著者らは、有益な意見を提供してくれたColler研究室のすべてのメンバーに感謝したいと思います。H.A.C.は、リタ・アレン財団のミルトン・E・カッセル奨学生でした。NIH / NCI 1 R01 CA221296-01A1、NIH 1 R01 AR070245-01A1、黒色腫研究アライアンスチーム科学賞、がん研究所臨床検査統合プログラム賞、アイリスカンターウィメンズヘルスセンター/ UCLA CTSI NIHグラントUL1TR000124、カリフォルニア大学がん研究調整委員会、デビッドゲフィン医学部代謝テーマ賞、臨床トランスレーショナルサイエンスインスティテュート、ジョンソン総合がんセンター、 Broad Stem Cell Research Center(Rose Hills and Ha Gaba)からのInnovation Award、UCLA SPORE in Prostate Cancer(National Cancer Institute of the National Cancer Institute of the National Institutes of the National Institutes、Award Number P50CA092131)、Broad Stem Cell Center、Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLAからのイノベーション賞、 腫瘍細胞生物学トレーニングプログラム(USHHSルースL.キルシュスタイン機関国立研究サービス賞#T32 CA009056)、UCLA AR32の皮膚科T071307プログラム、およびUCLA筋細胞生物学、病態生理学および治療T32トレーニングプログラム5 T32 AF 65972。

著者は開示するものは何もありません。

図1の実験では、ヒト皮膚線維芽細胞をヒトA2058メラノーマ細胞と同時導入すると、線維芽細胞を同時注入せずにメラノーマ細胞を導入した場合よりも大きな腫瘍が生じました。この差は、腫瘍の体積と腫瘍の重量に基づいて簡単に検出できます。結果は、癌関連線維芽細胞が腫瘍増殖を促進できるという複数の報告と一致しています5、6</...

