We thank Richard Lieber and Shannon Bremner for their ImageJ macro and instructions. While at Thomas Jefferson University, this work was supported by the Pennsylvania Department of Health CURE Grant TJU No. 080-37038-AI0801. Subsequently, this study was supported by a grant to AB from the National Institutes of Health (R21CA190028), and by grants to TAZ from the National Institutes of Health (R01CA122596, R01CA194593), the IU Simon Cancer Center, the Lustgarten Foundation, the Lilly Foundation, Inc., and the IUPUI Pancreas Signature Center.

<ol>
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The authors have nothing to disclose.

特にその最新の段階で、結腸直腸癌は、貧しい転帰と患者の生活の質の低下の原因である悪液質の開発と関連しています。多くの研究は、癌の二次的症状の治療に焦点を当てています。しかし、この方向に多くの努力にもかかわらず、癌性悪液質21には承認された治療法はまだありません。従って、動物モデルは、所見の変換を最大にするためにできるだけ近いヒト病理に似ているこ...

筋萎縮は、癌、敗血症、肝臓、肝硬変、心臓や腎不全、慢性閉塞性肺疾患、およびAIDSなどの様々な臨床症状の重篤な合併症です。特に、筋消耗、癌1患者の少なくとも50％において明らかです。原因および/またはからの骨格筋タンパク質分解システムの過剰活性化に増加したタンパク質分解からの癌の結果で骨格筋の損失は、タンパク質合成6を減少させました 。脂肪分解は、脂肪組織の枯渇をもたらすも明らかです。臨床的には、悪液質を低減し、品質及び寿命の長さと関連し、20における死亡の原因であると推定されている-癌患者7の30％。できるだけヒトの疾患に似ている実験モデルの使用は有益であろう。最適な動物モデルは、異なる治療法との予測不可能な要因から高い再現性により、同様に制限された干渉によって特徴づけられます通常、臨床状態8に関連付けられている食事、性別、遺伝的背景。新しい方法は、癌の発生に影響を受けやすい遺伝的に改変されたマウスを使用することであるが、これまで、癌性悪液質は、癌細胞または発癌物質の注入の移植によって特徴付けられる動物モデルにおいて主に研究されてきました。 C26癌を有するマウスは、癌性悪液質2,5のよく特徴付けられたと広範囲に使用されるモデルを表す（結腸-26および腺癌と呼ばれます）。主に強化脂肪及びタンパク質異化9を介して身体および筋肉重量損失C26腫瘍結果の成長。一般に、全体重に対して10％の腫瘍重量は、骨格筋重量の20％〜25％の減少および脂肪3,10の高い枯渇と関連しています。肝腫および脾腫も、急性期反応の活性化及びプロinflaの上昇に伴って、腫瘍の成長で観察されていますmmatoryサイトカインレベル3,11。これらの中でも、同様にこのサイトカインはおそらく、悪液質12の唯一の誘導因子ではないにもかかわらず、IL-6は、C26モデルにおいて筋肉消耗を媒介する重要な役割を果たしていることが知られています。上昇したIL-6は、JAK / STAT3経路の活性化を介して筋萎縮を引き起こし、この転写因子を阻害すること3,4筋肉消耗を防ぐことができます。 C26誘発性筋萎縮時には、筋萎縮の多くの条件のように、筋肉量がない細胞死または繊維13の損失を介して、主に筋線維全体の筋肉のタンパク質含有量の減少によって失われます。 C26の悪液質では、より小さな断面積へのシフトは、解糖および酸化繊維2の両方で観察されました。これはまた、減少筋力5と一致しています。多くのグループが、世界中のがんCACのための筋萎縮の新しいメディエーターまたは臨床的に関連する薬物を発見するために、C26モデルの利点をとっていますhexia。しかし、このモデルの使用のために多くの異なる手順は、得られたデータの整合性についての懸念を高め、異なる実験条件で再現性の障壁を装った、報告されています。ここでは、標準化と再現性のあるデータが得られ、癌性悪液質の研究のためのこのモデルの典型的な使用を報告しています。

