This research was supported by grants from the New Jersey Commission on Cancer Research (Pre-Doctoral Fellowship DFHS13PPCO17), the National Institutes of Health (CA049062), and the National Aeronautics and Space Administration (NNX15AD62G).

著者は、彼らは、競合や利害の対立がないことを宣言します。

ここで説明するプロトコルは、透過性の微多孔膜インサートが異細胞の共培養物から高度に濃縮された個々の細胞集団を生成するために利用する、単純なインビトロ手順の適合（ 図1）です。重要なことは、このモデルは細胞間コミュニケーションの様々なモードを調査するのに適しています。重要なステップは、上面側の第2の細胞集団を播種し、50％FBSを補充した...

腫瘍微小環境（TME）が共存するとホスト間質と一緒に進化する癌細胞で構成される非常に複雑なシステムです。この間質成分は、典型的には、線維芽細胞、筋線維芽細胞、内皮細胞、様々な免疫成分、ならびに細胞外マトリックス1で構成されています。重要な構成要素、この間質の、しばしば大部分は、活性化された線維芽細胞は、しばしば癌関連線維芽細胞または癌関連線維芽細胞（CAF）2,3と呼ばれています。通常、非活性化線維芽細胞とは異なり、CAFSは、乳房、前立腺、肺、膵臓、皮膚、結腸、食道癌などの多種多様な、腫瘍の開始、進行、血管新生、浸潤、転移、および再発4-11に寄与し、そして5,6,12-17卵巣。しかし、がんの病因全体でCAFSの寄与の正確な性質は、定義が不十分のまま。さらに、臨床的証拠は、CAFSの予後値を実証してきました、高品位の悪性疾患、治療の失敗、および全体的な予後不良10,18,19にその存在を関連付けます。 明らかに、CAF開発の開始イベントと同様に、TME内での役割を媒介する細胞間コミュニケーションの我々の理解を高め、患者の転帰を改善することができるエキサイティングな新しい治療標的と強化戦略を提供することができます。この目標に向かって、in vivoおよびin vitroモデルにおけるいくつかが開発されています。 in vivoでのアプローチで患者のTMEをより反映しているが、それらは腫瘍内との間の両方の巨大な複雑さと不均一性などの制限を、持っています。さらに、ヒト被験者からの腫瘍サンプルは、しばしば高度に発達したTMEを表し、TME開始イベントの理解を許可していません。実験動物研究は、しかし、ヒトへの動物のデータの一般化が原因でphysiの違いに注意して行う必要があります、いくつかの利点を提供しますこのような齧歯類（ 例えば、チオール化学20、 ​​代謝率21 などのストレス22、に対する耐性）などの人間と動物の間に学問。また、自然の中で遺伝的に不均質である人間集団とは異なり、実験動物は、一般的に均一になるまで飼育されています。また、一過性の生理的変動および細胞表現型の変化を調べるために、ならびにげっ歯類などの動物を使用して、特定の実験パラメータを制御することがしばしば困難です。したがって、 インビトロ 2-および3次元（2Dおよび3D）組織培養モデルで頻繁TME開発の基本的な理解を進めるために利用されます。 インビボでのシステムの複雑さの正確な描写の欠如にもかかわらず、これらのモデルは、非常に機械的調査を容易にする利点を提供する。 インビトロモデルは、TMEのより単純化着目し、費用対効果の分析のために、それによって統計学的に有意な可能データを、生成することができます動物で起こる全身ばらつきのない細胞。 インビトロ系のいくつかの種類があります。 2つの最も一般的に使用TME in vitroモデルは、混合単層又はスフェロイド細胞培養物で構成されています。両方の培養方法は、細胞間の相互作用の基本的な研究（腫瘍細胞と、例えば、正常細胞）および種々のTME特定の細胞表現型の変化（正常線維芽細胞からの癌関連線維芽細胞の、例えば 、出現）の分析のために有利です。加えて、スフェロイドはTMEをより反映組織様構造を作成することができ、腫瘍異質23を表すことができます。しかし、スフェロイドは、多くの場合、実験の結論24を複雑にする可能性がある層全体で幅広く変化する酸素張力勾配を作り出します。残念ながら、両方のモデルは非常に共同以下のさらなる特性評価および研究のために純粋な細胞集団を分離する能力が制限されています文化。これを行うには、蛍光タグ付きまたは特定メーカーで標識するために少なくとも1つの細胞型を必要とし、その後、細胞集団を分離するために仕分け広範な処理し、細胞に混合共培養を施すことになります。セルソーターではなく、純粋な細胞集団を単離することができるが、1は、細胞ストレスと潜在的な微生物汚染25を危険を認識しなければなりません。 細胞間コミュニケーションの理解を容易にするため、多大な努力を開発し、簡単なアプローチを可能にしながら密接に、 生体内環境を模倣するインビトロシステムに最適化に向けて専念してきました。そのようなツールの一つは、透過性の微多孔性インサート、最初の1953年に26を開発し、その後、多様なアプリケーションとの研究（ 例えば 、細胞極性27、エンドサイトーシス28、薬物輸送29、組織モデリング30に適合された膜基材、FERTですilization 31、バイスタンダー効果32,33、 など ）。このシステムは、34,35、インビボマーカーの インビボ様の解剖学的および機能的分化、ならびに多くの発現を有する細胞の増殖を可能にするときに不浸透性のプラスチック容器で培養観察されません。また、非常に薄い多孔質膜（厚さ10μm）は、 生体内環境をシミュレートし、両方の頂端および側底細胞ドメインで独立した細胞機能を可能にする分子と平衡時間の急速な普及を可能にします。 TMEシステムなどの挿入物の有用性のさらなる利点は、膜の孔を通って細胞間コミュニケーションの様々なモードを維持しながら、同じ環境条件で膜の両側に成長させた2異型細胞集団の物理的な分離です。物理的に分離されているが、2つの細胞集団は、代謝的にデとして、分泌された要素を介して結合され、また、ギャップジャンクショ​​ンチャネルを介して、ここにスクライビング。さらに、in vivoでの部分的な酸素分圧（PO 2）で挿入を維持することによって、モデルは、他のシステムで観察された酸素と化学的勾配の合併症を減らすことができます。むしろ、それはTMEを制御する自然のメカニズムの理解を向上させます。注目すべきことに、2つの細胞集団を容易に共培養の長期間以下の蛍光タグ付けおよび/または細胞選別せずに、高純度で単離することができます。 ここでは、膜の孔を通って連続的な双方向通信ではまだヒト乳癌細胞透過性微孔質膜インサートの両側に、それぞれ、成長したヒト線維芽細胞からなるインビトロ TMEプロトコルを説明するが、。我々は、開発に異なる孔径を有する膜を使用することによって、細胞間コミュニケーションの特定のタイプの寄与（ 例えば 、ギャップ結合対因子を分泌した）ことを示していますTMEの調査することができます。

