ドイツがんセンター(DKFZ)のシングルセルオープンラボ(scOpenLab)に感謝します。この研究は、ドイツのがん援助、マックス・イーダー・プログラムの助成金番号70111964(S.T.)によって支援されました。

新鮮な凍結グリオーマサンプルは、ドイツのハイデルベルクにある国立腫瘍疾患センター(NCT)組織バンクから入手した。患者材料の使用は、ハイデルベルクの医学部の機関審査委員会によって承認され、インフォームド・コンセントは、研究に含まれるすべての患者から得られました。
1. 実験準備
<ol><li>氷上または4°Cですべてのステップを実行します。</li>
	<li>プレチルチューブ、食器、カミソリの刃、ドウンサー、害虫を4°Cに。</li>
	<li>すべてのバッファを事前に準備します。これらのバッファーは室温で安定しています。滅菌濾過は、特にスクロースのために、推奨されます。ストック準備は、コルセスら19. 表 1-7 を参照してください。</li>
	<li>液体窒素または-80°C冷凍庫の貯蔵からサンプルを取り出し、ステップ2.1までドライアイスに保管してください。</li>
</ol>2. 組織解剖と解離
<ol><li>新鮮な凍結組織サンプル(10〜60mg)を冷やされたペトリ皿に移します。氷の上の小片にカミソリの刃で新鮮な凍結組織をミンチ/チョップ。</li>
	<li>冷蔵1.5mLチューブに500μLの冷蔵核リシスバッファーを加えます。核ライシスバッファーを含む1.5mLチューブに組織片を入れ、Douncer(材料表)に移す。 注:2つのDounceホモジナイザー害虫があります:初期サンプル還元のための「緩い」または「A」害虫と完全なサンプル均質化のための「タイト」または「B」害虫。</li>
	<li>摩擦が減少するまで、約20ストロークの「緩い」害虫で組織片を捨てる。破片が存在する場合、試料は100mmストレーナーで濾過することによって予めクリアされ得る。</li>
	<li>完全な組織均質化を達成するために20ストロークのための「タイトな」害虫でダウンス。</li>
	<li>ホモジネート(約500μL)を冷やした2mLチューブに移します。冷やしたライシスバッファーを1 mL加えます。やさしく混ぜ、氷の上で5分間インキュベートします。広孔ピペットチップを軽く混ぜ、インキュベーション中に1~2回繰り返します。</li>
	<li>30 μm のストレーナー メッシュを使用してホモゲネート全体をフィルターし、15 mL ファルコン チューブに集めて、新しい冷やされた 2 mL チューブに戻します。単一のストレーナーは、典型的には、全体のホモジネートのために十分です。</li>
	<li>軽い顕微鏡で大きな破片の除去と核膜の無傷性を確認します。核は丸くする必要があり、核膜は歪んではなりません。破片が存在する場合、核を再濾過することができる。</li>
	<li>ベンチトップ遠心分離機で4°Cで5分間500xgで核を遠心する。上清を取り除き、核を含むペレットで〜50μLの後ろに残します。別の1 mLの核リシスバッファーでペレットを徐々に再懸濁し、氷上で5分間インキュベートします。</li>
