozen sections.</li>
	<li>Bake the post dissected tissue slides at 60 &#176;C for 30 min and then stain with H&#38;E using routine protocols.</li>
	<li>Scan the post dissected H&#38;E stained slides on a whole slide imager at 20x magnification and/or archive for a reference of what tissue was not collected and remains on the slide. 
	&#8203;NOTE: See step 1.5 above for alternative scanning options.</li>
</ol>
3. Nucleic acid extraction
<ol>
	<li>Pool and pellet the tissue. Perform the nucleic acid extraction using a commercially available kit and following the manufacturer&#39;s instructions.</li>
</ol>

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Charles A Havnar, Oliver Zill, Jeff Eastham, Jeffrey Hung, Jennifer Giltnane, Nicolas Lounsbury, Daniel Oreper, Sarajane Saturnio, and Amy A Lo are employees and stockholders of Genentech and Roche and Mana Javey and Emmanuel Naouri are employees and stockholders of Roche.

Presented here is a protocol for the application of digital annotation and automated dissection to dissect tumor regions from low tumor content FFPE or fresh frozen tissues for tumor enrichment and use in WES. Combining digital annotation and mask creation with automated dissection significantly reduces the required hands-on time and expertise common to classical methods of tumor enrichment inclusive of manual macrodissection and LCM. The protocol demonstrates a potentially important mid-range tumor enrichment option tha...

Next generation sequencing (NGS) is increasingly utilized for both patient care and in cancer research to help guide treatments and facilitate scientific discovery. Tissue is often limited and small specimens with variable tumor content are routinely used. Tumor adequacy and integrity, therefore, remain a barrier to obtaining meaningful data. Samples with lower tumor percentages may cause difficulty in distinguishing true variants from sequencing artifacts and are often ineligible for NGS1. Tumor enrichment of low tumor content cases, those below 20%, has been shown to help yield sufficient material in order to generate reproducible sequencing data and ensure low frequency variants are not missed2,3. However, limits will vary depending on the platforms utilized and planned use of the data generated.
Traditionally, enrichment of tumor regions for extraction is performed by manual macrodissection or laser capture microdissection (LCM) of formalin fixed paraffin embedded (FFPE) slides. Manual macrodissection, or scraping specified tissue areas from slides, allows tumor regions to be removed for use in downstream assays with relatively low cost, but with low accuracy and low precision2,4. Minimal technical accuracy can be very effective with higher tumor content cases where large swaths of tumor are present and/or minimal tissue loss does not significantly impact results, but low tumor content cases or cases with more dispersed tumor require increased precision. LCM was therefore invented in the 1990s and became a valuable way to precisely remove small, defined, microscopic regions of tissue from formalin fixed paraffin embedded (FFPE) slides5,6,7,8. LCM can be utilized to collect single cell populations when complex heterogeneity of the sample exists9 allowing for collection of previously difficult to separate cell populations. However, LCM requires costly machinery that requires extensive technical expertise and hands-on time10,11,12,13,14.
The instrument used for automated tissue dissection has precision in between that of LCM (~10 &#181;m) and macrodissections (~1 mm)15. Additionally, it exhibits both cost and technical expertise requirements between that of macrodissection and LCM and is designed to perform rapid tissue enrichment from sequential FFPE slides to alleviate the disadvantages of previous methods15. Automated dissection in this fashion utilizes digital annotations or on-stage slide reference image overlays onto serially sectioned unstained tissue slides for dissecting and enriching regions of interest. The instrument uses plastic spinning blade milling tips, 1.5 mL collection tubes and can be used with a number of different fluids for dissection to collect regions of interest for downstream assays inclusive of nucleic extraction and sequencing. The spinning plastic milling tip utilizes inner and outer syringe barrel reservoirs and a plunger to collect buffer, then mills and collects tissue16. The variable milling tip size diameter (250 &#181;m, 525 &#181;m, 725 &#181;m) can allow for dissection of separate tissue areas for comparison, multifocal regions that can be pooled or individual small areas from single or multiple FFPE slides. Section thicknesses used for harvest can be adjusted based on individual experiment needs and users can ensure regions of interest have not been depleted by performing an additional H&#38;E on one serial section immediately after the last section used for harvest.
Automated dissection was identified as a way to enrich tumor content in low tumor content cases and we tested and expanded the intended functionality of an automated tissue dissection instrument, which is currently marketed for use on FFPE clinical specimens up to 10 &#181;m in thickness. The work shows that automated dissection can be applied to both FFPE and fresh frozen human or animal tissue sections up to 20 &#181;m in thickness for research purposes. The protocol also demonstrates an approach to digitally annotate and automate dissection for tumor enrichment in tissues with low tumor content and/or cases with nested, dispersed tumor where meaningful macrodissection is challenging or not feasible and show both quality and yield of nucleic acid sufficient for NGS. Automated dissection can therefore provide mid-level precision and increased throughput for tumor enrichment and could also be applied to enrich other regions of interest or combined with other platforms to answer research or clinical questions.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agilent SureSelectXT</td>
			<td>Agilent</td>
			<td>G9611A</td>
			<td></td>
		</tr>
		<tr>
			<td>AVENIO Millisect Fill Station</td>
			<td>Roche</td>
			<td>8106533001</td>
			<td></td>
		</tr>
		<tr>
			<td>AVENIO Millisect Instrument, Base</td>
			<td>Roche</td>
			<td>8106568001</td>
			<td></td>
		</tr>
		<tr>
			<td>AVENIO Millisect Instrument, Head</td>
			<td>Roche</td>
			<td>8106550001</td>
			<td></td>
		</tr>
		<tr>
			<td>AVENIO Millisect Milling Tips Small</td>
			<td>Roche</td>
			<td>8106509001</td>
			<td></td>
		</tr>
		<tr>
			<td>AVENIO Millisect PC</td>
			<td>Roche</td>
			<td>8106495001</td>
			<td></td>
		</tr>
		<tr>
			<td>BioAnalyzer</td>
			<td>Agilent</td>
			<td>G2939BA</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf 5427R</td>
			<td>Eppendorf</td>
			<td>22620700</td>
			<td>Micro-centrifuge</td>
		</tr>
		<tr>
			<td>Incubation Buffer</td>
			<td>Promega</td>
			<td>D920D</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica Autostainer XL</td>
			<td>Leica</td>
			<td>ST5010</td>
			<td>Automated stainer</td>
		</tr>
		<tr>
			<td>Molecular Grade Mineral Oil</td>
			<td>Sigma</td>
			<td>M5904-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Promega</td>
			<td>V302B</td>
			<td>Digestion buffer</td>
		</tr>
		<tr>
			<td>Qiagen AllPrep DNA/RNA Mini Kit</td>
			<td>Qiagen</td>
			<td>80284</td>
			<td></td>
		</tr>
		<tr>
			<td>RLT Plus buffer</td>
			<td>Qiagen</td>
			<td>80204</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Plus positively charged microscope slides</td>
			<td>Thermo Scientific</td>
			<td>6776214</td>
			<td></td>
		</tr>
	</tbody>
</table>