<table border="0" cellpadding="0" cellspacing="0" width="886">
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			<td height="21" style="height:21px;width:289px;">For Cell Culture</td>
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			<td height="42" style="height:42px;width:289px;">AG01522 (i.e., AG1522) human diploid fibroblast</td>
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			<td height="63" style="height:63px;width:289px;">Transwell Insert (i.e., permeable microporous membrane insert) (3 &#956;m pore)</td>
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			<td height="21" style="height:21px;width:289px;">75 cm2 cell culture flask</td>
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			<td style="width:139px;">658 170</td>
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			<td height="21" style="height:21px;width:289px;">Phosphate-Buffered Saline (PBS), 1X</td>
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			<td style="width:139px;">21-040-CV</td>
			<td style="width:279px;">without calcium &#38; magnesium</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:289px;">0.25% (vol/vol) Trypsin, 2.21 mM EDTA, 1X</td>
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			<td style="width:139px;">25-053-Cl</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:289px;">15 mL Centrifuge Tube</td>
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			<td height="21" style="height:21px;width:289px;">35 x 10 mm Cell Culture Dish</td>
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			<td style="width:139px;"></td>
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			<td height="42" style="height:42px;width:289px;">Mouse anti-Caveolin 1</td>
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			<td style="width:139px;">610406</td>
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			<td height="42" style="height:42px;width:289px;">Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Triton X-100</td>
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			<td height="21" style="height:21px;width:289px;">SlowFade Gold antifade reagent with DAPI</td>
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			<td style="width:139px;">S36938</td>
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			<td style="width:139px;"></td>
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			<td height="21" style="height:21px;width:289px;">Calcein, AM</td>
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		<tr height="21">
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			<td style="width:139px;">14025-076</td>
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			<td style="width:180px;"></td>
			<td style="width:139px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:289px;">Mouse anti-Caveolin 1</td>
			<td style="width:180px;">BD Transduction Laboratories</td>
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			<td style="width:279px;">Western Blot - 1:10000</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Tween-20</td>
			<td style="width:180px;">BioRad</td>
			<td style="width:139px;">170-6531</td>
			<td style="width:279px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Nitrocellulose Membrane (0.2 &#956;m)</td>
			<td style="width:180px;">BioRad</td>
			<td>162-0112</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Western Lightning Plus-ECL</td>
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			<td style="width:139px;">NEL104001EA</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:289px;">BioRad DC Protein Assay</td>
			<td style="width:180px;">BioRad</td>
			<td style="width:139px;">500-0116</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Sodium dodecyl sulfate (SDS)</td>
			<td style="width:180px;">BioRad</td>
			<td style="width:139px;">161-0302</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Sodium deoxycholate monohydrate (DOC)</td>
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			<td style="width:139px;">D5670</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:289px;">IGEPAL CA-630 (NP40)</td>
			<td style="width:180px;">Sigma</td>
			<td style="width:139px;">I8896</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">30% Acrylamide/Bis Solution, 37.5:1</td>
			<td style="width:180px;">BioRad</td>
			<td style="width:139px;">161-0158</td>
			<td></td>
		</tr>
	</tbody>
</table>