	<li>4°Cで5分間500xgで核を遠心分離する。 ペレットを乱さずに上清を取り除き、500mLの1x均質化バッファー(HB)(表4)を加え、再中断することなく5分間インキュベートする。次いで、1x HBの別の1.0mLで核を再懸濁する。</li>
	<li>4°Cで5分間500xgで核を遠心分離する。 上清を取り除き、1x HBの200 μLの核を新しい2.0 mLチューブに静かに再懸濁します。</li>
</ol>3. グラデーション遠心
<ol><li>200 μLの 50% イオデキサノール溶液 (表 5) を加え、25% イオデキサノールの最終濃度を与える。300mLにピペットセットを10回よく混ぜます。</li>
	<li>29%のイオジキサノール溶液(表6)の300μLを25%混合物下に加える。P1000の細かい先端を使用して、層の混合を避けてください。</li>
	<li>300 μlの35%のイオデキサノール溶液(表7)を29%混合物下に加える。P1000の細かい先端を使用して、層の混合を避けてください。 注意: このステップでは、過度の体積変位を避けるために、ピペットの間にピペットチップを徐々に除去する必要があります。</li>
	<li>サンプルを振るバケツの遠心分離機に入れ、ブレーキをオフにして4°Cで3,500 x g で20分間回転します。</li>
	<li>揺れることなくサンプルを静かに取り出し、光の下で観察します。純核95%の透明な白色帯は、第2層と第3層の間に見えるべきである(図1)。</li>
</ol>4. 核の単離
<ol><li>29%〜35%の間相で白核帯まで最上層を吸引する。</li>
	<li>200 mLのボリュームで核バンドを収集し、20 μmのフィルター(材料表)で新鮮なチューブとフィルターに移します。 注: 核はろ過する前に再懸濁する必要はありません。</li>
	<li>軽い顕微鏡で大きな破片の除去と核膜の無傷性を確認します。核は丸くする必要があり、核膜は歪んではなりません。</li>
	<li>血球計上のトリパンブルー染色とsnRNA-seq/snATAC-seqのアリコート核を使用して核を数えます。</li>
</ol>5. 単核RNAとATACセク
<ol><li>単一細胞遺伝子発現および単一細胞ATAC試薬キット(材料表)を用いて核を直ちに処理する。 注:10xゲノミクス系の核サンプル濃度は、snRNA-seqの場合はμLあたり1500-3000核、snATAC-seqの場合は1μLあたり3500-7000核です。核は、1x PBSを用いて希釈することができる。</li>
	<li>生成されたライブラリーをゲノミクス・コア・ファシリティでシーケンスします。</li>
	<li>データの品質管理分析を実行します。核は、一意分子識別子(UMI)>1000、>500の遺伝子数、ミトコンドリア遺伝子の割合<5%を含み、UMIおよび遺伝子の平均+3つの標準偏差内にある場合、さらなる分析のために含まれています。</li>
</ol>