<table border="0" cellpadding="0" cellspacing="0" width="661">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:167px;">Cell culture Flasks</td>
			<td style="width:205px;">Falcon - Becton Dickinson</td>
			<td style="width:104px;">35-5001</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:167px;">DMEM</td>
			<td style="width:205px;">Cellgro</td>
			<td style="width:104px;">10-017-CV</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:167px;">FBS</td>
			<td style="width:205px;">Gibco</td>
			<td style="width:104px;">26140</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:167px;">Streptomycin-Penicillin&#160;</td>
			<td style="width:205px;">Cellgro</td>
			<td style="width:104px;">30-002-CI</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:167px;">CD2F1 mice</td>
			<td>Harlan</td>
			<td>060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:167px;">Anesthesia apparatus</td>
			<td style="width:205px;">EZ-Anesthesia</td>
			<td style="width:104px;">EZ-7000</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:167px;">2-Methyl Butane</td>
			<td style="width:205px;">Sigma-Aldrich</td>
			<td style="width:104px;">M32631</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:167px;">OCT</td>
			<td style="width:205px;">Tissue-Tek</td>
			<td style="width:104px;">4583</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:167px;">Cryostat</td>
			<td style="width:205px;">Leica</td>
			<td style="width:104px;">CM1850</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:167px;">Cork disks</td>
			<td>Electron Microscopy Sciences</td>
			<td>63305</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:167px;">Superfrost plus glass slides</td>
			<td>VWR</td>
			<td>48311-703</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:167px;">Anti-Laminin Rabbit polyclonal antibody</td>
			<td>Sigma-Aldrich</td>
			<td>L9393</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:167px;">Anti-Dystrophin Mouse Monoclonal antibody</td>
			<td>Vector Laboratories</td>
			<td>VP-D508</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:167px;">Alexa Flour 594 anti-mouse IgG</td>
			<td>Life Technologies</td>
			<td>A11062</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:167px;">Alexa Flour 594 anti-rabbit IgG</td>
			<td>Life Technologies</td>
			<td>A21211</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hematoxylin</td>
			<td>Sigma-Aldrich</td>
			<td>GHS216</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eosin</td>
			<td>Sigma-Aldrich</td>
			<td>HT110332</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylene</td>
			<td>Acros Organics</td>
			<td>422680025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:167px;">Cytoseal-XYL</td>
			<td style="width:205px;">Thermo</td>
			<td style="width:104px;">8312-4</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:167px;">Microscope</td>
			<td style="width:205px;">Zeiss</td>
			<td style="width:104px;">Observer.Z1&#160;</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:167px;">Bamboo Tablet</td>
			<td style="width:205px;">Wacom</td>
			<td style="width:104px;">CTH-661</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:167px;">Prism 7.0 for Mac OS X</td>
			<td style="width:205px;">GraphPad Software, Inc.</td>
			<td style="width:104px;"></td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:167px;">Excel for Mac 2011</td>
			<td style="width:205px;">Microsoft Corp.</td>
			<td style="width:104px;"></td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:167px;">Image J</td>
			<td style="width:205px;">US National Institutes of Health</td>
			<td style="width:104px;">IJ1.46</td>
			<td style="width:185px;">http://rsbweb.nih.gov/ij/download.html</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:167px;">Microtainer</td>
			<td style="width:205px;">BD</td>
			<td style="width:104px;">365873</td>
			<td style="width:185px;"></td>
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the colon-26 carcinoma tumor-bearing mouse as a model for the study of cancer cachexia

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and activation of lipolysis and proteolysis systems. Cancer patients with cachexia benefit less from anti-neoplastic therapies and show increased mortality1. Several animal models have been established in order to investigate the molecular causes responsible for body and muscle wasting as a result of tumor growth. Here, we describe methodologies pertaining to a well-characterized model of cancer cachexia: mice bearing the C26 carcinoma2-4. Although this model is heavily used in cachexia research, different approaches make reproducibility a potential issue. The growth of the C26 tumor causes a marked and progressive loss of body and skeletal muscle mass, accompanied by reduced muscle cross-sectional area and muscle strength3-5. Adipose tissue is also lost. Wasting is coincident with elevated circulating levels of pro-inflammatory cytokines, particularly Interleukin-6 (IL-6)3, which is directly, although not entirely, responsible for C26 cachexia. It is well-accepted that a primary mechanism by which the C26 tumor induces muscle tissue depletion is the activation of skeletal muscle proteolytic systems. Thus, expression of muscle-specific ubiquitin ligases, such as atrogin-1/MAFbx and MuRF-1, represent an accepted method for the evaluation of the ongoing muscle catabolism2. Here, we present how to execute this model in a reproducible manner and how to excise several tissues and organs (the liver, spleen, and heart), as well as fat and skeletal muscles (the gastrocnemius, tibialis anterior, and quadriceps). We also provide useful protocols that describe how to perform muscle freezing, sectioning, and fiber size quantification.

