THC is supported by an International Research Training Program Scholarship funded by the Australian Government. NB is supported by International Macquarie University Research Excellence Scholarships funded by Macquarie University. AC is supported by a Research Training Program scholarship funded by the Australian Government. RK was supported by the Cancer Institute of New South Wales (ECF181259). MTA is the recipient of an Australian Research Council Future Fellowship (FT210100455).

Profiling of Colorectal Cancer Tissues Using Glycomics-Guided Glycoproteomics

The authors declare no conflict of interest.

Critical steps in the protocol 
In this protocol paper, we have outlined step-by-step the glycomics-guided glycoproteomics method, which provides a comprehensive view of the glycoproteome&#39;s heterogeneity and dynamics in the complex TME.
A critical step in the protocol is to ensure complete proteolysis without over-digesting the protein mixture. Non-specific cleavages of proteins inherently increase the peptide search space, leading to longer search times and a higher ...

Protein glycosylation is a prevalent and complex type of co- and post-translational modification of proteins produced by species across the phylogenetic tree of life1,2,3. The protein-linked glycans are known to impact a wide span of biological processes important for human health, including mediation and regulation of cellular interactions and communication events4,5. Aberrant protein glycosylation has been thought to be a cause of malignant transformation, tumor progression, and spread6,7,8. This implicates glycans in key tumorigenic processes in the tumor microenvironment (TME) and offers a considerable and largely untapped potential for the discovery of glycan-based biomarkers and therapeutic applications. Glycobiology is, therefore, receiving increasing attention in cancer research and across life science9.
Powered by key developments in glycoanalytics and informatics over the past decade10,11,12,13,14,15,16,17, liquid chromatography-tandem mass spectrometry (LC-MS/MS)-driven glycomics and glycoproteomics have been established as powerful tools for the system-wide profiling of liberated glycans and intact glycopeptides from complex mixtures of glycoproteins extracted directly from their biological sample origins such as various types of cancer patient specimens (e.g., tumor tissues, cell populations, bodily fluids)3,13,18,19,20.
Glycomics provides information on the structure and quantity of the glycan repertoire (the glycome), which is dynamically produced by cells, tissues, and even entire organisms. On the other hand, glycoproteomics provides quantitative information about the protein carriers and their site(s) of glycosylation, the site occupancy rates (macro-heterogeneity), the site-specific glycan compositions and their site distribution pattern (micro-heterogeneity) with limited glycan structural information (often limited to glycan composition)15,16,21. Hence, glycomics and glycoproteomics are complementary approaches to survey the biochemical details of the glycoproteome22.
A myriad of methodological designs and analytical strategies for glycomics and glycoproteomics have been developed and applied across laboratories depending on the glycoanalyte of interest (e.g., N-/O-/C-linked, glycan/glycopeptide/glycoprotein, labeled/unlabeled), the nature of the sample (e.g., cells, tissues, bodily fluids), amount (nanogram/microgram/milligram protein levels) and the analytical instrument available23,24,25,26,27,28,29,30,31,32. Despite the recognized benefits of their parallel use, glycomics and glycoproteomics are not commonly applied together but rather&#160;as stand-alone approaches in glycobiological and cancer research studies due to their high demand for analytical expertise and specialized instrumentation.
Recognizing this technology gap, more than a decade ago, we introduced the glycomics-assisted glycoproteomics method, which is capable of integrating glycomics and glycoproteomics data obtained from complex biological specimens33. In short, the glycomics-assisted (or glycomics-guided as in this protocol) glycoproteomics technology profiles the N- and/or O-glycans through an established porous graphitized carbon (PGC) LC-MS/MS method34,35, which are then used for the analysis of intact N- and/or O-glycopeptide data acquired from the same sample(s) by defining the boundaries of the glycan search space36. PGC-LC-MS/MS is a particularly powerful glycomics method as it provides quantitative insight into the glycome with high sensitivity and structural resolution, yielding information on topology (monosaccharide sequence and branching pattern) and glycosidic linkages of glycans even within complex mixtures35,37,38. The glycoproteomics workflow involves peptide generation, optional peptide labeling, multiplexing, and prefractionation, as well as enrichment before reversed-phase LC-MS/MS analysis that ideally employs different fragmentation methods including stepped collision energy/higher-energy collisional dissociation (sceHCD, for N-glycopeptides) and electron transfer/higher-energy collisional dissociation (EThcD, for O-glycopeptides) for analyte identification. For N-glycoproteome analysis, parallel de-N-glycoproteomics can guide the intact N-glycopeptide data analysis by defining the protein/peptide search space and by providing more details on the occupied N-glycosylation sites and their rate of site occupancy while the parallel analysis of non-modified peptides reveals any protein level changes between the studied conditions.