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著者らは開示するものは何もない。

腫瘍内の異質性の分野は、既存の知識に挑戦し、拡大するために新しいアッセイやプラットフォームが開発され、エキサイティングな段階にあります。腫瘍内不均一性は、神経膠腫28における疾患の進行および現在の治療モダリティに対する耐性に寄与する重要な因子である。脳腫瘍に関する最近の研究は、同じ腫瘍内の細胞異質性をより良く特徴付けるために単一細胞転?...

成人で最も一般的な原発性脳腫瘍であるびまん性低悪性神経膠腫(LGG)は、大脳半球でしばしば生じる浸潤性新生物である。LGGsは腫瘍間と腫瘍内の不均一性を示し、腫瘍集団によって駆動されるだけでなく、腫瘍の発達と進行に複雑に関与する非悪性細胞によっても1、2、3、4、5を示す。
過去10年間、神経膠腫の分野でゲノムデータが集まっていました。これらのデータは、主にバルク腫瘍シーケンシング研究から来て、分子特性評価に非常に貢献している、と脳腫瘍の現在の分類5、6、7、8、9、10、11。しかし、これらの研究は、神経膠腫に関連する広範な分子景観を明らかにしたにもかかわらず、治療介入に関する進歩の残念な欠如がまだある。脳腫瘍における治療抵抗性の障害の1つは、腫瘍内不均一性である。この問題に対処するために、様々な研究は、ゲノム、トランスクリプトーム、プロテオミクス、および単一細胞レベル12、13、14、15、16、17で腫瘍内に存在するエピジェネティックな異種性に焦点を当ててきました。
ここ数年、単一細胞分野で顕著な技術進歩がありましたが、主な制限要因の1つは、細胞を単離し、これらの実験を行うために必要な新鮮な標本の入手可能性です。この制限を克服するために、凍結組織からのsnRNA-seqおよびsnATAC-seqのようなアッセイを、細胞18、19ではなく核を用いてアッセイを行う上で成功した試みがいくつか行われている。これらの方法の大部分は、蛍光活性化細胞選別(FACS)または濾過戦略のいずれかに依存しています。単一細胞と単一核アプローチの両方に長所と短所があります。単一細胞のアプローチは、ミトコンドリアの転写物を維持し、有益であるかもしれないが、その豊富さのためにトランスクリプトームのカバレッジを減らすことができる。単一核単離アプローチは、ミトコンドリア画分の高い割合を排除し、それによって核転写物20のより詳細なカバレッジを可能にする。
RNA-seqやATAC-seqを含む単一細胞ゲノミクスデータのアッセイに近年使用されている様々な市販のプラットフォームがあります。最も顕著なプラットフォームの1つは、単一細胞遺伝子発現および単一細胞ATACプロファイリングのための10xゲノミクスクロムプラットフォームである。プラットフォームはマイクロ流体室の助けを借りて動作するように、任意の破片や凝集物は、データ、試薬、および貴重な臨床サンプルの損失につながるシステムを詰まらせる可能性があります。したがって、単一細胞研究の成功は、単一細胞/核の正確な単離に大きく依存する。
ここで示すプロトコルは、DroNc-seqとOmni-ATAC-seqプロトコルのわずかに変更された組み合わせであり、snRNA-seqを利用してヒト脳18、19、21、22、23、24の神経疾患および神経細胞タイプを理解する最近の研究と同様のアプローチを利用している。このプロトコルは、凍結サンプルの酵素/機械的解離の組み合わせを使用し、その後ろ過および勾配遠心分離を行い、新鮮な凍結グリオーマ組織からの単一核の迅速かつ正確な単一の分離を可能にする。我々は、このプロトコルを使用して、脳腫瘍標本から同じ核調製物からsnRNA-seqおよびsnATAC-seqデータを生成することに成功した。

<table>
	<tbody>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma</td>
			<td>M6250</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>21115-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Dounce Homogenizer</td>
			<td>Active motif</td>
			<td>40401</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA (0.5 M)</td>
			<td>Thermo Scientific</td>
			<td>R1021</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 5 mL Round Bottom Polystyrene Test Tube</td>
			<td>Corning</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodixanol (aka Optiprep)</td>
			<td>Stem cell technologies</td>
			<td>07820</td>
			<td></td>
		</tr>
		<tr>
			<td>MACs Smart Strainers (30 &#181;m)</td>
			<td>Miltenyi Biotec</td>
			<td>130-098-458</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS SmartStrainers (100 &#181;m)</td>
			<td>Miltenyi Biotec</td>
			<td>130-098-463</td>
			<td></td>
		</tr>
		<tr>
			<td>Mg(Ac)2</td>
			<td>Sigma</td>
			<td>63052-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>NP-40</td>
			<td>Abcam</td>
			<td>ab142227</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclei Isolation Kit: Nuclei EZ Prep</td>
			<td>Sigma</td>
			<td>NUC101-1KT</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylmethanesulfonyl fluoride (PMSF)</td>
			<td>Sigma</td>
			<td>P7626</td>
			<td></td>
		</tr>
		<tr>
			<td>Pre-Separation Filters (20 &#181;m)</td>
			<td>Miltenyi Biotec</td>
			<td>130-101-812</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>Single Cell ATAC</td>
			<td>10x Genomics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Single Cell Gene Expression</td>
			<td>10x Genomics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S0389</td>
			<td></td>
		</tr>
		<tr>
			<td>Wide Bore pipette tips (1000 &#181;L)</td>
			<td>Themo Fisher Scientific</td>
			<td>2079GPK</td>
			<td></td>
		</tr>
		<tr>
			<td>Wide Bore pipette tips (200 &#181;L)</td>
			<td>Themo Fisher Scientific</td>
			<td>2069GPK</td>
			<td></td>
		</tr>
	</tbody>
</table>

nuclei isolation from fresh frozen brain tumors for single-nucleus rna-seq and atac-seq