（ - 5 D 4）指数関数的な細胞増殖が続く注射後8 D、 - C26腫瘍増殖動態は、最初の7のための誘導期を示しました。腫瘍塊は、最終的に（; 図 1A-B約2g）〜体重の10％に達します。最初のフェーズでは、腫瘍は触診により配置することができ、皮膚の小さな突起として表示されます。第二段階では、腫瘍は、皮膚の下の塊として観察されます。ま?...

Watch this Scientific Journal Video about The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia at JoVE.com

The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia

Mice bearing the Colon-26 (C26) carcinoma represent a classical model of cancer cachexia. Progressive muscle wasting occurs in association with tumor growth, over-expression of muscle-specific ubiquitin ligases, and reductions in muscle cross-sectional area. Fat loss is also observed. Cachexia is studied in a time-dependent manner with increasing severity of wasting.

大腸-26癌腫瘍を有するがん悪液質の研究のためのモデルとしてマウスを

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and ...

the-colon-26-carcinoma-tumor-bearing-mouse-as-model-for-study-cancer

Research

JoVE Journal

Cancer Research

16.0K ビュー.  Indiana University School of Medicine. Mice bearing the Colon-26 (C26) carcinoma represent a classical model of cancer cachexia. Progressive muscle wasting occurs in association with tumor growth, over-expression of muscle-specific ubiquitin ligases, and reductions in muscle cross-sectional area. Fat loss is also observed. Cachexia is studied in a time-dependent manner with increasing severity of wasting. 

ビデオ: The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia

Study of Viral Vectors in a Three-dimensional Liver Model Repopulated with the Human Hepatocellular Carcinoma Cell Line HepG2

This protocol describes the generation of a three-dimensional (3D) ex vivo liver model and its application to the study and development of viral vector systems. The model is obtained by repopulating the extracellular matrix of a decellularized rat liver with a human hepatocyte cell line. The model permits studies in a vascularized 3D cell system, replacing potentially harmful experiments with living animals. Another advantage is the humanized nature of the model, which is closer to human physiology than animal models.
In this study, we demonstrate the transduction of this liver model with a viral vector derived from adeno-associated viruses (AAV vector). The perfusion circuit that supplies the 3D liver model with media provides an easy means to apply the vector. The system permits monitoring of the major metabolic parameters of the liver. For final analysis, tissue samples can be taken to determine the extent of recellularization by histological techniques. Distribution of the virus vector and expression of the delivered transgene can be analyzed by quantitative PCR (qPCR), Western blotting and immunohistochemistry. Numerous applications of the vector model in basic research and in the development of gene therapeutic applications can be envisioned, including the development of novel antiviral therapeutics, cancer research, and the study of viral vectors and their potential side effects.

The recellularized extracellular matrix of a decellularized rat liver can be used as a humanized, three-dimensional ex vivo model to study the distribution and transgene expression of a virus or viral vector.

ヒト肝細胞癌細胞株HepG2細胞と三次元肝モデルに再増殖におけるウイルスベクターの研究

This protocol describes the generation of a three-dimensional (3D) ex vivo liver model and its application to the study and development of viral vector ...

がん研究

A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, radiation, and chemotherapy, locoregional recurrences and distant metastases occur at higher rates, and overall survival is decreased by 40% compared to HNSCC without PNI. In vitro studies of the pathways involved in HNSCC PNI have historically been challenging given the lack of a consistent, reproducible assay. Described here is the adaptation of the dorsal root ganglion (DRG) assay for the examination of PNI in HNSCC. In this model, DRG are harvested from the spinal column of a sacrificed nude mouse and placed within a semisolid matrix. Over the subsequent days, neurites are generated and grow in a radial pattern from the cell bodies of the DRG. HNSCC cell lines are then placed peripherally around the matrix and invade preferentially along the neurites toward the DRG. This method allows for rapid evaluation of multiple treatment conditions, with very high assay success rates and reproducibility.

Perineural invasion (PNI) is a common feature of head and neck squamous cell carcinoma (HNSCC), conferring lower survival rates. Its mechanisms are poorly understood. Utilizing neurites generated from murine dorsal root ganglia confined to a semisolid matrix, the pathways involved in the PNI of HNSCC cell lines can be investigated.

頭頸部扁平上皮癌における神経周囲の浸潤のためのモデル

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, ...

