<meta charset="utf8"></head><body>グライコミクスガイド下グライコプロテオミクスを用いた大腸がん組織のプロファイリング

<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

<meta charset="utf8"></head><body>グライコミクスガイド下グライコプロテオミクスは、複雑な腫瘍微小環境におけるグリコプロテオームの包括的なプロファイリングを容易にします

グライコミクスガイド下グライコプロテオミクスは、複雑な腫瘍微小環境におけるグリコプロテオームの包括的なプロファイリングを容易にします 

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

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

766 ビュー.  Macquarie University. このプロトコールでは、がんの糖鎖生物学をより深く理解するために必要な複雑な腫瘍微小環境における不均一なグリコプロテオームに関する包括的な洞察を提供する統合オミクス技術である、グライコミクスガイド下グライコプロテオミクス法について詳しく説明します。

ビデオ: グライコミクスガイド下グライコプロテオミクスは、複雑な腫瘍微小環境におけるグリコプロテオームの包括的なプロファイリングを容易にします

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.

ザ<em&gt;ショウジョウバエ</em&gt;成虫盤腫瘍モデル：遺伝的モザイクを用いた遺伝子発現と腫瘍侵襲性の可視化と定量化

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

がん研究

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.

評価するための総合的な手順<em&gt;インビボ</em&gt;がんNanomedicinesのパフォーマンス

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.

濃縮と確立された皮下マウス腫瘍における腫瘍免疫と免疫のない微小のキャラクタリゼーション

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.

チップ チューブ形式を使用して散発的な家族性大腸腺腫症患者の遺伝的変異のデジタル ポリメラーゼ連鎖反応分析

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.

腫瘍免疫プロファイル用のマウスおよび治療応答評価における扁平上皮癌細胞の粘膜接種

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.

腫瘍マクロファージ相互作用を研究するインビトロアッセイ

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.

透過性膜支持体との共培養を用いたマクロファージ活性化に及ぼす腫瘍分泌パラクリンリガンドの効果の研究

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.

腫瘍細胞の横尾静脈注入後の肺転移の病理学的解析

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.

がん患者の血漿サンプルにおける無細胞DNAの検出

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.

マウス心肺複合体の脱細胞化

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 ...