成人びまん性神経膠腫は、腫瘍間および腫瘍内不均一性を示す。最近まで、大規模な分子プロファイリングの大部分は、脳腫瘍の分子分類につながったバルクアプローチに焦点を当ててきた。過去5年間で、単一細胞シーケンシングアプローチは、神経膠腫のいくつかの重要な特徴を強調してきました。これらの研究の大半は、フローサイトメトリーまたは抗体ベースの分離方法を使用して単一細胞を単一細胞に分離するために新鮮な脳腫瘍標本を利用している。今後、バイオバンクからの新鮮な凍結組織サンプルを使用すると、単一細胞の用途に対する柔軟性が向上します。さらに、単一細胞分野が進むにつれて、次の課題は、腫瘍の複雑性をより良く解明するために、単一の細胞または同じサンプル調製物からマルチオミックスデータを生成することです。したがって、単核RNAシーケンシング(snRNA-seq)や、高スループットシーケンシング(snATAC-seq)を備えたトランスポサーゼ対応クロマチン用単核アッセイなどの様々な方法のデータ生成を可能にするシンプルで柔軟なプロトコルが重要になります。
10xゲノムプラットフォームのようなアクセス可能なマイクロ流体機器と結合された単一細胞分野の最近の進歩は、研究室での単一細胞の適用を促進している。脳腫瘍の不均一性を研究するために、我々は新鮮な凍結神経膠腫からの単一核の単一の分離のための強化されたプロトコルを開発した。このプロトコルは、既存の単一セルプロトコルをマージし、均質化ステップに続いて濾過およびバッファー媒介勾配遠心分離を組み合わせます。得られたサンプルは、同じ核調製物から単一の核遺伝子発現およびクロマチンのアクセシビリティデータを生成するために使用することができる純粋な単一核懸濁液である。

単一核ゲノミクスは、限られたデータとプロトコルを持つ進化分野です。単一核アッセイの結果に影響を与える重要な要因は、純粋で無傷の核の単離である。我々は、2つの公表されたプロトコル(DroNc-seqおよびOmni-ATAC-seqプロトコル)を組み合わせて、新鮮な凍結グリオーマ組織ブロックから高品質で純粋な核を比較的短時間で分離し、トランスクリプトの安定性を維持した(図1)...

Watch this Scientific Journal Video about Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq at JoVE.com

Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq

腫瘍内不均一性は、神経膠腫を含む腫瘍の固有の特徴である。我々は、単核RNAおよびATACシーケンシング研究のために、新鮮な凍結したグリオーマ組織から単一核を単一核に分離するために、バッファーと勾配遠心分離の組み合わせを利用するシンプルで効率的なプロトコルを開発した。

単核RNA-seqおよびATAC-seqのための新鮮な凍結脳腫瘍からの核分離

Adult diffuse gliomas exhibit inter- and intra-tumor heterogeneity. Until recently, the majority of large-scale molecular profiling efforts have focused ...

nuclei-isolation-from-fresh-frozen-brain-tumors-for-single-nucleus

Research

JoVE Journal

Cancer Research

11.9K ビュー.  University Hospital Heidelberg. 腫瘍内不均一性は、神経膠腫を含む腫瘍の固有の特徴である。我々は、単核RNAおよびATACシーケンシング研究のために、新鮮な凍結したグリオーマ組織から単一核を単一核に分離するために、バッファーと勾配遠心分離の組み合わせを利用するシンプルで効率的なプロトコルを開発した。

ビデオ: Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq

Next Generation Sequencing for the Detection of Actionable Mutations in Solid and Liquid Tumors

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific molecular profiles of a tumor may predict responsiveness to therapeutic agents or provide knowledge about prognosis. At our institution a tumor genotyping program was established as part of routine clinical care, screening both hematologic and solid tumors for a wide spectrum of mutations using two next-generation sequencing (NGS) panels: a custom, 33 gene hematological malignancies panel for use with peripheral blood and bone marrow, and a commercially produced solid tumor panel for use with formalin-fixed paraffin-embedded tissue that targets 47 genes commonly mutated in cancer. Our workflow includes a pathologist review of the biopsy to ensure there is adequate amount of tumor for the assay followed by customized DNA extraction is performed on the specimen. Quality control of the specimen includes steps for quantity, quality and integrity and only after the extracted DNA passes these metrics an amplicon library is generated and sequenced. The resulting data is analyzed through an in-house bioinformatics pipeline and the variants are reviewed and interpreted for pathogenicity. Here we provide a snapshot of the utility of each panel using two clinical cases to provide insight into how a well-designed NGS workflow can contribute to optimizing clinical outcomes.