This paper provides a detailed and easy-to-follow step-by-step protocol for the glycomics-guided glycoproteomics method (see Figure 1 for workflow overview). The protocol provides experimental details of the sample preparation steps and the LC-MS/MS-based glycomics and glycoproteomics experiments and presents representative results demonstrating the application of the method to formalin-fixed paraffin-embedded (FFPE) tissue sections of tumors resected from colorectal cancer (CRC) patients, enabling insights into the glycobiology of the TME in a valuable sample type that is archived in cancer biobanks around the world. The analysis and integration of glycomics and glycoproteomics data are also briefly discussed.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67405/67405fig01v2.jpg" /> 
Figure 1:&#160;Overview of the glycomics-guided glycoproteomics method. The overview shows detailed experimental steps in glycomics (left) and glycoproteomics (right) workflows, with an overview of the information obtained (bolded) and how the two approaches are integrated through improved data analysis and interpretation. Insert: Home-made SPE micro-columns used for the (i) glycomics and (ii-iii) glycoproteomics workflows. <a href="https://www.jove.com/files/ftp_upload/67405/67405fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium acetate&#160;</td>
			<td>Sigma Aldrich</td>
			<td>A1542</td>
			<td>Alternatives available</td>
		</tr>
		<tr>
			<td>Ammonium bicarbonate, purity &#8805;99.0%</td>
			<td>Sigma Aldrich</td>
			<td>A6141</td>
			<td>Alternatives available</td>
		</tr>
		<tr>
			<td>Anhydrous acetonitrile (ACN), LC-MS grade</td>
			<td>Sigma Aldrich</td>
			<td>34851</td>
			<td>Alternatives available</td>
		</tr>
		<tr>
			<td>Bovine fetuin</td>
			<td>Sigma Aldrich</td>
			<td>F3004</td>
			<td>Alternatives available</td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>Sigma Aldrich</td>
			<td>D0632</td>
			<td>Alternatives available</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma Aldrich</td>
			<td>E7023</td>
			<td>Alternatives available</td>
		</tr>
		<tr>
			<td>Formic acid, LC-MS grade</td>
			<td>Sigma Aldrich</td>
			<td>00940</td>
			<td>Alternatives available</td>
		</tr>
		<tr>
			<td>Glacial acetic acid</td>
			<td>Sigma Aldrich</td>
			<td>A6283</td>
			<td>Alternatives available</td>
		</tr>
		<tr>
			<td>Iodoacetamide (IAA)</td>
			<td>Sigma Aldrich</td>
			<td>I1149</td>
			<td>Alternatives available</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma Aldrich</td>
			<td>M1775</td>
			<td>Alternatives available</td>
		</tr>
		<tr>
			<td>Peptide-N-glycosidase F (PNGase F)</td>
			<td>Promega</td>
			<td>V4831</td>
			<td>Elizabethkingia miricola PNGase F (10 U/&#956;L) recombinantly expressed in Escherichia coli&#160;</td>
		</tr>
		<tr>
			<td>Polyvinylpyrrolidone 40 (PVP)</td>
			<td>Sigma Aldrich</td>
			<td>PVP40</td>
			<td>Alternatives available</td>
		</tr>
		<tr>
			<td>Potassium hydroxide (KOH)</td>
			<td>Sigma Aldrich</td>
			<td>484016</td>
			<td>Alternatives available</td>
		</tr>
		<tr>
			<td>Sequencing-grade porcine trypsin&#160;</td>
			<td>Promega</td>
			<td>V5113</td>
			<td>Alternatives available</td>
		</tr>
		<tr>
			<td>Sodium borohydride (NaBH4)</td>
			<td>Sigma Aldrich</td>
			<td>213462</td>
			<td>Alternatives available</td>
		</tr>
		<tr>
			<td>Triethylammonium bicarbonate (TEAB), LC-MS grade</td>
			<td>Sigma Aldrich</td>
			<td>T7408</td>
			<td>Alternatives available</td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid (TFA), LC-MS grade</td>
			<td>Sigma Aldrich</td>
			<td>T6508</td>
			<td>Alternatives available</td>
		</tr>
		<tr>
			<td>Tools/Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C18 disks</td>
			<td>Empore</td>
			<td>66883-U</td>
			<td></td>
		</tr>
		<tr>
			<td>C8 disks</td>
			<td>Empore</td>
			<td>66882-U</td>
			<td></td>
		</tr>
		<tr>
			<td>Flat-bottom polypropylene 96-well plate&#160;</td>
			<td>Corning</td>
			<td>3364</td>
			<td></td>
		</tr>
		<tr>
			<td>Immobilon-PSQ polyvinylidene difluoride (PVDF) membrane</td>
			<td>Millipore</td>
			<td>IPVH20200</td>
			<td>Pore size: 0.45 &#956;m&#160;</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Eppendorf</td>
			<td>5452000069</td>
			<td>Alternatives available</td>
		</tr>
		<tr>
			<td>Microcentrifuge adapters&#160;</td>
			<td>The Nest Group, Inc., MA</td>
			<td>SS18V</td>
			<td>MiniSpin Column Collar, comes with Microspin Columns</td>
		</tr>
		<tr>
			<td>Oligo R3 resin</td>
			<td>Thermo</td>
			<td>1133903</td>
			<td>Particle size 30 &#956;m</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Parafilm M</td>
			<td>PM996</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein LoBind tubes 1.5 mL</td>
			<td>Eppendorf</td>
			<td>0030108450</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein LoBind tubes 2.0 mL</td>
			<td>Eppendorf</td>
			<td>0030108442</td>
			<td></td>
		</tr>
		<tr>
			<td>Safe-lock tubes 1.5 mL</td>
			<td>Eppendorf</td>
			<td>0030120086</td>
			<td></td>
		</tr>
		<tr>
			<td>Safe-lock tubes 2.0 mL</td>