A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of diagnosis. Metastatic RCC (mRCC) is incurable, with a median survival time of only 18 months. Immune-based interventions (e.g., interferon (IFN) and interleukin (IL)-2) induce durable responses in a fraction of mRCC patients, and multikinase inhibitors (e.g., sunitinib or sorafenib) or anti-VEGF receptor monoclonal antibodies (mAb) are largely palliative, as complete remissions are rare. Such shortcomings in current therapies for mRCC patients provide the rationale for the development of novel treatment protocols. A key component in the preclinical testing of new therapies for mRCC is a suitable animal model. Beneficial features that recapitulate the human condition include a primary renal tumor, renal tumor metastases, and an intact immune system to investigate any therapy-driven immune effector responses and the formation of tumor-induced immunosuppressive factors. This report describes an orthotopic mRCC mouse model that has all of these features. We describe an intrarenal implantation technique using the mouse renal adenocarcinoma cell line Renca, followed by the assessment of tumor growth in the kidney (primary site) and lungs (metastatic site).

Implementation of an orthotopic model of renal cell carcinoma in immunocompetent mice affords the investigator a clinically-relevant system defined by the presence of a primary renal tumor and lung metastases in the same animal. This system can be used to preclinically test a variety of treatments in vivo.

定量的および前臨床治療の縦評価のための転移性腎細胞癌の同系マウスモデル

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of ...

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce decellularized tongue extracellular matrix (TEM) which provides functional scaffolds for TSCC construction. TEM provides an in vitro niche for cell growth, differentiation, and cell migration. The microstructures of native extracellular matrix (ECM) and biochemical compositions retained in the decellularized matrix provide tissue-specific niches for anchoring cells. The fabrication of TEM can be realized by deoxyribonuclease (DNase) digestion accompanied with a serious of organic or inorganic pretreatment. This protocol is easy to operate and ensures high efficiency for the decellularization. The TEM showed favorable cytocompatibility for TSCC cells under static or stirred culture conditions, which enables the construction of the TSCC model. A self-made bioreactor was also used for the persistent stirred condition for cell culture. Reconstructed TSCC using TEM showed the characteristics and properties resembling clinical TSCC histopathology, suggesting the potential in TSCC research.

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

A method is shown here for the preparation of the tongue extracellular matrix (TEM) with efficient decellularization. The TEM can be used as functional scaffolds for the reconstruction of a tongue squamous cell carcinoma (TSCC) model under static or stirred culture conditions.

舌の細胞外マトリックスと舌扁平上皮癌生体外の再構成の作製

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce ...

A Syngeneic Pancreatic Cancer Mouse Model to Study the Effects of Irreversible Electroporation

Pancreatic cancer (PC), a disease which kills approximately 40,000 patients each year in the US, has successfully evaded several therapeutic approaches including the promising immunotherapeutic strategies. Irreversible electroporation (IRE) is a non-thermal ablation technique that induces tumor cell death without destruction of adjacent collagenous structures, thus enabling the procedure to be performed in tumors very close to blood vessels. Unlike thermal ablation techniques, IRE results in gradual apoptotic cell death, along with immediate ablation induced necrosis, and is currently in clinical use for selected patients with locally advanced PC. An ablative, non-target specific procedure like IRE can induce a myriad of responses in the tumor microenvironment. A few studies have addressed the effects of IRE on tumor growth in other tumor types, but none have focused on PC. We have developed a syngeneic mouse model of PC in which subcutaneous (SQ) and orthotopic tumors can be successfully treated with IRE in a highly controlled setting, facilitating various longitudinal studies post procedure. This animal model serves as a robust system to study the effects of IRE and ways to improve the clinical efficacy of IRE.

Irreversible electroporation (IRE) is a non-thermal ablation technique used for the treatment of locally advanced pancreatic cancer. Being a relatively new technique, the effects of IRE on the tumor growth are poorly understood. We have developed a syngeneic mouse model that facilitates studying the effects of IRE on pancreatic cancer.

不可逆エレクトロポレーションの効果を研究する同系膵癌マウス モデル

Pancreatic cancer (PC), a disease which kills approximately 40,000 patients each year in the US, has successfully evaded several therapeutic approaches ...

Yeast As a Chassis for Developing Functional Assays to Study Human P53

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of different functional assays to study the impacts of 1) binding site [i.e., response element (RE)] sequence variants on P53 transactivation specificity or 2) TP53 mutations, co-expressed cofactors, or small molecules on P53 transactivation activity. Different basic and translational research applications have been developed. Experimentally, these approaches exploit two major advantages of the yeast model. On one hand, the ease of genome editing enables quick construction of qualitative or quantitative reporter systems by exploiting isogenic strains that differ only at the level of a specific P53-RE to investigate sequence-specificity of P53-dependent transactivation. On the other hand, the availability of regulated systems for ectopic P53 expression allows the evaluation of transactivation in a wide range of protein expression. Reviewed in this report are extensively used systems that are based on color reporter genes, luciferase, and the growth of yeast to illustrate their main methodological steps and to critically assess their predictive power. Moreover, the extreme versatility of these approaches can be easily exploited to study different TFs including P63 and P73, which are other members of TP53 gene family.