This manuscript describes clinical protocols for two next-generation sequencing panels. One panel interrogates hematologic malignancies while the other panel targets genes commonly mutated in solid tumors. Molecular classification of driver mutations in human malignancies offers valuable prognostic and predictive information.

固体と液体の腫瘍におけるアクショナブル変異の検出のための次世代シーケンシング

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific ...

がん研究

Transplantation of Zebrafish Pediatric Brain Tumors into Immune-competent Hosts for Long-term Study of Tumor Cell Behavior and Drug Response

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to assess potential patient response to therapy. Current methods largely depend on using syngeneic or immune-compromised animals to avoid rejection of the tumor graft. Such methods require the use of specific genetic strains that often prevent the analysis of immune-tumor cell interactions and/or are limited to specific genetic backgrounds. An alternative method in zebrafish takes advantage of an incompletely developed immune system in the embryonic brain before 3 days, where tumor cells are transplanted for use in short-term assays (i.e., 3 to 10 days). However, these methods cause host lethality, which prevents the long-term study of tumor cell behavior and drug response. This protocol describes a simple and efficient method for the long-term orthotopic transplantation of zebrafish brain tumor tissue into the fourth ventricle of a 2-day-old immune-competent zebrafish. This method allows: 1) long-term study of tumor cell behaviors, such as invasion and dissemination; 2) durable tumor response to drugs; and 3) re-transplantation of tumors for the study of tumor evolution and/or the impact of different host genetic backgrounds. In summary, this technique allows cancer researchers to assess engraftment, invasion, and growth at distant sites, as well as to perform chemical screens and cell competition assays over many months. This protocol can be extended to studies of other tumor types and can be used to elucidate mechanisms of chemoresistance and metastasis.

The transplantation of cancer cells is an important tool for the identification of cancer mechanisms and therapeutic responses. Current techniques depend on immune-incompetent animals. Here, we describe a method to transplant zebrafish tumor cells into immune-competent embryos for the long-term analysis of tumor cell behavior and in vivo drug responses.

腫瘍細胞の挙動および薬物応答の長期的研究のためのゼブラフィッシュ小児脳腫瘍の免疫適格宿主への移植

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to ...

Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We developed an in vitro fully human preclinical model of a co-culture of breast cancer cells and monocytes undergoing differentiation towards osteoclasts. We optimized a model of osteoclastogenesis starting from a sample of peripheral blood collected from healthy donors. Peripheral blood mononuclear cells (PBMCs) were first separated by density gradient centrifugation, seeded at a high density and induced to differentiate by adding two growth factors (GFs): receptor activator of nuclear factor-&#954;B ligand (RANKL) and macrophage colony-stimulating factor (MCSF). The cells were left in culture for 14 days and then fixed and analyzed by downstream analysis. In osteolytic bone metastases, one of the effects of cancer cell arrival in bone is the induction of osteoclastogenesis. We thus challenged our model with co-cultures of breast cancer cells to study the differentiation power of cancer cells with respect to GFs. A straightforward way of studying cancer cell-osteoclast interaction is to perform indirect co-cultures based on the use of conditioned medium collected from breast cancer cell cultures and mixed with fresh medium. This mixture is then used to induce osteoclast differentiation. We also optimized a method of direct co-culture in which cancer cells and monocytes undergoing differentiation share the medium and exchange secreted factors. This is a significant improvement over the original indirect co-culture method as researchers can observe the reciprocal interactions of the two cell types and perform downstream analyses for both cancer cells and osteoclasts. This method enables us to study the effect of drugs on the metastatic bone microenvironment and to seed cell lines other than those derived from breast cancer. The model can also be used to study other diseases such as osteoporosis or other bone conditions.