automated dissection protocol for tumor enrichment in low tumor content tissues

Tumor enrichment in low tumor content tissues, those below 20% tumor content depending on the method, is required to generate quality data reproducibly with many downstream assays such as next generation sequencing. Automated tissue dissection is a new methodology that automates and improves tumor enrichment in these common, low tumor content tissues by decreasing the user-dependent imprecision of traditional macro-dissection and time, cost, and expertise limitations of laser capture microdissection by using digital image annotation overlay onto unstained slides. Here, digital hematoxylin and eosin (H&#38;E) annotations are used to target small tumor areas using a blade that is 250 &#181;m2 in diameter in unstained formalin fixed paraffin embedded (FFPE) or fresh frozen sections up to 20 &#181;m in thickness for automated tumor enrichment prior to nucleic acid extraction and whole exome sequencing (WES). Automated dissection can harvest annotated regions in low tumor content tissues from single or multiple sections for nucleic acid extraction. It also allows for capture of extensive pre- and post-harvest collection metrics while improving accuracy, reproducibility, and increasing throughput with utilization of fewer slides. The described protocol enables digital annotation with automated dissection on animal and/or human FFPE or fresh frozen tissues with low tumor content and could also be used for any region of interest enrichment to boost adequacy for downstream sequencing applications in clinical or research workflows.

FFPE and FF mouse liver sections containing metastatic colorectal cancer in xenografts were selected. Sections were H&#38;E stained (Figure 1A,E,I) and scanned on a whole slide imager at 20x magnification. A pathologist digitally annotated tumor regions of interest and a mask was generated using commercial software and formatted as a digital png reference image (Figure 1B,F,J). Serial 10 &#181;m and 20 &#181;m thick unstained sa...