腫瘍微小環境の中で、最も顕著な細胞型の1つは、がん関連線維芽細胞(CAF)1です。これらの癌腫関連線維芽細胞は、腫瘍抑制の役割を果たすことができる2,3。例えば、S100A発現線維芽細胞は、発がん物質をカプセル化し、癌腫形成から保護することができるコラーゲンを分泌する4。さらに、膵臓がんにおけるα平滑筋アクチン(SMA)陽性筋線維芽細胞の枯渇は免疫抑制を引き起こし、膵臓がんの進行を加速させます2。CAFはまた、癌細胞と共進化し、腫瘍の進行を促進することができます5、6、7、8。線維芽細胞は、腫瘍促進環境を作り出す細胞外マトリックスタンパク質を合成して分泌することができる8。これらの細胞外マトリックスタンパク質は、組織の機械的硬化を引き起こす可能性があり、これは腫瘍の進行に関連する9,10。沈着した細胞外マトリックスは、免疫浸潤11を阻害する物理的障壁として作用することができる。CAFsによるマトリックス沈着は、CAFsによって生成されるフィブロネクチンが腫瘍浸潤を促進することが示されているため、腫瘍浸潤とも関連している12。CAFは、トランスフォーミング成長因子β(TGF-β)、血管内皮増殖因子(VEGF)、インターロイキン-6(IL-6)、およびCXC-ケモカインリガンド12(CXCL12)を分泌することにより、血管新生を促進し、免疫抑制細胞を腫瘍微小環境に動員します13,14,15。腫瘍増殖の促進における中心的な役割のために、癌関連線維芽細胞は抗癌療法の新たな標的である6,16,17,18。
以下のプロトコルは、十分に確立され広く使用されている腫瘍増殖のマウスモデルにおいて、線維芽細胞が腫瘍の成長にどのように影響するかをテストするための方法について説明しています。腫瘍微小環境における線維芽細胞の重要性を理解するために、マウスに癌細胞を導入してその成長を監視するための標準プロトコルは、癌細胞導入を伴う線維芽細胞を含むように変更されました。癌細胞は、皮下または皮内に導入することができる。皮内導入は、皮膚自体から発生する腫瘍をもたらすであろう。癌細胞と線維芽細胞をマウスに同時注射する異種移植片は、癌増殖を促進する能力における線維芽細胞、線維芽細胞の亜集団、およびタンパク質因子の役割を分析するための重要な方法論的ツールを表しています19,20,21。癌細胞と線維芽細胞をマウスに同時注射するための詳細なプロトコルが提供されています。この方法は、線維芽細胞の存在または非存在を比較するために、異なる供給源からの線維芽細胞を比較するために20、または特定のタンパク質の発現の有無にかかわらず線維芽細胞を比較するために使用することができる19。癌細胞および線維芽細胞が導入された後、腫瘍の大きさを経時的にモニターすることができる。実験の終わりに、腫瘍を解剖して計量することができます。腫瘍の成長を経時的に監視することにより、さまざまな要因の重要性を分析できます。
腫瘍増殖における線維芽細胞の役割を研究するための可能な代替アプローチがあります。一例として、線維芽細胞で優先的に発現されるドライバーを有する遺伝子の組織特異的ノックアウトを提供するCre-loxedベースのモデルがある。このようなアプローチは、腫瘍の進行に対する線維芽細胞における特定の遺伝子および経路の役割を調査する機会も提供する。Cre-loxベースのアプローチと比較して、提供されたプロトコルは、腫瘍の成長がわずか数週間で監視されるため、線維芽細胞の役割を監視するためのはるかに迅速なアプローチを表します。提供されたアプローチはまた、遺伝子操作されたマウスのコロニーを生成および収容する必要がないため、大幅に安価である。提供されたプロトコルは、マウスコロニーを開発する必要なしに、shRNAを使用して異なる遺伝子のノックダウンの効果を迅速にテストするために使用できます。提供されたアプローチは、異なる数の線維芽細胞の比較、異なる癌細胞と線維芽細胞の比率、異なる遺伝子のノックダウン、さらには異なる組織部位または種からの線維芽細胞の比較を可能にするため、より柔軟である。Cre-loxアプローチは、線維芽細胞がより生理学的な状況でマウス内に存在するという利点を有するであろう。
ここで報告されたプロトコルは、腫瘍の成長に対する線維芽細胞の影響を迅速かつ費用効果の高い方法で監視しようとする科学者にとって価値があります。このプロトコルは、腫瘍増殖に対する腫瘍増殖に関する線維芽細胞または異なるソースからの線維芽細胞の異なるサブセットを調査する科学者にとって特に価値があります。腫瘍の開始が生理学的な状況で起こることが重要であるならば、遺伝子操作されたマウスモデルが考慮されるべきです。
これらの実験を実行するには、いくつかの可能なアプローチがあります。免疫コンピテントマウスを宿主として使用することができ、線維芽細胞と免疫細胞の相互作用の調査が可能になります。免疫コンピテントマウスモデルの場合、マウスがん細胞とマウス胚性線維芽細胞(MEF)を注射する必要があります。MEFを使用することで、研究者は広範囲のノックアウトマウス系統を利用して、目的の遺伝子の有無を試験することもできます。あるいは、免疫不全マウスを使用して、ヒト癌細胞に由来するマウスにおける腫瘍の増殖の促進におけるヒト線維芽細胞の役割を試験することができる。癌細胞の導入は、皮下または同所的に行うことができる。黒色腫の場合、後述するように、腫瘍-線維芽細胞混合物を皮内注射して、黒色腫が発症する皮膚内の位置をより厳密にシミュレートする同所性注射を行うことができます。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>26G Needles</td>
			<td>Fisher Scientific</td>
			<td>14-826-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol swabs</td>
			<td>Fisher Scientific</td>
			<td>326895</td>
			<td></td>
		</tr>
		<tr>
			<td>Animal clipper miniARCO with surgical blade #40</td>
			<td>WAHL Professional</td>
			<td>8787-450A</td>
			<td></td>
		</tr>
		<tr>
			<td>Athymic nude mice (NU/J)</td>
			<td>The Jackson labs</td>
			<td>002019</td>
			<td>These mice are immunocompromised and can be used for experiments in which human cells are introduced. Immunocompetent mice can also be used if mouse cancer cells and fibroblasts will be introduced.</td>
		</tr>
		<tr>
			<td>Cancer cells</td>
			<td>ATCC</td>
			<td>ATCC&#174; CRL-11147&#8482;</td>
			<td>This is the catalog number for a primary human melanoma cell line. Other cancer cell types can also be used.</td>
		</tr>
		<tr>
			<td>Cell Culture Multi Flasks</td>
			<td>Fisher Scientific</td>
			<td>14-826-95</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge for conical tubes capable of reaching 180 x g</td>
			<td>Fisher Scientific</td>
			<td>14-432-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess Cell Counting Chamber</td>
			<td>Fisher Scientific</td>
			<td>C10228</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Fisher Scientific</td>
			<td>11965-118</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Fisher Scientific</td>
			<td>MT35010CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibroblasts&#160;</td>
			<td>ATCC</td>
			<td>PCS-201-012</td>
			<td>We isolate fibroblasts from skin in our lab. This is a catalog number for an adult primary human dermal fibroblast cell line. MEFs and fibroblasts derived from other sites can also be used.</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Henry Schein Animal Health</td>
			<td>NDC 11695-6776-2</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS USP grade for injection into mice</td>
			<td>Fisher Scientific</td>
			<td>50-751-7476</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 10 ml serological pipet</td>
			<td>Celltreat</td>
			<td>667210B</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 5 ml serological pipet</td>
			<td>Celltreat</td>
			<td>229005B</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 50 ml centrifuge tubes</td>
			<td>Genesee Scientific</td>
			<td>28-108</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Syringe Filters pore size 0.2 microns</td>
			<td>Fisher Scientific</td>
			<td>09-740-61A</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile tissue culture-grade Trypsin-EDTA</td>
			<td>Fisher Scientific</td>
			<td>15400054</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile tissue-culture grade PBS</td>
			<td>Fisher Scientific</td>
			<td>50-751-7476</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterle 25 ml serological pipet</td>
			<td>Celltreat</td>
			<td>667225B</td>
			<td></td>
		</tr>
		<tr>
			<td>TC treated 100 x 20 mm dishes</td>
			<td>Genesee Scientific</td>
			<td>25-202</td>
			<td></td>
		</tr>
		<tr>
			<td>TC treated 150 x 20 mm dishes</td>
			<td>Genesee Scientific</td>
			<td>25-203</td>
			<td></td>
		</tr>
		<tr>
			<td>TC treated 60 x 15 mm dishes</td>
			<td>Genesee Scientific</td>
			<td>25-260</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Fisher Scientific</td>
			<td>C10228</td>
			<td></td>
		</tr>
	</tbody>
</table>