a mimic of the tumor microenvironment: a simple method for generating enriched cell populations and investigating intercellular communication

癌細胞と周囲の非癌性間質との間の早期の異な相互作用を理解することは、間質の活性化と腫瘍の微小環境（TME）の設立をもたらす事象を解明する上で重要です。 インビトロおよびTMEのインビボモデルにおけるいくつかが開発されています。しかし、一般的にこれらのモデルは、容易にさらなる研究のために、非摂動条件の下で、個々の細胞集団の単離を許可していません。この問題を回避するために、我々は、拡張共同用挿入物の膜の両側に、別々に密接成長高度に濃縮された細胞集団の簡単な生成を可能にする、まだ透過性微多孔膜インサートからなる細胞成長用基板を用いたin vitro TMEモデルを採用しています-culture回。このモデルを使用することによって、我々は以下の正常な二倍体のヒト線維芽細胞から大きく富化癌関連線維芽細胞（CAF）の集団を生成することができます蛍光タグ及び/又は細胞選別を使用せずに高転移性ヒト乳癌細胞との共培養（120時間）。さらに、インサートの細孔サイズを調節することによって、我々は開発の根底にあるメカニズムの調査を可能にする2異細胞集団間の細胞間コミュニケーション（ 例えば 、ギャップ結合コミュニケーション、分泌因子）のモード、のために制御することができますギャップ結合透過性の役割を含むTME、。このモデルは、癌間質開始、TMEの初期進化、および治療薬に対する癌細胞の応答に対する間質の調節作用につながる初期のイベントの我々の理解を高める上で貴重なツ​​ールとして機能します。

ここでは、in vivoでの腫瘍微小環境（ 図1）を模倣するインビトロ異細胞共培養システムを開発するために透過性の微多孔膜インサートを適応しました。このシステムは、（私達の使用中の120時間まで、）長時間のインサートの多孔質膜の両側に成長させることには、2つの異なる細胞集団を可能にします。 0.4〜1-または3 M-細孔インサートの上...

Watch this Scientific Journal Video about A Mimic of the Tumor Microenvironment: A Simple Method for Generating Enriched Cell Populations and Investigating Intercellular Communication at JoVE.com

A Mimic of the Tumor Microenvironment: A Simple Method for Generating Enriched Cell Populations and Investigating Intercellular Communication

We adapted a permeable microporous membrane insert to mimic the tumor microenvironment (TME). The model consists of a mixed cell culture, allows simplified generation of highly enriched individual cell populations without using fluorescent tagging or cell sorting, and permits studying intercellular communication within the TME under normal or stress conditions.

濃縮細胞集団を生成し、細胞間コミュニケーションを調べるための簡単​​な方法：腫瘍微小環境のミミック

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events ...

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10.1K ビュー.  Rutgers University. We adapted a permeable microporous membrane insert to mimic the tumor microenvironment (TME). The model consists of a mixed cell culture, allows simplified generation of highly enriched individual cell populations without using fluorescent tagging or cell sorting, and permits studying intercellular communication within the TME under normal or stress conditions.

