、この作品は、スコットランドの財政審議会（承認番号HR07005によって部分的にサポートされていました<a href="http://www.sfc.ac.uk/">http://www.sfc.ac.uk/</a>医学研究スコットランド（、） <a href="http://www.medicalresearchscotland.org.uk/">http://www.medicalresearchscotland.org.uk/</a> ）、がん研究英国の実験がん医学センター（ <a href="http://www.cancerresearchuk.org/">http://www.cancerresearchuk.org/~~V</a> ）、および画期的な乳がん（ <a href="http://breakthrough.org.uk/">http://breakthrough.org.uk/</a> ）。

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J., Li, X., Landis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Residual+breast+cancers+after+conventional+therapy+display+mesenchymal+as+well+as+tumor-initiating+features.">Residual breast cancers after conventional therapy display mesenchymal as well as tumor-initiating features.</a> Proc. Natl. Acad. Sci. U. S. A. 106, 13820-13825 (2009).</li><li>Aitken, S. J., Thomas, J. S., Langdon, S. P., Harrison, D. J., Faratian, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+changes+in+ER,+PR+and+HER2+expression+in+primary+breast+cancer+and+paired+nodal+metastases.">Quantitative analysis of changes in ER, PR and HER2 expression in primary breast cancer and paired nodal metastases.</a> Ann. Oncol. 21, 1254-1261 (2010).</li><li>Sayagues, J. M., Abad, M. M., Melchor, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intratumoural+cytogenetic+heterogeneity+of+sporadic+colorectal+carcinomas+suggests+several+pathways+to+liver+metastasis.">Intratumoural cytogenetic heterogeneity of sporadic colorectal carcinomas suggests several pathways to liver metastasis.</a> J. Pathol. 221, 308-319 (2010).</li></ol>