Watch this Scientific Journal Video about Automated Dissection Protocol for Tumor Enrichment in Low Tumor Content Tissues at JoVE.com

Automated Dissection Protocol for Tumor Enrichment in Low Tumor Content Tissues

Digital annotation with automated tissue dissection provides an innovative approach to enriching tumor in low tumor content cases and is adaptable to both paraffin and frozen tissue types. The described workflow improves accuracy, reproducibility and throughput and could be applied to both research and clinical settings.

Tumor enrichment in low tumor content tissues, those below 20% tumor content depending on the method, is required to generate quality data reproducibly ...

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2.4K Views.  Genentech. Digital annotation with automated tissue dissection provides an innovative approach to enriching tumor in low tumor content cases and is adaptable to both paraffin and frozen tissue types. The described workflow improves accuracy, reproducibility and throughput and could be applied to both research and clinical settings.

Video: Automated Dissection Protocol for Tumor Enrichment in Low Tumor Content Tissues

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 ...

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently developed method uses a medical wire functionalized with anti-epithelial cell adhesion molecule (EpCAM) antibodies for in vivo isolation of circulating tumor cells (CTCs)1. A patient-matched cohort in non-metastatic prostate cancer showed that the in vivo isolation technique resulted in a higher percentage of patients positive for CTCs as well as higher CTC counts as compared to the current gold standard in CTC enumeration. As cells cannot be recovered from current medical devices, a new functionalized wire (referred to as Device) was manufactured allowing capture and subsequent detachment of cells by enzymatic treatment. Cells are allowed to attach to the Device, visualized on a microscope and detached using enzymatic treatment. Recovered cells are cytocentrifuged onto membrane-coated slides and harvested individually by means of laser microdissection or micromanipulation. Single-cell samples are then subjected to single-cell whole genome amplification allowing multiple downstream analysis including screening and target-specific approaches. The procedure of isolation and recovery yields high quality DNA from single cells and does not impair subsequent whole genome amplification (WGA). A single cell&#39;s amplified DNA can be forwarded to screening and/or targeted analysis such as array comparative genome hybridization (array-CGH) or sequencing. The device allows ex vivo isolation from artificial rare cell samples (i.e. 500 target cells spiked into 5 mL of peripheral blood). Whereas detachment rates of cells are acceptable (50 - 90%), the recovery rate of detached cells onto slides spans a wide range dependent on the cell line used (&#60;10 - &#62;50%) and needs some further attention. This device is not cleared for the use in patients.

This protocol is to recover and prepare rare target cells from a mixture with non-target background cells for molecular genetic characterization at the single-cell level. DNA quality is equal to non-treated single cells and allows for single-cell application (both screening based and targeted analysis).

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently ...

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by CTCs critically contributes to early lung metastatic processes, has been vigorously investigated. As such, animal models are the only approach that captures the full systemic process of metastasis. Given that problems occur in previous experimental designs for examining the contributions of CTCs to blood vessel extravasation, we established an in vivo lung colonization assay in which a long-term-fluorescence cell-tracer, carboxyfluorescein succinimidyl ester (CFSE), was used to label suspended tumor cells and lung perfusion was performed to clear non-specifically trapped CTCs prior to lung removal, confocal imaging, and quantification. Polymeric fibronectin (polyFN) assembled on CTC surfaces has been found to mediate lung colonization in the final establishment of metastatic tumor tissues. Here, to specifically test the requirement of polyFN assembly on CTCs for lung colonization and extravasation, we performed short term lung colonization assays in which suspended Lewis lung carcinoma cells (LLCs) stably expressing FN-shRNA (shFN) or scramble-shRNA (shScr) and pre-labeled with 20 &#956;M of CFSE were intravenously inoculated into C57BL/6 mice. We successfully demonstrated that the abilities of shFN LLC cells to colonize the mouse lungs were significantly diminished in comparison to shScr LLC cells. Therefore, this short-term methodology may be widely applied to specifically demonstrate the ability of CTCs within the circulation to colonize the lungs.

An animal model is needed to decipher the role of circulating tumor cells (CTCs) in promoting lung colonization during cancer metastasis. Here, we established and successfully performed an in vivo assay to specifically test the requirement of polymeric fibronectin (polyFN) assembly on CTCs for lung colonization.