a mouse model to investigate the role of cancer-associated fibroblasts in tumor growth

がん関連線維芽細胞(CAF)は、腫瘍を促進する微小環境を作り出すことにより、腫瘍の成長に重要な役割を果たすことができます。腫瘍微小環境におけるCAFの役割を研究するためのモデルは、線維芽細胞、さまざまな組織からの線維芽細胞、および線維芽細胞の特定の遺伝的要因の機能的重要性を理解するのに役立ちます。マウスモデルは、in vivoの状況における腫瘍の成長と進行の寄与を理解するために不可欠です。ここでは、がん細胞を線維芽細胞と混合し、マウスに導入して腫瘍を発生させるプロトコールを提供する。経時的な腫瘍サイズと最終的な腫瘍重量が決定され、グループ間で比較されます。記載されたプロトコルは、腫瘍の成長および進行におけるCAFの機能的役割についてより多くの洞察を提供することができる。

A2058ヒト黒色腫細胞および初代ヒト皮膚線維芽細胞を無菌条件下で培養した。細胞を回収し、PBSで3回洗浄した。免疫不全マウス(NU/J-Foxn1ヌード系統)を25万個のA2058メラノーマ細胞のみで片側腹部に皮下注射した。もう一方の側面では、マウスに25万個のA2058メラノーマ細胞と75万個の線維芽細胞の混合物を注射した。細胞を12匹の免疫不全マウスに注射した。左右の脇腹?...

Watch this Scientific Journal Video about A Mouse Model to Investigate the Role of Cancer-Associated Fibroblasts in Tumor Growth at JoVE.com

A Mouse Model to Investigate the Role of Cancer-Associated Fibroblasts in Tumor Growth

癌細胞と線維芽細胞を同時注入し、腫瘍の成長を経時的に監視するためのプロトコルが提供されます。このプロトコルは、腫瘍増殖の調節因子としての線維芽細胞の役割の分子基盤を理解するために使用できます。

腫瘍増殖における癌関連線維芽細胞の役割を調べるためのマウスモデル

Cancer-associated fibroblasts (CAFs) can play an important role in tumor growth by creating a tumor-promoting microenvironment. Models to study the role ...

a-mouse-model-to-investigate-role-cancer-associated-fibroblasts-tumor

Research

JoVE Journal

Cancer Research

4.3K ビュー.  University of California, Los Angeles. 癌細胞と線維芽細胞を同時注入し、腫瘍の成長を経時的に監視するためのプロトコルが提供されます。このプロトコルは、腫瘍増殖の調節因子としての線維芽細胞の役割の分子基盤を理解するために使用できます。

ビデオ: A Mouse Model to Investigate the Role of Cancer-Associated Fibroblasts in Tumor Growth