JC、MG、CJ、そしてCSはHistoRx、（株）の従業員です

方法はここに腫瘍材料の標準的なホルマリン固定、パラフィン包埋組織学セクションの分子多様性の定量化を可能にする説明。方法は、これはその後、バイオマーカーの開発と分析の変数として考慮することができるように、組織切片の不均質性の度合いに割り当てられる値を許可します。一般的には、組織のバイオマーカーのアッセイは、サンプリングエラーやバイアスの影響を受けにくくなるように、表現に関して均質であることが好ましいが、それはいくつかの状況下でのパラメータとして不均一性を定量化するために有用かもしれません。 ERとHER2のための例示の例に示すように、例えば、一般的なバイオマーカーは発現のかなりの異質性を示すが、このことを、それぞれ臨床転帰や治療への応答に関して、独立した予後または予測因子を、表しているかどうか現時点では不明です。同様に、図に示すように、治療自体が動的に変更するかもしれない構成する細胞の集団、そしてターゲット濃縮の測定は、治療上の意思決定を導くのに有用かもしれません。 しかし、技術は従来の病理組織学的またはバイオマーカー（など免疫組織化学など）の分析を制限する同じ要因によって制限されます。その結果、タイプ、サイズ、および分析される組織の品質に依存し、したがって、単一の組織切片では、腫瘍全体を代表できない場合があります。実際、シンプソンの指数は、不当に組織の大きさ（キャプチャとAQUAのスコアが測定されたフレームの数）によってバイアスされることがあります。測定されるエピトープの免疫原性は、人為的に組織切片（例えば冷阻血時間、または固定の長さ、およびエッジのアーティファクトなど）を介してそれらの発現を変化させる制御できない分析前の要因が適用される場合があります。これは特に悪名高い不安定な14、15であるリン酸化エピトープ、のための問題です。そのための手法は適しているかもしれません。切除標本ではなく、小さな生検に、というだけのものよりも、複数のセクションで実行されます。最終的に、3Dイメージング技術は、より良い、このパラメータを定量化する機会を提供する場合があります。 提示された技術はまた、ケラチンマスキングのエピトープの発現の変動性などの生物学的要因によって制限される場合があります。これは、卵巣と上皮間葉移行（EMT）を受けるか、非上皮成分または幹細胞16に濃縮されている乳がん、を含む、これらのがんに特に懸念されるなど、最近で発生することが示されているかもしれない化学療法- 試験管17、および治療​​18〜応答における乳癌患者の卵巣癌を治療。この制限は、EMT 16にアップレギュレートされるサイトケラチンプラスビメンチン、などの代替マスキング抗体（または抗体のカクテル）、使用して克服することができる。加えて、組織の他のコンポーネント（マイクからroenvironment）、線維芽細胞や血管内皮細胞はまた、ますます生物学的治療の標的とされているような、技術はまた、血管内皮細胞に対して染色するなどPECAM / CD31のような関心の区画のための特定のマスクを、使用して、これらのコンポーネントを獲得するために拡張される。 シンプソンの指数の適応は、その単純さのため、現在のプロトコルの不均一性の尺度として使用されているが、異質のその他の措置は、中心傾向の単純な測定値（平均、分散、中央値など）として、使用することができる。また、シンプソンの指数の計算は、より良い特定のテストの人口に合わせて変更することができます。 Z -スコアの変換の使用は、異質性がデータセットの平均値周りの偏差に​​基づいているので、得点が複数のマーカーのためのより適切にすることができます。しかし、得点の全体の範囲は、変換のこのタイプに制限することができます。設計によっては、方が適切な場合があります。単に特定のマーカーセットのすべてのサンプルのため実際のAQUAスコアをbinに。ビンとカットオフの数の選択もつながると代替実験的な設計に最適化される値の範囲の制限です。 我々は、彼らがすでに診療所で使用されているので、標的療法の有効性に関して、関連する臨床上の疑問に答えるため、バイオマーカー候補として現在の研究でERとHER2を使用している、と主と遠くにかなりの異質性と変化を示すことが知られている病気19。しかしながら、異質性の最適なマーカーは決定されていない。他の可能性は、以前に間期FISHの研究20で使用されるようなクローンの細胞遺伝学的マーカーが含まれる場合があります。アプローチは、さらにがんの生物学では、このパラメータの妥当性を確立するために大規模な、よく注釈付きの臨床コホートまたは臨床試験のさらなる検証が必要です。

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td> ScanScope FL </td><td>アペリオ</td><td> ScanScope FL </td><td>使用されるようにベンダーから装備</td></tr><tr><td>スペクトラム</td><td>アペリオ</td><td>スペクトラム</td><td>構成とカタログ番号は、地元の機関がそのデータベースとIT構造のセットアップ方法によって異なります。 </td></tr><tr><td> AQUAjustmentと一緒にAQUAnalysis、 </td><td> HistoRx </td><td> V2.3 </td><td> AQUAjustmentパッケージはAQUAnalysisがスペクトラムに格納されているイメージを使用することができます。 </td></tr><tr><td> HER2 </td><td> Dako社製</td><td> A0485 </td><td> 400希釈の1 </td></tr><tr><td>マウス抗パンサイトケラチン</td><td> Dako社製</td><td> M3515 </td><td> 50希釈の1 </td></tr><tr><td>抗体の希釈剤</td><td> Dako社製</TD&gt; <td> S0809 </td><td></td></tr><tr><td>ヤギ抗ウサギHRPを思い描く</td><td> Dako社製</td><td> K4003 </td><td></td></tr><tr><td>タンパク質のブロック</td><td> Dako社製</td><td> X0909 </td><td></td></tr><tr><td>ヤギ抗マウスAlexa555 </td><td>インビトロジェン</td><td> A21422 </td><td></td></tr><tr><td> DAPIは、培地を取り付け</td><td>インビトロジェン</td><td> P36931 </td><td></td></tr><tr><td> Cy5標識チラミド</td><td> HistoRx </td><td> AQ - EMR1 - 00001 </td><td></td></tr><tr><td> Sequenzaの免疫染色センター</td><td>サーモサイエンティフィックシャンドン</td><td> 73300001 </td><td>ベンチトップ免疫蛍光染色のためにここで使用</td></tr></tbody></table>

heterogeneity mapping of protein expression in tumors using quantitative immunofluorescence