This protocol describes development of an in vitro human preclinical model of osteoclastogenesis from peripheral blood monocytes cultured with breast cancer cell lines to mimic the cancer cell-osteoclast interaction. The model could be used to further our understanding of bone metastasis formation and improve therapeutic options.

破骨細胞形成乳房癌細胞と共培養末梢血単球からの人間の前臨床モデルの開発

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We ...

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR

CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal gastric mucosa. Recently, we detected CDH1a transcript variant in fresh-frozen tumor tissues obtained from patients with GC. The expression of this variant in tissue samples was investigated by the chip-based digital PCR (dPCR) approach presented here. dPCR offers the potential for an accurate, robust, and highly sensitive measurement of nucleic acids and is increasingly utilized for many applications in different fields. dPCR is capable of detecting rare targets; in addition, dPCR offers the possibility for absolute and precise quantification of nucleic acids without the need for calibrators and standard curves. In fact, the reaction partitioning enriches the target from the background, which improves amplification efficiency and tolerance to inhibitors. Such characteristics make dPCR an optimal tool for the detection of the CDH1a rare transcript.

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR

The manuscript describes a chip-based digital PCR assay to detect a rare CDH1 transcript variant (CDH1a) in fresh-frozen normal and tumor tissues obtained from patients with gastric cancer.

チップを使ったデジタル PCR による新鮮凍結胃癌組織内CDH1まれなトラン スクリプト変種の検出

CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal ...

Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and recurrent disease. This population can be identified by surface markers, enzymatic activity and a functional profile. These approaches per se are limited, due to phenotypic heterogeneity and CSC plasticity. Here, we update the sphere-forming protocol to obtain CSC spheres from breast and gynecological cancers, assessing functional properties, CSC markers and protein expression. The spheres are obtained with single-cell seeding at low density in suspension culture, using a semi-solid methylcellulose medium to avoid migration and aggregates. This profitable protocol can be used in cancer cell lines but also in primary tumors. The tridimensional non-adherent suspension culture thought to mimic the tumor microenvironment, particularly the CSC-niche, is supplemented with epidermal growth factor and basic fibroblast growth factor to ensure CSC signaling. Aiming for robust identification of CSC, we propose a complementary approach, combining functional and phenotypic evaluation. Sphere-forming capacity, self-renewal and sphere projection area establish CSC functional properties. Additionally, characterization comprises flow cytometry evaluation of the markers, represented by CD44+/CD24- and CD133, and Western blot, considering ALDH. The presented protocol was also optimized for primary tumor samples, following a sample digestion procedure, useful for translational research.

The aim of this methodology is to identify cancer stem cells (CSC) in cancer cell lines and primary human tumor samples with the sphere-forming protocol, in a robust manner, using functional assays and phenotypic characterization with flow cytometry and Western blot.

婦人科腫瘍および乳がん腫瘍から癌幹細胞球体を取得する

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and ...

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 ...

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. Immortalized cell lines are easy to grow and genetically modify to study molecular mechanisms, yet the selective pressure from cell culture often leads to genetic and epigenetic alterations over time. Patient-derived xenograft (PDX) models faithfully recapitulate the heterogeneity and drug response of human breast tumors. PDX models exhibit a relatively short latency after orthotopic transplantation that facilitates the investigation of breast tumor biology and drug response. The transplantable genetically engineered mouse models allow the study of breast tumor immunity. The current protocol describes the method to orthotopically transplant breast tumor fragments into the mammary fat pad followed by drug treatments. These preclinical models provide valuable approaches to investigate breast tumor biology, drug response, biomarker discovery and mechanisms of drug resistance.

Patient-derived xenograft (PDX) models and transplantable genetically engineered mouse models faithfully recapitulate human disease and are preferred models for basic and translational breast cancer research. Here, a method is described to orthotopically transplant breast tumor fragments into the mammary fat pad to study tumor biology and evaluate drug response.

乳癌の前臨床モデルとしての乳房腫瘍の移植性

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. ...