ビデオ: A Mimic of the Tumor Microenvironment: A Simple Method for Generating Enriched Cell Populations and Investigating Intercellular Communication

Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, in the case that the KD of the protein of interest has a lethal effect on cells or the anticipated effect of the KD is time-dependent, unconditional KD methods are not appropriate. Conditional systems are more suitable in these cases and have been the subject of much interest. These include Ecdysone-inducible overexpression systems, Cytochrome P-450 induction system1, and the tetracycline regulated gene expression systems. 
The tetracycline regulated gene expression system enables reversible control over protein expression by induction of shRNA expression in the presence of tetracycline. In this protocol, we present an experimental design using functional Tet-ON system in human cancer cell lines for conditional regulation of gene expression. We then demonstrate the use of this system in the study of tumor cell-monocyte interaction.

This protocol serves as a scheme for setting up a functional Tet-ON system in cancer cell lines and its subsequent use, in particular for studying the role of tumor cell-derived proteins in recruitment of monocytes/macrophages to the tumor microenvironment.

単球/マクロファージにおける腫瘍微小環境の採用を検討する癌細胞株における遺伝子発現の条件付きのノックダウン

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, ...

がん研究

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic probes to provide quantitative information on the chemical tumor microenvironment (TME), including pO2, pH, redox status, concentrations of interstitial inorganic phosphate (Pi), and intracellular glutathione (GSH). In particular, an application of a recently developed soluble multifunctional trityl probe provides unsurpassed opportunity for in vivo concurrent measurements of pH, pO2 and Pi in Extracellular space (HOPE probe). The measurements of three parameters using a single probe allow for their correlation analyses independent of probe distribution and time of the measurements.

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

Low-field (L-band, 1.2 GHz) electron paramagnetic resonance using soluble nitroxyl and trityl probes is demonstrated for assessment of physiologically important parameters in the tumor microenvironment in mouse models of breast cancer.

生体内でPH、 pO2、酸化還元状態とリン酸濃度および腫瘍微小環境におけるグルタチオンの EPR 評価

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

Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. In the clinical setting, formalin-fixed paraffin-embedded (FFPE) tissues are widely used for genetic testing. However, FFPE DNA is generally damaged and fragmented during the fixation process with formalin. Therefore, FFPE DNA is sometimes not adequate for genetic testing because of low quality and quantity of DNA. Here we present a method of touch imprint cytology (TIC) to obtain genomic DNA from cancer cells, which can be observed under a microscope. Cell morphology and cancer cell numbers can be evaluated using TIC specimens. Furthermore, the extraction of genomic DNA from TIC samples can be completed within two days. The total amount and quality of TIC DNA obtained using this method was higher than that of FFPE DNA. This rapid and simple method allows researchers to obtain high-quality DNA for genetic testing (e.g., next generation sequencing analysis, digital PCR, and quantitative real time PCR) and to shorten the turnaround time for reporting results.

Obtaining high-quality genomic DNA from tumor tissues is an essential first step for analyzing genetic alterations using next generation sequencing. In this article, we present a simple and rapid method to enrich tumor cells and obtain intact DNA from touch imprint cytology specimens.

タッチ捺印細胞診を用いた臨床病理標本から質の高い腫瘍 DNA を取得する簡易法

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. ...

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 ...

Measuring Bone Remodeling and Recreating the Tumor-Bone Microenvironment Using Calvaria Co-culture and Histomorphometry

Bone is a connective tissue constituted of osteoblasts, osteocytes, and osteoclasts and a mineralized extracellular matrix, which gives it its strength and flexibility and allows it to fulfill its functions. Bone is continuously exposed to a variety of stimuli, which in pathological conditions can deregulate bone remodeling. To study bone biology and diseases and evaluate potential therapeutic agents, it has been necessary to develop in vitro and in vivo models.
This manuscript describes the dissection process and culture conditions of calvarias isolated from neonatal mice to study bone formation and the bone tumor microenvironment. In contrast to in vitro and in vivo models, this ex vivo model allows preservation of the three-dimensional environment of the tissue as well as the cellular diversity of the bone while culturing under defined conditions to simulate the desired microenvironment. Therefore, it is possible to investigate bone remodeling and its mechanisms, as well as the interactions with other cell types, such as the interactions between cancer cells and bone.
The assays reported here use calvarias from 5-7 day old BALB/C mice. The hemi-calvarias obtained are cultured in the presence of insulin, breast cancer cells (MDA-MB-231), or conditioned medium from breast cancer cell cultures. After analysis, it was established that insulin induced new bone formation, while cancer cells and their conditioned medium induced bone resorption. The calvarial model has been successfully used in basic and applied research to study bone development and cancer-induced bone diseases. Overall, it is an excellent option for an easy, informative, and low-cost assay.