			<td>Eppendorf</td>
			<td>0030120094</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaker</td>
			<td>Eppendorf</td>
			<td>5382000066</td>
			<td>Alternatives available</td>
		</tr>
		<tr>
			<td>Single hole puncher</td>
			<td>Swingline</td>
			<td>74005</td>
			<td></td>
		</tr>
		<tr>
			<td>SpeedVac concentrator</td>
			<td>Martin Christ</td>
			<td>RVC 2-25 CDplus</td>
			<td>Alternatives available</td>
		</tr>
		<tr>
			<td>Supelclean ENVI-Carb SPE resin</td>
			<td>Supelco</td>
			<td>57088</td>
			<td>Particle size 120-400 mesh. Resin can be manually extracted from cartridges, and can be stored long-term in 50% (v/v) methanol in MilliQ water</td>
		</tr>
		<tr>
			<td>Syringe 5 mL</td>
			<td>Sigma Aldrich</td>
			<td>Z116866</td>
			<td>Alternatives available</td>
		</tr>
		<tr>
			<td>Temperature-controlled incubator</td>
			<td>Thermoline</td>
			<td>E7.30</td>
			<td>Alternatives available</td>
		</tr>
		<tr>
			<td>Ultrasonic bath</td>
			<td>Unisonics</td>
			<td>FXP</td>
			<td>Alternatives available</td>
		</tr>
		<tr>
			<td>ZIC-HILIC resin</td>
			<td>Merck</td>
			<td>150455</td>
			<td>Particle/pore size, 5 &#956;m/200 &#197;. Resin can be manually extracted from LC column, and can be stored long-term in 50% (v/v) methanol in MilliQ water</td>
		</tr>
		<tr>
			<td>ZipTip C18 solid-phase extraction (SPE) micro-columns&#160;</td>
			<td>Millipore</td>
			<td>ZTC18S096</td>
			<td>Alternatively, home-made C18-SPE micro-columns can replace commercial product</td>
		</tr>
		<tr>
			<td>LC-MS/MS Analysis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1260 Infinity Capillary HPLC system&#160;</td>
			<td>Agilent</td>
			<td></td>
			<td>Alternatives available</td>
		</tr>
		<tr>
			<td>Analytical LC column pre-packed with ReproSil-Pur C18 AQ resin</td>
			<td>Dr Maisch, Ammerbuch-Entringen, Germany</td>
			<td>r13.aq.s2570</td>
			<td>Particle/pore size: 3 &#956;m/120 &#197;, length/inner diameter: 25 cm/75 &#956;m. Commercial C18 nano-LC columns also available/&#160;</td>
		</tr>
		<tr>
			<td>Dionex UltiMate 3000 RSLCnano LC System&#160;</td>
			<td>Thermo</td>
			<td></td>
			<td>Alternatives available</td>
		</tr>
		<tr>
			<td>HyperCarb KAPPA PGC capillary LC column&#160;</td>
			<td>Thermo</td>
			<td>Discontinued</td>
			<td>Particle/pore size, 3 &#956;m/250 &#197;; column length, 30 mm; inner diameter, 0.180 mm. Larger geometries of the HyperCarb PGC-LC columns, producing similar quality data are commercially available (e.g., Thermo #35003-031030). SupelTMCarb LC column from Merck with a particle/pore size, 2.7 &#956;m/200 and with different column lengths and internal diameter are also available (e.g., Merck #59994-U).&#160;</td>
		</tr>
		<tr>
			<td>LCMS-grade acetonitrile</td>
			<td>LiChrosolv</td>
			<td>100029</td>
			<td>Alternatives available</td>
		</tr>
		<tr>
			<td>Linear trap quadrupole (LTQ) Velos Pro ion trap mass spectrometer (glycomics)&#160;</td>
			<td>Thermo</td>
			<td></td>
			<td>Alternatives available</td>
		</tr>
		<tr>
			<td>Orbitrap Fusion Lumos Tribrid Mass Spectrometer (glycoproteomics)</td>
			<td>Thermo</td>
			<td></td>
			<td>Other high-resolution MS systems with or without ETD (only required for O-glycoproteomics) are also available (e.g., Q-Exactive HF-X Hybrid Quadrupole-Orbitrap, Thermo, TIMS-ToF-MS, Bruker or similar high performance mass spectrometers from other vendors)</td>
		</tr>
		<tr>
			<td>Total Recovery Clear Glass screw vials 0.9 mL</td>
			<td>Thermo</td>
			<td>THC11093563</td>
			<td>Alternatives available</td>
		</tr>
		<tr>
			<td>Total Recovery Clear Glass screw vials matching caps</td>
			<td>Thermo</td>
			<td>THC09150869</td>
			<td>Alternatives available</td>
		</tr>
		<tr>
			<td>Trap LC column packed in-house with ReproSil-Pur C18 AQ resin&#160;</td>
			<td>Dr Maisch, Ammerbuch-Entringen, Germany</td>
			<td>r15.aq.s0202</td>
			<td>Particle/pore size: 5 &#956;m/120 &#197;, length/inner diameter: 2 cm/100 &#956;m. Commercial C18 trap LC columns also available.&#160;</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Byonic</td>
			<td>Protein Metrics Inc</td>
			<td>v2.6.46 or higher</td>
			<td>Commercial glycopeptide and PTM search engine, accepting LC-MS/MS raw data from most MS vendors</td>
		</tr>
		<tr>
			<td>Byos</td>
			<td>Protein Metrics Inc</td>
			<td>v3.9-7 or higher</td>
			<td>Commercial software for manual inspection of glycopeptide candidates and the automatic annotation of glycan MS/MS spectra</td>
		</tr>
		<tr>
			<td>GlycoMod</td>
			<td>Expasy</td>
			<td></td>
			<td>Open access software, assisting the annotation of glycomics data (https://web.expasy.org/glycomod/)</td>
		</tr>
		<tr>
			<td>GlycoWorkBench</td>
			<td>EUROCarbDB</td>
			<td>v2.1</td>
			<td>Open access software, assisting the annotation of glycomics data and the drawing of glycan cartoons</td>
		</tr>
		<tr>
			<td>RawMeat</td>
			<td>VAST Scientific</td>
			<td>v2.1</td>
			<td>Open access software, extracting m/z of glycan precursor ions from raw spectral data</td>
		</tr>
		<tr>
			<td>Skyline</td>
			<td>Brendan X. MacLean</td>
			<td>v21.2 or higher</td>
			<td>Open access software for relative quantitation of glycans</td>
		</tr>
		<tr>
			<td>Xcalibur</td>
			<td>Thermo</td>
			<td>v2.2 or higher</td>
			<td>For browsing raw LC-MS/MS data</td>
		</tr>
	</tbody>
</table>