個々の腫瘍内の形態学的不均一性は外科診療におけるhistopathologistsでよく認識されています。これはしばしば認識組織学的サブタイプへの明確な差別化の領域の形態、または別の病理学的悪性度をとる一方で、多くの場合、正確な分類（図1）逆らう表現型でより多くの微妙な違いがあります。最終的に、形態が根底にある分子表現型によって規定されているので、目に見える違いはある地域では、外観の細胞機能と動作を調整し、従って、タンパク質の発現の違いを添付する可能性があります。予後のための可視と不可視の（分子）不均一性の意義は不明であるが、最近の証拠は、少なくとも遺伝子レベルで、異質性が、原発腫瘍の1,2に存在する、そしてこれらのサブクローンの一部が転移を生じさせる、ことを示唆している（したがって、致命的な）疾患。 また、いくつかのタンパク質は彼らは治療の目標（タモキシフェンとトラスツズマブ（ハーセプチン）のインスタンスのERとHER2、それぞれのための）であるため、バイオマーカーとして測定。これらのタンパク質は、腫瘍内の変数式を表示する場合は、治療反応も変数があります。免疫組織化学用に広く使用されている病理組織学的スコアリングのスキームは、どちらか無視するか、数値的にタンパク質発現の定量化をホモジナイズする。同様に、腫瘍サンプルをホモジナイズしている破壊的な技術、（そのような遺伝子発現プロファイリングなど）で、定量的な情報を明らかにすることができますが、空間情報は失われます。膵臓癌の遺伝的異質性のマッピングのアプローチは、単一の細胞懸濁液3の世代上で、またはmacrodissection 4に頼ってきた。最近の研究では、形態と分子のマッピングが実現可能であることを原理の証明を提供し、前立腺癌組織5の形態学的および分子多様性をマッピングするために量子ドットを使用するが、sを落下している異質性を定量化するのホルト。免疫組織化学では唯一、最高の状態で、半定量的および内および間の観察者バイアスの対象となるので、より高感度かつ定量的手法は、正確にその場で組織の不均一性をマッピングし、定量化するために必要です。 我々は（AQUA）自動定量分析に基づいて、体系的に腫瘍の全体の組織切片でのタンパク質発現の不均一性を定量化するためにシステム6を実験的および統計的方法論を開発し、適用しています。組織切片を蛍光標識二次抗体に結合サイトケラチンと興味の対象に対して向けられた特異的な抗体で標識されています。スライドは全体のスライド蛍光スキャナーを用いて画像化している。画像は、タイルの数百〜数千に細分化され、各タイルは、組織の上皮（腫瘍）コンポーネント内のタンパク質濃度の尺度であるAQUAのスコアが割り当てられます。 HEAtmapsはSimpsonの多様性のインデックス7に基づいて、もともとは生態学で使用される不均一性の統計的尺度を用いて、タンパク質の組織発現と割り当てられている不均一性のスコアを表すために生成されます。 現在までに組織学的製剤で、体系的に蛋白質の発現と並行してこの変動をマッピングし、定量化する試みがなされていない。ここで、我々は、ERおよび卵巣癌におけるHER2バイオマーカーの発現に適用されるメソッドの最初の使用例を示します。このメソッドを使用すると、予後と治療への応答の予測の不均一性の重要性を確立するために、トランスレーショナル研究におけるバイオマーカーの発現の研究​​の独立変数としての異質性を分析するための道を開く。

Watch this Scientific Journal Video about Heterogeneity Mapping of Protein Expression in Tumors using Quantitative Immunofluorescence at JoVE.com

Heterogeneity Mapping of Protein Expression in Tumors using Quantitative Immunofluorescence

ここでは定量的免疫蛍光法、画像解析、および不均質性の統計的尺度を用いて腫瘍材料の組織切片の分子多様性を定量化する方法を説明します。方法は、臨床バイオマーカーの開発と分析に使用することを目的としています。

定量的免疫蛍光法を用いて腫瘍におけるタンパク質発現の不均一性のマッピング

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of ...