The ex vivo culture of bone explants can be a valuable tool for the study of bone physiology and the potential evaluation of drugs in bone remodeling and bone diseases. The presented protocol describes the preparation and culture of calvarias isolated from newborn mice skulls, as well as its applications.

カルバリア共培養と組織病量を用いた骨リモデリングと腫瘍-骨微小環境の再現

Bone is a connective tissue constituted of osteoblasts, osteocytes, and osteoclasts and a mineralized extracellular matrix, which gives it its strength ...

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response.
This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 &#181;L basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFN&#947; and TNF&#945;) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFN&#947;+, TNF&#945;+ and CD107a+ CD4+ and CD8+ T cells was quantified.
This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

直交トピック膵腫瘍の生成と腫瘍浸潤T細胞細胞傷害のEx生体内特性評価

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

Analysis of Side Population in Solid Tumor Cell Lines

Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great significance for tumor research. Currently, several techniques are used for the identification and purification of CSCs from tumor tissues and tumor cell lines. Separation and analysis of side population (SP) cells are two of the commonly used methods. The methods rely on the ability of CSCs to rapidly expel fluorescent dyes, such as Hoechst 33342. The efflux of the dye is associated with the ATP-binding cassette (ABC) transporters and can be inhibited by ABC transporter inhibitors. Methods for staining cultured tumor cells with Hoechst 33342 and analyzing the proportion of their SP cells by flow cytometry are described. This assay is convenient, fast, and cost-effective. Data generated in this assay can contribute to a better understanding of the effect of genes or other extracellular and intracellular signals on the stemness properties of tumor cells.

A convenient, fast, and cost-effective method to measure the proportion of side population cells in solid tumor cell lines is presented.

固形腫瘍細胞株における側集団の解析

Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great ...

Isolation of Proximal Fluids to Investigate the Tumor Microenvironment of Pancreatic Adenocarcinoma

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death, and soon to become the second. There is an urgent need of variables associated to specific pancreatic pathologies to help preoperative differential diagnosis and patient profiling. Pancreatic juice is a relatively unexplored body fluid, which, due to its close proximity to the tumor site, reflects changes in the surrounding tissue. Here we describe in detail the intraoperative collection procedure. Unfortunately, translating pancreatic juice collection to murine models of PDAC, to perform mechanistic studies, is technically very challenging. Tumor interstitial fluid (TIF) is the extracellular fluid, outside blood and plasma, which bathes tumor and stromal cells. Similarly to pancreatic juice, for its property to collect and concentrate molecules that are found diluted in plasma, TIF can be exploited as an indicator of microenvironmental alterations and as a valuable source of disease-associated biomarkers. Since TIF is not readily accessible, various techniques have been proposed for its isolation. We describe here two simple and technically undemanding methods for its isolation: tissue centrifugation and tissue elution.

Pancreatic juice is a precious source of biomarkers for human pancreatic cancer. We describe here a method for intraoperative collection procedure. To overcome the challenge of adopting this procedure in murine models, we suggest an alternative sample, tumor interstitial fluid, and describe here two protocols for its isolation.

膵臓腺癌の腫瘍微小環境を調べるための近位液の分離

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death, and soon to become the second. There is an urgent need of variables ...

A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor microenvironment is an important step toward understanding mechanisms that regulate tumor progression. While this can be accomplished through current intravital imaging techniques, it remains challenging due to the heterogeneous nature of tissues and the need for spatial context within the experimental observation. To this end, we have developed an intravital imaging workflow that pairs collagen second harmonic generation imaging, endogenous fluorescence from the metabolic co-factor NAD(P)H, and fluorescence lifetime imaging microscopy (FLIM) as a means to non-invasively compartmentalize the tumor microenvironment into basic domains of the tumor nest, the surrounding stroma or ECM, and the vasculature. This non-invasive protocol details the step-by-step process ranging from the acquisition of time-lapse images of mammary tumor models to post-processing analysis and image segmentation. The primary advantage of this workflow is that it exploits metabolic signatures to contextualize the dynamically changing live tumor microenvironment without the use of exogenous fluorescent labels, making it advantageous for human patient-derived xenograft (PDX) models and future clinical use where extrinsic fluorophores are not readily applicable.

The intravital imaging method described here utilizes collagen second harmonic generation and endogenous fluorescence from the metabolic co-factor NAD(P)H to non-invasively segment an unlabeled tumor microenvironment into tumor, stromal, and vascular compartments for in-depth analysis of 4D intravital images.