glycomics-guided glycoproteomics facilitates comprehensive profiling of the glycoproteome in complex tumor microenvironments

Glycosylation is a common and structurally diverse protein modification that impacts a wide range of tumorigenic processes. Mass spectrometry-driven glycomics and glycoproteomics have emerged as powerful approaches to analyze liberated glycans and intact glycopeptides, respectively, offering a deeper understanding of the heterogeneous glycoproteome in the tumor microenvironment (TME). This protocol details the glycomics-guided glycoproteomics method, an integrated omics technology, which firstly employs porous graphitized carbon-LC-MS/MS-based glycomics to elucidate the glycan structures and their quantitative distribution in the glycome of tumor tissues, cell populations, or bodily fluids being investigated. This allows for a comparative glycomics analysis to identify altered glycosylation across patient groups, disease stages, or conditions, and, importantly, serves to enhance the downstream glycoproteomics analysis of the same sample(s) by creating a library of known glycan structures, thus reducing the data search time and the glycoprotein misidentification rate. Focusing on N-glycoproteome profiling, the sample preparation steps for the glycomics-guided glycoproteomics method are detailed in this protocol, and key aspects of the data collection and analysis are discussed. The glycomics-guided glycoproteomics method provides quantitative information on the glycoproteins present in the TME and their glycosylation sites, site occupancy, and site-specific glycan structures. Representative results are presented from formalin-fixed paraffin-embedded tumor tissues from colorectal cancer patients, demonstrating that the method is sensitive and compatible with tissue sections commonly found in biobanks. Glycomics-guided glycoproteomics, therefore, offers a comprehensive view into the heterogeneity and dynamics of the glycoproteome in complex TMEs, generating robust biochemical data required to better understand the glycobiology of cancers.