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18.5K ビュー.  University of Edinburgh. ここでは定量的免疫蛍光法、画像解析、および不均質性の統計的尺度を用いて腫瘍材料の組織切片の分子多様性を定量化する方法を説明します。方法は、臨床バイオマーカーの開発と分析に使用することを目的としています。

ビデオ: Heterogeneity Mapping of Protein Expression in Tumors using Quantitative Immunofluorescence

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

免疫不全マウスにおけるヒト網膜芽細胞腫の腫瘍の確立と伝播

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

医学

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

生体内イメージングを使ってリアルタイムで腫瘍におけるナノ粒子の取り込みの評価

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development1, apoptosis2, and cell cycle regulation3. MiRNAs are estimated to regulate the expression of 30% to 90% of human genes4 by binding to their target messenger RNAs (mRNAs)5. Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes6. Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets.
Methods used to investigate miRNA expression include SYBR green I dye- based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies7. The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis8.
Quantification of miRNA levels in prostate tumor samples. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences. 
The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this protocol, RNU6B is used as the reference gene.

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR qPCR

Quantitative Real Time polymerase chain reaction (qPCR) is a rapid and sensitive method to investigate the expression levels of various microRNA (miRNA) molecules in tumor samples. Using this method expression of hundreds of different miRNA molecules can be amplified, quantified, and analyzed from the same cDNA template.

定量的リアルタイムPCR（定量PCR）による前立腺腫瘍におけるマイクロRNAの検出

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process ...

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

Genomic, proteomic, transcriptomic, and epigenomic analyses of human tumors indicate that there are thousands of anomalies within each cancer genome compared to matched normal tissue. Based on these analyses it is evident that there are many undiscovered genetic drivers of cancer1. Unfortunately these drivers are hidden within a much larger number of passenger anomalies in the genome that do not directly contribute to tumor formation. Another aspect of the cancer genome is that there is considerable genetic heterogeneity within similar tumor types. Each tumor can harbor different mutations that provide a selective advantage for tumor formation2. Performing an unbiased forward genetic screen in mice provides the tools to generate tumors and analyze their genetic composition, while reducing the background of passenger mutations. The Sleeping Beauty (SB) transposon system is one such method3. The SB system utilizes mobile vectors (transposons) that can be inserted throughout the genome by the transposase enzyme. Mutations are limited to a specific cell type through the use of a conditional transposase allele that is activated by Cre Recombinase. Many mouse lines exist that express Cre Recombinase in specific tissues. By crossing one of these lines to the conditional transposase allele (e.g. Lox-stop-Lox-SB11), the SB system is activated only in cells that express Cre Recombinase. The Cre Recombinase will excise a stop cassette that blocks expression of the transposase allele, thereby activating transposon mutagenesis within the designated cell type. An SB screen is initiated by breeding three strains of transgenic mice so that the experimental mice carry a conditional transposase allele, a concatamer of transposons, and a tissue-specific Cre Recombinase allele. These mice are allowed to age until tumors form and they become moribund. The mice are then necropsied and genomic DNA is isolated from the tumors. Next, the genomic DNA is subjected to linker-mediated-PCR (LM-PCR) that results in amplification of genomic loci containing an SB transposon. LM-PCR performed on a single tumor will result in hundreds of distinct amplicons representing the hundreds of genomic loci containing transposon insertions in a single tumor4. The transposon insertions in all tumors are analyzed and common insertion sites (CISs) are identified using an appropriate statistical method5. Genes within the CIS are highly likely to be oncogenes or tumor suppressor genes, and are considered candidate cancer genes. The advantages of using the SB system to identify candidate cancer genes are: 1) the transposon can easily be located in the genome because its sequence is known, 2) transposition can be directed to almost any cell type and 3) the transposon is capable of introducing both gain- and loss-of-function mutations6. The following protocol describes how to devise and execute a forward genetic screen using the SB transposon system to identify candidate cancer genes (Figure 1).