Tumor Engraftment in a Xenograft Mouse Model of Human Mantle Cell Lymphoma

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when stimulated by T lymphocytes, oncogenic B cells survive and expand autonomously in undefined organ niches. Mantle cell lymphoma (MCL) is one such B cell disorder, where the median survival rate of patients is 4 - 5 years. This calls for the need of effective mechanisms by which the homing and engraftment of these cells are blocked in order to increase the survival and longevity of patients. Therefore, the effort to develop a xenograft mouse model to study the efficacy of MCL therapeutics by blocking the homing mechanism in vivo is of utmost importance. Development of animal recipients for human cell xenotransplantation to test early stage drugs have long been pursued, as relevant preclinical mouse models are crucial to screen new therapeutic agents. This animal model is developed to avoid human graft rejection and to establish a model for human diseases, and it may be an extremely useful tool to study disease progression of different lymphoma types and to perform preclinical testing of candidate drugs for hematologic malignancies, like MCL. We established a xenograft mouse model that will serve as an excellent resource to study and develop novel therapeutic approaches for MCL.

Mantle cell lymphoma (MCL) is a difficult to treat B cell disorder and it is equally difficult to establish a xenograft mouse model of primary MCL to study and develop therapeutics. Here, we describe the successful establishment of MCL xenografts in mice to help understand its underlying biology.

人間マントル細胞リンパ腫の異種移植マウス モデルにおける腫瘍生着

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when ...

がん研究

A Method to Define the Effects of Environmental Enrichment on Colon Microbiome Biodiversity in a Mouse Colon Tumor Model

Several recent studies have illustrated the beneficial effects of living in an enriched environment on improving human disease. In mice, environmental enrichment (EE) reduces tumorigenesis by activating the mouse immune system, or affects tumor bearing animal survival by stimulating the wound repair response, including improved microbiome diversity, in the tumor microenvironment. Provided here is a detailed procedure to assess the effects of environmental enrichment on the biodiversity of the microbiome in a mouse colon tumor model. Precautions regarding animal breeding and considerations for animal genotype and mouse colony integration are described, all of which ultimately affect microbial biodiversity. Heeding these precautions may allow more uniform microbiome transmission, and consequently will alleviate non-treatment dependent effects that can confound study findings. Further, in this procedure, microbiota changes are characterized using 16S rDNA sequencing of DNA isolated from stool collected from the distal colon following long-term environmental enrichment. Gut microbiota imbalance is associated with the pathogenesis of inflammatory bowel disease and colon cancer, but also of obesity and diabetes among others. Importantly, this protocol for EE and microbiome analysis can be utilized to study the role of microbiome pathogenesis across a variety of diseases where robust mouse models exist that can recapitulate human disease.

Environmental Enrichment (EE) is an animal housing environment that is used to reveal mechanisms that underlie the connections between lifestyle, stress, and disease. This protocol describes a procedure that uses a mouse model of colon tumorigenesis and EE to specifically define alterations in microbiota biodiversity that may impact animal mortality.

マウスの大腸腫瘍モデルにおける結腸マイクロバイ生物多様性環境エンリッチメントの効果を定義する方法

Several recent studies have illustrated the beneficial effects of living in an enriched environment on improving human disease. In mice, environmental ...

Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in Prostate Cancer

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction pathways and is tightly regulated by kinases and phosphatases. Therefore, characterizing the phosphoproteome may provide insights into identifying novel targets and biomarkers for oncologic therapy. Mass spectrometry provides a way to globally detect and quantify thousands of unique phosphorylation events. However, phosphopeptides are much less abundant than non-phosphopeptides, making biochemical analysis more challenging. To overcome this limitation, methods to enrich phosphopeptides prior to the mass spectrometry analysis are required. We describe a procedure to extract and digest proteins from tissue to yield peptides, followed by an enrichment for phosphotyrosine (pY) and phosphoserine/threonine (pST) peptides using an antibody-based and/or titanium dioxide (TiO2)-based enrichment method. After the sample preparation and mass spectrometry, we subsequently identify and quantify phosphopeptides using liquid chromatography-mass spectrometry and analysis software.

This protocol describes a procedure to extract and enrich phosphopeptides from prostate cancer cell lines or tissues for an analysis of the phosphoproteome via mass spectrometry-based proteomics.

前立腺癌におけるリン酸化プロテオームを調べる定量的ラベル無料質量と相まってリン酸化ペプチド濃縮

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction ...

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 ...