Presented here are four protocols to construct and exploit yeast Saccharomyces cerevisiae reporter strains to study human P53 transactivation potential, impacts of its various cancer-associated mutations, co-expressed interacting proteins, and the effects of specific small molecules.

ヒトP53を研究する機能アッセイを開発するシャーシとしての酵母

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of ...

Transfer of Manipulated Tumor-associated Neutrophils into Tumor-Bearing Mice to Study their Angiogenic Potential In Vivo

The contribution of neutrophils to the regulation of tumorigenesis is getting increased attention. These cells are heterogeneous, and depending on the tumor milieu can possess pro- or anti-tumor capacity. One of the important cytokines regulating neutrophil functions in a tumor context are type I interferons. In the presence of interferons, neutrophils gain anti-tumor properties, including cytotoxicity or stimulation of the immune system. Conversely, the absence of an interferon signaling results in prominent pro-tumor activity, characterized with strong stimulation of tumor angiogenesis. Recently, we could demonstrate that pro-angiogenic properties of neutrophils depend on the activation of nicotinamide phosphoribosyltransferase (NAMPT) signaling pathway in these cells. Inhibition of this pathway in tumor-associated neutrophils leads to their potent anti-angiogenic phenotype. Here, we demonstrate our newly established model allowing in vivo evaluation of tumorigenic potential of manipulated tumor-associated neutrophils (TANs). Shortly, pro-angiogenic tumor-associated neutrophils can be isolated from tumor-bearing interferon-deficient mice and repolarized into anti-angiogenic phenotype by blocking of NAMPT signaling. The angiogenic activity of these cells can be subsequently evaluated using an aortic ring assay. Anti-angiogenic TANs can be transferred into tumor-bearing wild type recipients and tumor growth should be monitored for 14 days. At day 14 mice are sacrificed, tumors removed and cut with their vascularization assessed. Overall, our protocol provides a novel tool to in vivo evaluate angiogenic capacity of primary cells, such as tumor-associated neutrophils, without a need to use artificial neutrophil cell line models. vc

Here, we show therapeutic potential of anti-angiogenic tumor-associated neutrophils after their transfer into tumor-bearing mice. This protocol can be used to manipulate neutrophil activity ex vivo and to subsequently evaluate their functionality in vivo&#160;in developing tumors. It is an appropriate model for studying potential neutrophil-based immunotherapies.

操作された腫瘍関連好中球を腫瘍性マウスに移植し、生体内の血管新生電位を研究する

The contribution of neutrophils to the regulation of tumorigenesis is getting increased attention. These cells are heterogeneous, and depending on the ...

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 ...

Portal Vein Injection of Colorectal Cancer Organoids to Study the Liver Metastasis Stroma

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the tumor microenvironment, play a crucial role in metastatic CRC progression and predict poor patient prognosis. However, there is a lack of satisfactory mouse models to study the crosstalk between metastatic cancer cells and CAFs. Here, we present a method to investigate how liver metastasis progression is regulated by the metastatic niche and possibly could be restrained by stroma-directed therapy. Portal vein injection of CRC organoids generated a desmoplastic reaction, which faithfully recapitulated the fibroblast-rich histology of human CRC liver metastases. This model was tissue-specific with a higher tumor burden in the liver when compared to an intra-splenic injection model, simplifying mouse survival analyses. By injecting luciferase-expressing tumor organoids, tumor growth kinetics could be monitored by in vivo imaging. Moreover, this preclinical model provides a useful platform to assess the efficacy of therapeutics targeting the tumor mesenchyme. We describe methods to examine whether adeno-associated virus-mediated delivery of a tumor-inhibiting stromal gene to hepatocytes could remodel the tumor microenvironment and improve mouse survival. This approach enables the development and assessment of novel therapeutic strategies to inhibit hepatic metastasis of CRC.

Portal vein injection of colorectal cancer (CRC) organoids generates stroma-rich liver metastasis. This mouse model of CRC hepatic metastasis represents a useful tool to study tumor-stroma interactions and develop novel stroma-directed therapeutics such as adeno-associated virus-mediated gene therapies.

肝転移間質を研究するための結腸直腸癌オルガノイドの門脈注入

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the ...

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 ...