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by ...

An Integrated Platform for Genome-wide Mapping of Chromatin States Using High-throughput ChIP-sequencing in Tumor Tissues

Histone modifications constitute a major component of the epigenome and play important regulatory roles in determining the transcriptional status of associated loci. In addition, the presence of specific modifications has been used to determine the position and identity non-coding functional elements such as enhancers. In recent years, chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) has become a powerful tool in determining the genome-wide profiles of individual histone modifications. However, it has become increasingly clear that the combinatorial patterns of chromatin modifications, referred to as Chromatin States, determine the identity and nature of the associated genomic locus. Therefore, workflows consisting of robust high-throughput (HT) methodologies for profiling a number of histone modification marks, as well as computational analyses pipelines capable of handling myriads of ChIP-Seq profiling datasets, are needed for comprehensive determination of epigenomic states in large number of samples. The HT-ChIP-Seq workflow presented here consists of two modules: 1) an experimental protocol for profiling several histone modifications from small amounts of tumor samples and cell lines in a 96-well format; and 2) a computational data analysis pipeline that combines existing tools to compute both individual mark occupancy and combinatorial chromatin state patterns. Together, these two modules facilitate easy processing of hundreds of ChIP-Seq samples in a fast and efficient manner. The workflow presented here is used to derive chromatin state patterns from 6 histone mark profiles in melanoma tumors and cell lines. Overall, we present a comprehensive ChIP-seq workflow that can be applied to dozens of human tumor samples and cancer cell lines to determine epigenomic aberrations in various malignancies.

Here, we describe an optimized high-throughput ChIP-sequencing protocol and computational analyses pipeline for the determination of genome-wide chromatin state patterns from frozen tumor tissues and cell lines.

Histone modifications constitute a major component of the epigenome and play important regulatory roles in determining the transcriptional status of ...

Radionuclide-fluorescence Reporter Gene Imaging to Track Tumor Progression in Rodent Tumor Models

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis remains incomplete. In vivo models are paramount for metastasis research, but require refinement. Tracking spontaneous metastasis by non-invasive in vivo imaging is now possible, but remains challenging as it requires long-time observation and high sensitivity. We describe a longitudinal combined radionuclide and fluorescence whole-body in vivo imaging approach for tracking tumor progression and spontaneous metastasis. This reporter gene methodology employs the sodium iodide symporter (NIS) fused to a fluorescent protein (FP). Cancer cells are engineered to stably express NIS-FP followed by selection based on fluorescence-activated cell sorting. Corresponding tumor models are established in mice. NIS-FP expressing cancer cells are tracked non-invasively in vivo at the whole-body level by positron emission tomography (PET) using the NIS radiotracer [18F]BF4-. PET is currently the most sensitive in vivo imaging technology available at this scale and enables reliable and absolute quantification. Current methods either rely on large cohorts of animals that are euthanized for metastasis assessment at varying time points, or rely on barely quantifiable 2D imaging. The advantages of the described method are: (i) highly sensitive non-invasive in vivo 3D PET imaging and quantification, (ii) automated PET tracer production, (iii) a significant reduction in required animal numbers due to repeat imaging options, (iv) the acquisition of paired data from subsequent imaging sessions providing better statistical data, and (v) the intrinsic option for ex vivo confirmation of cancer cells in tissues by fluorescence microscopy or cytometry. In this protocol, we describe all steps required for routine NIS-FP-afforded non-invasive in vivo cancer cell tracking using PET/CT and ex vivo confirmation of in vivo results. This protocol has applications beyond cancer research whenever in vivo localization, expansion and long-time monitoring of a cell population is of interest.

We describe a protocol for preclinical in vivo tracking of cancer metastasis. It is based on a radionuclide-fluorescence reporter combining the sodium iodide symporter, detected by non-invasive [18F]tetrafluoroborate-PET, and a fluorescent protein for streamlined ex vivo confirmation. The method is applicable for preclinical in vivo cell tracking beyond tumor biology.

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis ...

Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for immunotherapies. Recent clinical advances in immunotherapy highlight the need for reproducible methods to accurately and thoroughly characterize the tumor and its associated immune infiltrate. Tumor enzymatic digestion and flow cytometric analysis allow broad characterization of numerous immune cell subsets and phenotypes; however, depth of analysis is often limited by fluorophore restrictions on panel design and the need to acquire large tumor samples to observe rare immune populations of interest. Thus, we have developed an effective and high throughput method for separating and enriching the tumor immune infiltrate from the non-immune tumor components. The described tumor digestion and centrifugal density-based separation technique allows separate characterization of tumor and tumor immune infiltrate fractions and preserves cellular viability, and thus, provides a broad characterization of the tumor immunologic state. This method was used to characterize the extensive spatial immune heterogeneity in solid tumors, which further demonstrates the need for consistent whole tumor immunologic profiling techniques. Overall, this method provides an effective and adaptable technique for the immunologic characterization of subcutaneous solid murine tumors; as such, this tool can be used to better characterize the tumoral immunologic features and in the preclinical evaluation of novel immunotherapeutic strategies.

Here we describe a method to separate and enrich components of the tumor immune and non-immune microenvironment in established subcutaneous tumors. This technique allows for the separate analysis of tumor immune infiltrate and non-immune tumor fractions which can permit comprehensive characterization of the tumor immune microenvironment.

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as tumors. Because digital polymerase chain reactions (PCR) enable the precise quantification of DNA copy number variants, they are becoming an essential tool for detecting rare genetic variations, such as drug-resistant mutations. It is expected that molecular diagnoses using digital PCR (dPCR) will be available in clinical practice in the near future; thus, how to efficiently conduct dPCR with human genetic material is a hot topic. Here, we introduce a method to detect Adenomatous polyposis coli (APC) somatic mosaicism using dPCR with the chip-in-a-tube format, which allows eight dPCR reactions to be simultaneously conducted. Care should be taken when filling and sealing the reaction mixture on the chips. This article demonstrates how to avoid the over- and underestimation of positive partitions. Furthermore, we present a simple procedure for collecting the dPCR product from the partitions on the chips, which can then be used to confirm the specific amplification. We hope that this methods report will help promote the dPCR with the chip-in-a-tube method in genetic research.

Digital polymerase chain reaction (PCR) is a useful tool for the high-sensitivity detection of single nucleotide variants and DNA copy number variants. Here, we demonstrate key considerations for measuring rare variants in the human genome using digital PCR with the chip-in-a-tube format.

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

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 ...

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

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

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

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

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

Fluorescence Microscopy for ATP Internalization Mediated by Macropinocytosis in Human Tumor Cells and Tumor-xenografted Mice

Adenosine triphosphate (ATP), including extracellular ATP (eATP), has been shown to play significant roles in various aspects of tumorigenesis, such as drug resistance, epithelial-mesenchymal transition (EMT), and metastasis. Intratumoral eATP is 103 to 104 times higher in concentration than in normal tissues. While eATP functions as a messenger to activate purinergic signaling for EMT induction, it is also internalized by cancer cells through upregulated macropinocytosis, a specific type of endocytosis, to perform a wide variety of biological functions. These functions include providing energy to ATP-requiring biochemical reactions, donating phosphate groups during signal transduction, and facilitating or accelerating gene expression as a transcriptional cofactor. ATP is readily available, and its study in cancer and other fields will undoubtedly increase. However, eATP study remains at an early stage, and unresolved questions remain unanswered before the important and versatile activities played by eATP and internalized intracellular ATP can be fully unraveled.
These authors&#39; laboratories&#39; contributions to these early eATP studies include microscopic imaging of non-hydrolysable fluorescent ATP, coupled with high- and low-molecular weight fluorescent dextrans, which serve as macropinocytosis and endocytosis tracers, as well as various endocytosis inhibitors, to monitor and characterize the eATP internalization process. This imaging modality was applied to tumor cell lines and to immunodeficient mice, xenografted with human cancer tumors, to study eATP internalization in vitro and in vivo. This paper describes these in vitro and in vivo protocols, with an emphasis on modifying and finetuning assay conditions so that the macropinocytosis-/endocytosis-mediated eATP internalization assays can be successfully performed in different systems.

We developed a reproducible method to visualize the internalization of nonhydrolyzable fluorescent adenosine triphosphate (ATP), an ATP surrogate, with high cellular resolution. We validated our method using independent in vitro and in vivo assays-human tumor cell lines and immunodeficient mice xenografted with human tumor tissue.

Adenosine triphosphate (ATP), including extracellular ATP (eATP), has been shown to play significant roles in various aspects of tumorigenesis, such as ...