Representative results of the glycomics-guided glycoproteomics method applied to a FFPE tissue slide from a patient suffering from CRC in stage II are provided in this section.
To achieve sufficient protein starting material for the protocol, protein extracts from two slides (z stacks) were combined from the same FFPE tissue block following a published protocol and a TMT labeling approach was applied to increase the sensitivity of the glycoproteomics experiment (see optional steps below)<sup c...

Glycomics-Guided Glycoproteomics Facilitates Comprehensive Profiling of the Glycoproteome in Complex Tumor Microenvironments

This protocol details the glycomics-guided glycoproteomics method, an integrated omics technology that offers comprehensive insights into the heterogeneous glycoproteome in complex tumor microenvironments required to better understand the glycobiology of cancers.

Glycosylation is a common and structurally diverse protein modification that impacts a wide range of tumorigenic processes. Mass spectrometry-driven ...

glycomics-guided-glycoproteomics-facilitates-comprehensive-profiling

Research

JoVE Journal

Cancer Research

787 Views.  Macquarie University. This protocol details the glycomics-guided glycoproteomics method, an integrated omics technology that offers comprehensive insights into the heterogeneous glycoproteome in complex tumor microenvironments required to better understand the glycobiology of cancers.

Video: Glycomics-Guided Glycoproteomics Facilitates Comprehensive Profiling of the Glycoproteome in Complex Tumor Microenvironments

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 ...

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.

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.

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

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 ...

Intramucosal Inoculation of Squamous Cell Carcinoma Cells in Mice for Tumor Immune Profiling and Treatment Response Assessment

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop better therapeutic strategies, understanding tumor microenvironmental factors that contribute to the treatment resistance is important. A major impediment to understanding disease mechanisms and improving therapy has been a lack of murine cell lines that resemble the aggressive and metastatic nature of human HNSCCs. Furthermore, a majority of murine models employ subcutaneous implantations of tumors which lack important physiological features of the head and neck region, including high vascular density, extensive lymphatic vasculature, and resident mucosal flora. The purpose of this study is to develop and characterize an orthotopic model of HNSCC. We employ two genetically distinct murine cell lines and established tumors in the buccal mucosa of mice. We optimize collagenase-based tumor digestion methods for the optimal recovery of single cells from established tumors. The data presented here show that mice develop highly vascularized tumors that metastasize to regional lymph nodes. Single-cell multiparametric mass cytometry analysis shows the presence of diverse immune populations with myeloid cells representing the majority of all immune cells. The model proposed in this study has applications in cancer biology, tumor immunology, and preclinical development of novel therapeutics. The resemblance of the orthotopic model to clinical features of human disease will provide a tool for enhanced translation and improved patient outcomes.