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

A method of identifying unknown drivers of carcinogenesis using an unbiased approach is described. The method uses the Sleeping Beauty transposon as a random mutagen directed to specific tissues. Genomic mapping of transposon insertions that drive tumor formation identifies novel oncogenes and tumor suppressor genes

の同定<em&gt;眠れる森の美女</emリンカ媒介PCRを用いた固形腫瘍&gt;トランスポゾン挿入

Genomic, proteomic, transcriptomic, and epigenomic analyses of human tumors indicate that there are thousands of anomalies within each cancer genome ...

In vitro Organoid Culture of Primary Mouse Colon Tumors

Several human and murine colon cancer cell lines have been established, physiologic integrity of colon tumors such as multiple cell layers, basal-apical polarity, ability to differentiate, and anoikis are not maintained in colon cancer derived cell lines. The present study demonstrates a method for culturing primary mouse colon tumor organoids adapted from Sato T et al. 1, which retains important physiologic features of colon tumors. This method consists of mouse colon tumor tissue collection, adjacent normal colon epithelium dissociation, colon tumor cells digestion into single cells, embedding colon tumor cells into matrigel, and selective culture based on the principle that tumor cells maintain growth on limiting nutrient conditions compared to normal epithelial cells.
The primary tumor organoids if isolated from genetically modified mice provide a very useful system to assess tumor autonomous function of specific genes. Moreover, the tumor organoids are amenable to genetic manipulation by virus meditated gene delivery; therefore signaling pathways involved in the colon tumorigenesis could also be extensively investigated by overexpression or knockdown. Primary tumor organoids culture provides a physiologic relevant and feasible means to study the mechanisms and therapeutic modalities for colon tumorigenesis.

In vitro Organoid Culture of Primary Mouse Colon Tumors

A simple method to establish primary murine colon tumor organoid is described. This method utilizes the feature that colon tumor cells survive and grow into organoids in media containing limited growth factors, whereas normal colon epithelial do not.

プライマリマウス大腸腫瘍のin vitroオルガノイド培養で

Several  human and murine colon cancer cell lines have been established, physiologic  integrity of colon tumors such as multiple cell layers, basal-apical ...

The Use of Reverse Phase Protein Arrays (RPPA) to Explore Protein Expression Variation within Individual Renal Cell Cancers

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment resistance is thought to be the molecular complexity of the disease 1. Targeted therapy such as the tyrosine kinase inhibitor (TKI)-sunitinib have been utilized, but only 40% of patients will respond, with the overwhelming majority of these patients relapsing within 1 year 2. As such the question of intrinsic and acquired resistance in renal cell cancer patients is highly relevant 3.
In order to study resistance to TKIs, with the ultimate goal of developing effective, personalized treatments, sequential tissue after a specific period of targeted therapy is required, an approach which had proved successful in chronic myeloid leukaemia 4. However the application of such a strategy in renal cell carcinoma is complicated by the high level of both inter- and intratumoral heterogeneity, which is a feature of renal cell carcinoma5,6 as well as other solid tumors 7. Intertumoral heterogeneity due to transcriptomic and genetic differences is well established even in patients with similar presentation, stage and grade of tumor. In addition it is clear that there is great morphological (intratumoral) heterogeneity in RCC, which is likely to represent even greater molecular heterogeneity. Detailed mapping and categorization of RCC tumors by combined morphological analysis and Fuhrman grading allows the selection of representative areas for proteomic analysis.
Protein based analysis of RCC8 is attractive due to its widespread availability in pathology laboratories; however, its application can be problematic due to the limited availability of specific antibodies 9. Due to the dot blot nature of the Reverse Phase Protein Arrays (RPPA), antibody specificity must be pre-validated; as such strict quality control of antibodies used is of paramount importance. Despite this limitation the dot blot format does allow assay miniaturization, allowing for the printing of hundreds of samples onto a single nitrocellulose slide. Printed slides can then be analyzed in a similar fashion to Western analysis with the use of target specific primary antibodies and fluorescently labelled secondary antibodies, allowing for multiplexing. Differential protein expression across all the samples on a slide can then be analyzed simultaneously by comparing the relative level of fluorescence in a more cost-effective and high-throughput manner.