乳腺腫瘍微小環境の生体内イメージングのためのラベルフリーセグメンテーションアプローチ

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor ...

Industrialized, Artificial Intelligence-guided Laser Microdissection for Microscaled Proteomic Analysis of the Tumor Microenvironment

The tumor microenvironment (TME) represents a complex ecosystem comprised of dozens of distinct cell types, including tumor, stroma, and immune cell populations. To characterize proteome-level variation and tumor heterogeneity at scale, high-throughput methods are needed to selectively isolate discrete cellular populations in solid tumor malignancies. This protocol describes a high-throughput workflow, enabled by artificial intelligence (AI), that segments images of hematoxylin and eosin (H&#38;E)-stained, thin tissue sections into pathology-confirmed regions of interest for selective harvest of histology-resolved cell populations using laser microdissection (LMD). This strategy includes a novel algorithm enabling the transfer of regions denoting cell populations of interest, annotated using digital image software, directly to laser microscopes, thus enabling more facile collections. Successful implementation of this workflow was performed, demonstrating the utility of this harmonized method to selectively harvest tumor cell populations from the TME for quantitative, multiplexed proteomic analysis by high-resolution mass spectrometry. This strategy fully integrates with routine histopathology review, leveraging digital image analysis to support enrichment of cellular populations of interest and is fully generalizable, enabling harmonized harvests of cell populations from the TME for multiomic analyses.

This protocol describes a high-throughput workflow for artificial intelligence-driven segmentation of pathology-confirmed regions of interest from stained, thin tissue section images for enrichment of histology-resolved cell populations using laser microdissection. This strategy includes a novel algorithm enabling the transfer of demarcations denoting cell populations of interest directly to laser microscopes.

腫瘍微小環境のマイクロスケールプロテオミクス分析のための工業化された人工知能誘導レーザーマイクロダイセクション

The tumor microenvironment (TME) represents a complex ecosystem comprised of dozens of distinct cell types, including tumor, stroma, and immune cell ...

濃縮細胞集団を生成し、細胞間コミュニケーションを調べるための簡単な方法：腫瘍微小環境のミミック

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この動画の章

単球/マクロファージにおける腫瘍微小環境の採用を検討する癌細胞株における遺伝子発現の条件付きのノックダウン

生体内でPH、 pO₂、酸化還元状態とリン酸濃度および腫瘍微小環境におけるグルタチオンの EPR 評価

タッチ捺印細胞診を用いた臨床病理標本から質の高い腫瘍 DNA を取得する簡易法

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

カルバリア共培養と組織病量を用いた骨リモデリングと腫瘍-骨微小環境の再現

直交トピック膵腫瘍の生成と腫瘍浸潤T細胞細胞傷害のEx生体内特性評価

固形腫瘍細胞株における側集団の解析

膵臓腺癌の腫瘍微小環境を調べるための近位液の分離

乳腺腫瘍微小環境の生体内イメージングのためのラベルフリーセグメンテーションアプローチ

腫瘍微小環境のマイクロスケールプロテオミクス分析のための工業化された人工知能誘導レーザーマイクロダイセクション

濃縮細胞集団を生成し、細胞間コミュニケーションを調べるための簡単​​な方法：腫瘍微小環境のミミック

要約

さらに動画を探す

この動画の章

単球/マクロファージにおける腫瘍微小環境の採用を検討する癌細胞株における遺伝子発現の条件付きのノックダウン

生体内でPH、 pO2、酸化還元状態とリン酸濃度および腫瘍微小環境におけるグルタチオンの EPR 評価

タッチ捺印細胞診を用いた臨床病理標本から質の高い腫瘍 DNA を取得する簡易法

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

カルバリア共培養と組織病量を用いた骨リモデリングと腫瘍-骨微小環境の再現

直交トピック膵腫瘍の生成と腫瘍浸潤T細胞細胞傷害のEx生体内特性評価

固形腫瘍細胞株における側集団の解析

膵臓腺癌の腫瘍微小環境を調べるための近位液の分離

乳腺腫瘍微小環境の生体内イメージングのためのラベルフリーセグメンテーションアプローチ

腫瘍微小環境のマイクロスケールプロテオミクス分析のための工業化された人工知能誘導レーザーマイクロダイセクション

濃縮細胞集団を生成し、細胞間コミュニケーションを調べるための簡単な方法：腫瘍微小環境のミミック

生体内でPH、 pO₂、酸化還元状態とリン酸濃度および腫瘍微小環境におけるグルタチオンの EPR 評価

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