Here we present a reproducible method for developing an orthotopic murine model of head and neck squamous cell carcinoma. Tumors demonstrate clinically relevant histopathological features of the disease, including necrosis, poor differentiation, nodal metastases, and immune infiltration. Tumor-bearing mice develop clinically relevant symptoms including dysphagia, jaw displacement, and weight loss.

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop ...

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 ...

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

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.

Intravenous injection of cancer cells is often used in metastasis research, but the metastatic tumor burden can be difficult to analyze. Herein, we demonstrate a tail-vein injection model of metastasis and include a novel approach to analyze the resulting metastatic lung tumor burden.

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

Detection of Cell-Free DNA in Blood Plasma Samples of Cancer Patients

Identifying mutations in tumors of cancer patients is a very important step in disease management. These mutations serve as biomarkers for tumor diagnosis as well as for the treatment selection and its response in cancer patients. The current gold standard method for detecting tumor mutations involves a genetic test of tumor DNA by means of tumor biopsies. However, this invasive method is difficult to be performed repeatedly as a follow-up test of the tumor mutational repertoire. Liquid biopsy is a new and emerging technique for detecting tumor mutations as an easy-to-use and non-invasive biopsy approach.
Cancer cells multiply rapidly. In parallel, numerous cancer cells undergo apoptosis. Debris from these cells are released into a patient&#8217;s circulatory system, together with finely fragmented DNA pieces, called cell-free DNA (cfDNA) fragments, which carry tumor DNA mutations. Therefore, for identifying cfDNA based biomarkers using liquid biopsy technique, blood samples are collected from the cancer patients, followed by the separation of plasma and buffy coat. Next, plasma is processed for the isolation of cfDNA, and the respective buffy coat is processed for the isolation of a patient&#39;s genomic DNA. Both nucleic acid samples are then checked for their quantity and quality; and analyzed for mutations using next-generation sequencing (NGS) techniques.
In this manuscript, we present a detailed protocol for liquid biopsy, including blood collection, plasma, and buffy coat separation, cfDNA and germline DNA extraction, quantification of cfDNA or germline DNA, and cfDNA fragment enrichment analysis.

In this paper we present a detailed protocol for non-invasive liquid biopsy technique, including blood collection, plasma and buffy coat separation, cfDNA and germline DNA extraction, quantification of cfDNA or germline DNA, and cfDNA fragment enrichment analysis.

Identifying mutations in tumors of cancer patients is a very important step in disease management. These mutations serve as biomarkers for tumor diagnosis ...

Decellularization of the Murine Cardiopulmonary Complex

We present here a decellularization protocol for mouse heart and lungs. It produces structural ECM scaffolds that can be used to analyze ECM topology and composition. It is based on a microsurgical procedure designed to catheterize the trachea and aorta of a euthanized mouse to perfuse the heart and lungs with decellularizing agents. The decellularized cardiopulmonary complex can subsequently be immunostained to reveal the location of structural ECM proteins. The whole procedure can be completed in 4 days.
The ECM scaffolds resulting from this protocol are free of dimensional distortions. The absence of cells enables structural examination of ECM structures down to submicron resolution in 3D. This protocol can be applied to healthy and diseased tissue from mice as young as 4-weeks old, including mouse models of fibrosis and cancer, opening the way to determine ECM remodeling associated with cardiopulmonary disease.

This protocol aims to decellularize the heart and lungs of mice. The resulting extracellular matrix (ECM) scaffolds can be immunostained and imaged to map the location and topology of their components.

We present here a decellularization protocol for mouse heart and lungs. It produces structural ECM scaffolds that can be used to analyze ECM topology and ...