The Use of Reverse Phase Protein Arrays RPPA to Explore Protein Expression Variation within Individual Renal Cell Cancers

RPPA enables the protein expression of hundreds of samples, printed on nitrocellulose slides to be interrogated simultaneously, using fluorescently labelled antibodies. This technique has been applied to study the effect of drug treatment heterogeneity within clear cell renal carcinoma.

個々の腎細胞がんの中のタンパク質発現変動を探るために逆相タンパク質アレイ（RPPA）の使用

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

蛍光SAPC-DOPSナノベシクルを用いて脳腫瘍、関節炎のin vivo光イメージング中

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Coordinate Mapping of Hyolaryngeal Mechanics in Swallowing

Characterizing hyolaryngeal movement is important to dysphagia research. Prior methods require multiple measurements to obtain one kinematic measurement whereas coordinate mapping of hyolaryngeal mechanics using Modified Barium Swallow (MBS) uses one set of coordinates to calculate multiple variables of interest. For demonstration purposes, ten kinematic measurements were generated from one set of coordinates to determine differences in swallowing two different bolus types. Calculations of hyoid excursion against the vertebrae and mandible are correlated to determine the importance of axes of reference.
To demonstrate coordinate mapping methodology, 40 MBS studies were randomly selected from a dataset of healthy normal subjects with no known swallowing impairment. A 5 ml thin-liquid bolus and a 5 ml pudding swallows were measured from each subject. Nine coordinates, mapping the cranial base, mandible, vertebrae and elements of the hyolaryngeal complex, were recorded at the frames of minimum and maximum hyolaryngeal excursion. Coordinates were mathematically converted into ten variables of hyolaryngeal mechanics.
Inter-rater reliability was evaluated by Intraclass correlation coefficients (ICC). Two-tailed t-tests were used to evaluate differences in kinematics by bolus viscosity. Hyoid excursion measurements against different axes of reference were correlated. Inter-rater reliability among six raters for the 18 coordinates ranged from ICC = 0.90 - 0.97. A slate of ten kinematic measurements was compared by subject between the six raters. One outlier was rejected, and the mean of the remaining reliability scores was ICC = 0.91, 0.84 - 0.96, 95% CI. Two-tailed t-tests with Bonferroni corrections comparing ten kinematic variables (5 ml thin-liquid vs. 5 ml pudding swallows) showed statistically significant differences in hyoid excursion, superior laryngeal movement, and pharyngeal shortening (p &#60; 0.005). Pearson correlations of hyoid excursion measurements from two different axes of reference were: r = 0.62, r2 = 0.38, (thin-liquid); r = 0.52, r2 = 0.27, (pudding).
Obtaining landmark coordinates is a reliable method to generate multiple kinematic variables from video fluoroscopic images useful in dysphagia research.

Coordinate mapping is a method of documenting salient features of hyolaryngeal biomechanics in the pharyngeal phase of swallowing. This methodology uses image analysis software to record coordinates of anatomical landmarks. These coordinates are imported into an excel macro and translated into kinematic variables of interest useful in dysphagia research.