In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice

MicroRNAs (miRNAs) are important regulators of gene expression through their ability to destabilize mRNA and inhibit translation of target mRNAs. An ever-increasing number of studies have identified miRNAs as potential biomarkers for cancer diagnosis and prognosis, and also as therapeutic targets, adding an extra dimension to cancer evaluation and treatment. In the context of thyroid cancer, tumorigenesis results not only from mutations in important genes, but also from the overexpression of many miRNAs. Accordingly, the role of miRNAs in the control of thyroid gene expression is evolving as an important mechanism in cancer. Herein, we present a protocol to examine the effects of miRNA-inhibitor delivery as a therapeutic modality in thyroid cancer using human tumor xenograft and orthotopic mouse models. After engineering stable thyroid tumoral cells expressing GFP and luciferase, cells are injected into nude mice to develop tumors, which can be followed by bioluminescence. The in vivo inhibition of a miRNA can reduce tumor growth and upregulate miRNA gene targets. This method can be used to assess the importance of a determined miRNA in vivo, in addition to identifying new therapeutic targets.

This protocol describes xenograft and orthotopic mouse models of human thyroid tumorigenesis as a platform to test microRNA-based inhibitor treatments. This approach is ideal to study the function of non-coding RNAs and their potential as new therapeutic targets.

マウスの腫瘍増殖を減少させるマイクロRNAの生体内阻害

MicroRNAs (miRNAs) are important regulators of gene expression through their ability to destabilize mRNA and inhibit translation of target mRNAs. An ...

A 3D Spheroid Model as a More Physiological System for Cancer-Associated Fibroblasts Differentiation and Invasion In Vitro Studies

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor stroma, host fibroblasts are stimulated by cancer cells to differentiate. Thus, they acquire a phenotype that contributes to the tumor microenvironment and supports tumor progression. By using the spheroid model, we have set up such a 3D in vitro model system, in which we analyzed the role of laminin-332 and its receptor integrin &#945;3&#946;1 in this differentiation process. This spheroid model system not only reproduces the tumor microenvironment conditions in a more accurate way, but also is a very versatile model since it allows different downstream studies, such as immunofluorescent staining of both intra- and extracellular markers, as well as deposited extracellular matrix proteins. Moreover, transcriptional analyses by qPCR, flow cytometry and cellular invasion can be studied with this model. Here, we describe a protocol of a spheroid model to assess the role of CAFs&#39; integrin &#945;3&#946;1 and its ectopically deposited ligand, laminin-332, in differentiation and in supporting the invasion of pancreatic cancer cells.

The goal of this protocol is to establish a 3D in vitro model to study the differentiation of cancer-associated fibroblasts (CAFs) in a tumor bulk-like environment, which can be addressed in different analysis systems, such as immunofluorescence, transcriptional analysis and life cell imaging.

がん関連線維芽細胞分化とインビトロ研究における侵入のためのより生理学的システムとしての3Dスフェロイドモデル

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor ...

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 ...

Isolation of Primary Cancer-Associated Fibroblasts from a Syngeneic Murine Model of Breast Cancer for the Study of Targeted Nanoparticles

Cancer-associated fibroblasts (CAFs) are key actors in the context of the tumor microenvironment. Despite being reduced in number as compared to tumor cells, CAFs regulate tumor progression and provide protection from antitumor immunity. Emerging anticancer strategies aim to remodel the tumor microenvironment through the ablation of pro-tumorigenic CAFs or reprogramming of CAFs functions and their activation status. A promising approach is the development of nanosized delivery agents able to target CAFs, thus allowing the specific delivery of drugs and active molecules. In this context, a cellular model of CAFs may provide a useful tool for in vitro screening and preliminary investigation of such nanoformulations.
This study describes the isolation and culture of primary CAFs from the syngeneic 4T1 murine model of triple-negative breast cancer. Magnetic beads were used in a 2-step separation process to extract CAFs from dissociated tumors. Immunophenotyping control was performed using flow cytometry after each passage to verify the process yield. Isolated CAFs can be employed to study the targeting capability of different nanoformulations designed to tackle the tumor microenvironment. Fluorescently labeled H-ferritin nanocages were used as candidate nanoparticles to set up the method. Nanoparticles, either bare or conjugated with a targeting ligand, were analyzed for their binding to CAFs. The results suggest that ex vivo extraction of breast CAFs may be a useful system to test and validate nanoparticles for the specific targeting of tumorigenic CAFs.