A Rapid Screening Workflow to Identify Potential Combination Therapy for GBM using Patient-Derived Glioma Stem Cells

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in glioblastoma (GBM), the most prevalent and devastating primary brain tumor. The presence of GSCs makes the GBM very refractory to most of individual targeted agents, so high-throughput screening methods are required to identify potential effective combination therapeutics. The protocol describes a simple workflow to enable rapid screening for potential combination therapy with synergistic interaction. The general steps of this workflow consist of establishing luciferase-tagged GSCs, preparing matrigel coated plates, combination drug screening, analyzing, and validating the results.

患者由来グリオーマ幹細胞を用いたGBMの潜在的併用療法を同定するための迅速スクリーニングワークフロー

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in ...

ATAC-Seq Optimization for Cancer Epigenetics Research

The assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) probes deoxyribonucleic acid (DNA) accessibility using the hyperactive Tn5 transposase. Tn5 cuts and ligates adapters for high-throughput sequencing within accessible chromatin regions. In eukaryotic cells, genomic DNA is packaged into chromatin, a complex of DNA, histones, and other proteins, which acts as a physical barrier to the transcriptional machinery. In response to extrinsic signals, transcription factors recruit chromatin remodeling complexes to enable access to the transcriptional machinery for gene activation. Therefore, identifying open chromatin regions is useful when monitoring enhancer and gene promoter activities during biological events such as cancer progression. Since this protocol is easy to use and has a low cell input requirement, ATAC-seq has been widely adopted to define open chromatin regions in various cell types, including cancer cells. For successful data acquisition, several parameters need to be considered when preparing ATAC-seq libraries. Among them, the choice of cell lysis buffer, the titration of the Tn5 enzyme, and the starting volume of cells are crucial for ATAC-seq library preparation in cancer cells. Optimization is essential for generating high-quality data. Here, we provide a detailed description of the ATAC-seq optimization methods for epithelial cell types.

ATAC-seq is a DNA sequencing method that uses the hyperactive mutant transposase, Tn5, to map changes in chromatin accessibility mediated by transcription factors. ATAC-seq enables the discovery of the molecular mechanisms underlying phenotypic alterations in cancer cells. This protocol outlines optimization procedures for ATAC-seq in epithelial cell types, including cancer cells.

がんエピジェネティクス研究のためのATAC-Seq最適化

The assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) probes deoxyribonucleic acid (DNA) accessibility using the ...

新鮮組織からの膵臓癌由来腫瘍オルガノイドおよび線維芽細胞

Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. Patient-derived tumor organoids have been used in translational cancer research and can be applied to assess treatment sensitivity and resistance, cell-cell interactions, and tumor cell interactions with the tumor microenvironment. Tumor organoids are complex culture systems that require advanced cell culture techniques and culture media with specific growth factor cocktails and a biological basement membrane that mimics the extracellular environment. The ability to establish primary tumor cultures highly depends on the tissue of origin, the cellularity, and the clinical features of the tumor, such as the tumor grade. Furthermore, tissue sample collection, material quality and quantity, as well as correct biobanking and storage are crucial elements of this procedure. The technical capabilities of the laboratory are also crucial factors to consider. Here, we report a validated SOP/protocol that is technically and economically feasible for the culture of ex vivo tumor organoids from fresh tissue samples of pancreatic adenocarcinoma origin, either from fresh primary resected patient donor tissue or patient-derived xenografts (PDX). The technique described herein can be performed in laboratories with basic tissue culture and mouse facilities and is tailored for wide application in the translational oncology field.

著者スポットライト:新鮮組織からの膵臓がん由来腫瘍オルガノイドと線維芽細胞の確立  

Tumor organoids have revolutionized cancer research and the approach to personalized medicine. They represent a clinically relevant tumor model that allows researchers to stay one step ahead of the tumor in the clinic. This protocol establishes tumor organoids from fresh pancreatic tumor tissue samples and patient-derived xenografts of pancreatic adenocarcinoma origin.

膵臓癌由来腫瘍オルガノイドおよび線維芽細胞の新鮮組織からの樹立

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. ...