嚥下Hyolaryngeal力学のマッピングを調整する

Characterizing hyolaryngeal movement is important to dysphagia research. Prior methods require multiple measurements to obtain one kinematic measurement ...

Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic biomarkers. However, methods for quantitative proteomic characterization of patient-derived tumors and in particular their cellular subpopulations are largely lacking. Here we describe an experimental set-up that allows quantitative analysis of proteomes of cancer cell subpopulations derived from either liquid or solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry followed by bioinformatic data analysis. To enrich specific cellular subsets, liquid tumors are first immunophenotyped by flow cytometry followed by FACS-sorting; for solid tumors, laser-capture microdissection is used to purify specific cellular subpopulations. In a second step, proteins are extracted from the purified cells and subsequently combined with a tumor-specific, SILAC-labeled spike-in standard that enables protein quantification. The resulting protein mixture is subjected to either gel electrophoresis or Filter Aided Sample Preparation (FASP) followed by tryptic digestion. Finally, tryptic peptides are analyzed using a hybrid quadrupole-orbitrap mass spectrometer, and the data obtained are processed with bioinformatic software suites including MaxQuant. By means of the workflow presented here, up to 8,000 proteins can be identified and quantified in patient-derived samples, and the resulting protein expression profiles can be compared among patients to identify diagnostic proteomic signatures or potential drug targets.

In-depth analyses of cancer cell proteomes facilitate identification of novel drug targets and diagnostic biomarkers. We describe an experimental workflow for quantitative analysis of (phospho-)proteomes in cancer cell subpopulations derived from liquid and solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry.

液体および固形腫瘍由来の癌細胞のプロテオームの定量的質量分析プロファイリング

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic ...

Quantitative Mapping of Specific Ventilation in the Human Lung using Proton Magnetic Resonance Imaging and Oxygen as a Contrast Agent

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of the fresh gas entering a lung region divided by the region&#8217;s end-expiratory volume &#8213; in the human lung, using only inhaled oxygen as a contrast agent. Regional quantification of specific ventilation has the potential to help identify areas of pathologic lung function. Oxygen in solution in tissue shortens the tissue&#8217;s longitudinal relaxation time (T1), and thus a change in tissue oxygenation can be detected as a change in T1-weighted signal with an inversion recovery acquired image. Following an abrupt change between two concentrations of inspired oxygen, the rate at which lung tissue within a voxel equilibrates to a new steady-state reflects the rate at which resident gas is being replaced by inhaled gas. This rate is determined by specific ventilation. To elicit this sudden change in oxygenation, subjects alternately breathe 20-breath blocks of air (21% oxygen) and 100% oxygen while in the MRI scanner. A stepwise change in inspired oxygen fraction is achieved through use of a custom three-dimensional (3D)-printed flow bypass system with a manual switch during a short end-expiratory breath hold. To detect the corresponding change in T1, a global inversion pulse followed by a single shot fast spin echo sequence was used to acquire two-dimensional T1-weighted images in a 1.5&#160;T MRI scanner, using an eight-element torso coil. Both single slice and multi-slice imaging are possible, with slightly different imaging parameters. Quantification of specific ventilation is achieved by correlating the time-course of signal intensity for each lung voxel with a library of simulated responses to the air/oxygen stimulus. SVI estimations of specific ventilation heterogeneity have been validated against multiple breath washout and proved to accurately determine the heterogeneity of the specific ventilation distribution.

Specific ventilation imaging is a functional magnetic resonance imaging technique that allows for quantification of regional specific ventilation in the human lung, using inhaled oxygen as a contrast agent. Here, we present a protocol to collect and analyze specific ventilation imaging data.

造影剤としてのプロトン磁気共鳴イメージングと酸素を用いたヒト肺における特定の換気の定量的マッピング

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of ...