This paper aims to provide a protocol for the isolation and culture of primary cancer-associated fibroblasts from a syngeneic murine model of triple-negative breast cancer and their application for the preclinical study of novel nanoparticles designed to target the tumor microenvironment.&#160;

標的ナノ粒子の研究のための乳癌の同質性マウスモデルからの原発癌関連線維芽細胞の分離

Cancer-associated fibroblasts (CAFs) are key actors in the context of the tumor microenvironment. Despite being reduced in number as compared to tumor ...

An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth

Brain metastasis is a serious consequence of breast cancer for women as these tumors are difficult to treat and are associated with poor clinical outcomes. Preclinical mouse models of breast cancer brain metastatic (BCBM) growth are useful but are expensive, and it is difficult to track live cells and tumor cell invasion within the brain parenchyma. Presented here is a protocol for ex vivo brain slice cultures from xenografted mice containing intracranially injected breast cancer brain-seeking clonal sublines. MDA-MB-231BR luciferase tagged cells were injected intracranially into the brains of Nu/Nu female mice, and following tumor formation, the brains were isolated, sliced, and cultured ex vivo. The tumor slices were imaged to identify tumor cells expressing luciferase and monitor their proliferation and invasion in the brain parenchyma for up to 10 days. Further, the protocol describes the use of time-lapse microscopy to image the growth and invasive behavior of the tumor cells following treatment with ionizing radiation or chemotherapy. The response of tumor cells to treatments can be visualized by live-imaging microscopy, measuring bioluminescence intensity, and performing histology on the brain slice containing BCBM cells. Thus, this ex vivo slice model may be a useful platform for rapid testing of novel therapeutic agents alone or in combination with radiation to identify drugs personalized to target an individual patient&#39;s breast cancer brain metastatic growth within the brain microenvironment.

An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth

We introduce a protocol for measuring real-time drug and radiation response of breast cancer brain metastatic cells in an organotypic brain slice model. The methods provide a quantitative assay to investigate the therapeutic effects of various treatments on brain metastases from breast cancer in an ex vivo manner within the brain microenvironment interface.

乳がん脳転移性腫瘍増殖を研究し、標的とする Ex Vivo 脳スライスモデル

Brain metastasis is a serious consequence of breast cancer for women as these tumors are difficult to treat and are associated with poor clinical ...

A Three-Dimensional Spheroid Model to Investigate the Tumor-Stromal Interaction in Hepatocellular Carcinoma

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver cancer, rationalize its rank as the second most common cause of cancer-related death. Preclinical models need to be adapted to recapitulate the human conditions to select the best therapeutic candidates for clinical development and aid the delivery of personalized medicine. Three-dimensional (3D) cellular spheroid models show promise as an emerging in vitro alternative to two-dimensional (2D) monolayer cultures. Here, we describe a 3D tumor spheroid&#160;model which exploits the ability of individual cells to aggregate when maintained in hanging droplets, and is more representative of an in vivo environment than standard monolayers. Furthermore, 3D spheroids can be produced by combining homotypic or heterotypic cells, more reflective of the cellular heterogeneity in vivo, potentially enabling the study of environmental interactions that can influence progression and treatment responses. The current research optimized the cell density to form 3D homotypic and heterotypic tumor spheroids by immobilizing cell suspensions on the lids of standard 10 cm3 Petri dishes. Longitudinal analysis was performed to generate growth curves for homotypic versus heterotypic tumor/fibroblasts spheroids. Finally, the proliferative impact of fibroblasts (COS7 cells) and liver myofibroblasts (LX2) on homotypic tumor (Hep3B) spheroids was investigated. A seeding density of 3,000 cells (in 20 &#181;L media) successfully yielded Huh7/COS7 heterotypic spheroids, which displayed a steady increase in size up to culture day 8, followed by growth retardation. This finding was corroborated using Hep3B homotypic spheroids cultured in LX2 (human hepatic stellate cell line) conditioned medium (CM). LX2 CM triggered the proliferation of Hep3B spheroids compared to control tumor spheroids. In conclusion, this protocol has shown that 3D tumor spheroids can be used as a simple, economical, and prescreen in vitro tool to study tumor-stromal interactions more comprehensively.

Comprehensive in vitro models that faithfully recapitulate the relevant human disease are lacking. The current study presents three-dimensional (3D) tumor spheroid creation and culture, a reliable in vitro tool to study the tumor-stromal interaction in human hepatocellular carcinoma.

肝細胞癌における腫瘍間質相互作用を調べる三次元スフェロイドモデル

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver ...