La ricerca riportata in questa pubblicazione è stata supportata dal National Center for Advancing Translational Sciences con il premio UL1TR003096 (MDE), National Heart, Lung, and Blood Institute Predoctoral Fellowship in Lung Diseases Training Program 5T32HL134640 (MLD).

Tutti i metodi qui descritti sono stati approvati dall'Institutional Animal Care and Use Committee (IACUC) dell'Università dell'Alabama a Birmingham.
1. Protocollo sperimentale
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	<li>Hsia, C. C., Hyde, D. M., Ochs, M., Weibel, E. R., Structure, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+official+research+policy+statement+of+the+American+Thoracic+Society/European+Respiratory+Society:+standards+for+quantitative+assessment+of+lung+structure.">An official research policy statement of the American Thoracic Society/European Respiratory Society: standards for quantitative assessment of lung structure.</a> American Journal of Respiratory and Critical Care Medicine. 181 (4), 394-418 (2010).</li><li>Vasilescu, D. M., Knudsen, L., Ochs, M., Weibel, E. R., Hoffman, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+murine+lung+preparation+for+detailed+structural+evaluation+via+micro-computed+tomography.">Optimized murine lung preparation for detailed structural evaluation via micro-computed tomography.</a> Journal of Applied Physiology. 112 (1), 159-166 (2012).</li><li>Blumler, P., Acosta, R. H., Thomas-Semm, A., Reuss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+fixation+for+the+preservation+of+air+spaces.">Lung fixation for the preservation of air spaces.</a> Experimental Lung Research. 30 (1), 73-82 (2004).</li><li>Oldmixon, E. H., Suzuki, S., Butler, J. P., Hoppin, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfusion+dehydration+fixes+elastin+and+preserves+lung+air-space+dimensions.">Perfusion dehydration fixes elastin and preserves lung air-space dimensions.</a> Journal of Applied Physiology. 58 (1), 105-113 (1985).</li><li>Braber, S., Verheijden, K. A., Henricks, P. A., Kraneveld, A. D., Folkerts, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+fixation+methods+on+lung+morphology+in+a+murine+model+of+emphysema.">A comparison of fixation methods on lung morphology in a murine model of emphysema.</a> American Journal of Physiology-Lung Cellular and Molecular Physiology. 299 (6), 843-851 (2010).</li><li>Renne, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recommendation+of+optimal+method+for+formalin+fixation+of+rodent+lungs+in+routine+toxicology+studies.">Recommendation of optimal method for formalin fixation of rodent lungs in routine toxicology studies.</a> Toxicologic Pathology. 29 (5), 587-589 (2001).</li><li>Limjunyawong, N., Mock, J., Mitzner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Instillation+and+Fixation+Methods+Useful+in+Mouse+Lung+Cancer+Research.">Instillation and Fixation Methods Useful in Mouse Lung Cancer Research.</a> Journal of Visualized Experiments. (102), e52964 (2015).</li><li>Lum, H., Mitzner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+10%+formalin+fixation+on+fixed+lung+volume+and+lung+tissue+shrinkage.+A+comparison+of+eleven+laboratory+species.">Effects of 10% formalin fixation on fixed lung volume and lung tissue shrinkage. A comparison of eleven laboratory species.</a> American Review of Respiratory Disease. 132 (5), 1078-1083 (1985).</li><li>Edmonds, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-31+initiates+lung+tumorigenesis+and+promotes+mutant+KRAS-driven+lung+cancer.">MicroRNA-31 initiates lung tumorigenesis and promotes mutant KRAS-driven lung cancer.</a> Journal of Clinical Investigation. 126 (1), 349-364 (2016).</li><li>Zhao, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wogonin+suppresses+melanoma+cell+B16-F10+invasion+and+migration+by+inhibiting+Ras-medicated+pathways.">Wogonin suppresses melanoma cell B16-F10 invasion and migration by inhibiting Ras-medicated pathways.</a> PLoS One. 9 (9), 106458 (2014).</li></ol>

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La procedura sopra descritta per la perfusione, l'inflazione e la fissazione dei polmoni del topo è ideale per una preparazione rapida ed efficiente dei polmoni del topo per l'istologia del tumore polmonare e l'analisi della patologia. La procedura non richiede alcuna attrezzatura speciale e può essere eseguita in meno di 6 minuti per mouse. La procedura non richiede un volume fisso per l'inflazione né una pressione fluida costante. Poiché questa procedura non è standardizzata, non è raccomandata per coloro che des...

Esistono una varietà di modelli di topo di oncogenesi polmonare e metastasi tumorale al polmone che vanno dai complessi GEMM ai modelli indotti da cancerogeni ai modelli sigeneici e xenograftici, dove le cellule tumorali vengono iniettate tramite intracardiaco, intratoracico, vena di coda o altri metodi per stabilire tumori all'interno del polmone. Tutti questi modelli condividono la comune necessità di valutazione istologica dell'istologia e della patologia polmonare. Pertanto, è necessario avere un metodo robusto ma rapido per eseguire necropsie di topi mentre si perfondono i polmoni per rimuovere il sangue in eccesso e gonfiare e fissare i polmoni per visualizzare chiaramente l'architettura polmonare. La velocità è un componente critico di questa procedura in quanto potrebbe essere necessario raccogliere i polmoni da dozzine di topi in un singolo momento. Questa procedura può essere eseguita in meno di 6 minuti per mouse.
Sebbene questa procedura sia più che sufficiente per valutare l'istologia tumorale, non è raccomandata per coloro che desiderano eseguire misurazioni stereologiche o morfometriche dei polmoni. Tali misurazioni richiedono che l'inflazione polmonare sia standardizzata, così come il calcolo della superficie assoluta del polmone, del volume assoluto e delle dimensioni alveolari e delnumero 1. Questo metodo non è ottimale anche per alcuni approcci di imaging. Ad esempio, l'imaging dei polmoni tramite μCT per l'analisi morfometrica ex vivo richiede che i polmoni rimangano riempiti d'aria2. Quando la conservazione degli spazi e delle dimensioni dell'aria è la preoccupazione principale, si consiglia di fissare i polmoni con tecniche di disidratazioneperfusione 3,4. Una delle maggiori preoccupazioni di questo modello è il potenziale di rupturing delle pareti alveolari, che ne diminuisce l'uso negli studi sull'enfisema; tuttavia, la procedura raccomandata per la fissazione dei polmoni per lo studio dell'enfisema è ancora abbastanza simile, in quanto si raccomanda di fissare i polmoni per instillazione intratracheale del 10% di formalina (simile al protocollo qui descritto) sotto pressione costante del fluido o mediante fissazione in situ5.
Il vantaggio della procedura descritta qui è che non richiede una pressione costante del fluido, ma gonfia i polmoni fino a quando non si sono completamente espansi, diminuendo così il tempo necessario per la procedura. La procedura qui descritta assomiglia molto ai metodi raccomandati da un armamentario della Society of Toxicologic Pathology, dove è stato formato un sottocomitato per raccomandare i migliori metodi di fissazione polmonare per gli studi tossicologici. La maggior parte degli scienziati all'interno di questa sottocommissione ha raccomandato di fissare i polmoni mediante instillazione intratracheale con una siringa, anche se c'erano raccomandazioni variabili sul tempo in cui il polmone è stato lasciato nelfissatore 6. Pertanto, mentre esistono una varietà di metodi di inflazione polmonare e fissazione, il metodo descritto nel presente documento è proposto come metodo ottimale per gonfiare e fissare rapidamente i polmoni per la valutazione istologica del tumore a valle.

<table>
	<tbody>
		<tr>
			<td>10% buffered formalin</td>
			<td>Fisher</td>
			<td>23-245685</td>
			<td></td>
		</tr>
		<tr>
			<td>22 G Needle</td>
			<td>BD</td>
			<td>305155</td>
			<td></td>
		</tr>
		<tr>
			<td>3 mL syringe</td>
			<td>BD</td>
			<td>309656</td>
			<td></td>
		</tr>
		<tr>
			<td>70% Ethanol</td>
			<td>Decon</td>
			<td>2405</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Harvard Apparatus</td>
			<td>72-8595</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Fisher</td>
			<td>H19</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Corning</td>
			<td>21-030-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>Harvard Apparatus</td>
			<td>72-8428</td>
			<td></td>
		</tr>
	</tbody>
</table>

perfusion and inflation of the mouse lung for tumor histology

La capacità di valutare l'istologia polmonare è fondamentale per i campi della ricerca sul cancro ai polmoni e della metastasi tumorale. È altrettanto importante eseguire necrosi in modo rapido ed efficiente dagli studi senza sacrificare la qualità dei tessuti acquistati. L'obiettivo di questo protocollo è quello di presentare un metodo per perfondere, gonfiare e fissare rapidamente i polmoni del topo per l'analisi istologica a valle. Questo metodo non standardizza l'inflazione polmonare; pertanto, non richiede procedure o attrezzature speciali e invece infonde semplicemente fissativo direttamente attraverso la trachea che segue la perfusione attraverso il cuore. Ciò consente una stima sufficiente delle dimensioni del tumore, dell'istologia e del punteggio. Ciò consente anche la raccolta di tessuto congelato prima della fissazione del tessuto polmonare. Questo metodo è limitato in quanto non consente una successiva quantificazione morfometrica del polmone; tuttavia, è più che sufficiente per l'analisi del tumore polmonare da modelli di topi geneticamente modificati (GEMM), modelli sigeneici, così come studi sul tumore allo xenografto e sulla metastasi.

Il protocollo di cui sopra consente una rapida perfusione, inflazione e fissazione dei polmoni del topo. Le cifre che seguono rappresentano l'importanza di ogni fase. La figura 1 raffigura i polmoni macchiati H&E che sono stati perfusi con PBS e polmoni in cui la fase di perfusione è stata saltata o i polmoni non sono riusciti a perfondersi correttamente. Come mostrato, l'eccesso di sangue nei polmoni scarsamente perfusi crea un'istologia non ideale e può rendere difficile osservare appien...

Watch this Scientific Journal Video about Perfusion and Inflation of the Mouse Lung for Tumor Histology at JoVE.com

Perfusion and Inflation of the Mouse Lung for Tumor Histology

Lo scopo di questo metodo è quello di presentare un metodo semplice ed efficiente per la perfusione, l'inflazione e la fissazione dei polmoni del topo per l'esame della patologia tumorale polmonare e la valutazione delle metastasi al polmone.

Perfusione e inflazione del polmone del topo per l'istologia tumorale

The ability to evaluate lung histology is critical for the fields of lung cancer research and cancer metastasis. It is equally important to perform ...

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Research

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Cancer Research

17.5K Views.  University of Alabama at Birmingham. Lo scopo di questo metodo è quello di presentare un metodo semplice ed efficiente per la perfusione, l'inflazione e la fissazione dei polmoni del topo per l'esame della patologia tumorale polmonare e la valutazione delle metastasi al polmone.

Video: Perfusion and Inflation of the Mouse Lung for Tumor Histology

Lung Tumor Cell Recruitment Assay

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we demonstrate a simple assay to evaluate tumor cell extravasation or micrometastasis. In this assay, tumor cells were injected through the tail vein, and after a short period, the lungs were dissected and digested to count the accumulated labeled tumor cells. This assay skips the initial step of primary tumor invasion into the blood vessel and facilitates the study of events in the distant organ where tumor metastasis occurs. The number of cells injected into the blood vessel can be optimized to observe a limited number of metastases. It has been reported that stromal cells in the distant organ contribute to metastasis. Thus, this assay could be a useful tool to explore potential therapeutic drugs or devices for prevention of tumor metastasis.

This manuscript describes a method to quantify tumor cell accumulation in the lungs in an animal model of tumor metastasis.

Analisi di reclutamento delle cellule di tumore del polmone

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we ...

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Ricerca sul cancro

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.

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.

Il Colon-26 Carcinoma tumore-cuscinetto mouse come un modello per lo studio del cancro cachessia

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

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently developed method uses a medical wire functionalized with anti-epithelial cell adhesion molecule (EpCAM) antibodies for in vivo isolation of circulating tumor cells (CTCs)1. A patient-matched cohort in non-metastatic prostate cancer showed that the in vivo isolation technique resulted in a higher percentage of patients positive for CTCs as well as higher CTC counts as compared to the current gold standard in CTC enumeration. As cells cannot be recovered from current medical devices, a new functionalized wire (referred to as Device) was manufactured allowing capture and subsequent detachment of cells by enzymatic treatment. Cells are allowed to attach to the Device, visualized on a microscope and detached using enzymatic treatment. Recovered cells are cytocentrifuged onto membrane-coated slides and harvested individually by means of laser microdissection or micromanipulation. Single-cell samples are then subjected to single-cell whole genome amplification allowing multiple downstream analysis including screening and target-specific approaches. The procedure of isolation and recovery yields high quality DNA from single cells and does not impair subsequent whole genome amplification (WGA). A single cell&#39;s amplified DNA can be forwarded to screening and/or targeted analysis such as array comparative genome hybridization (array-CGH) or sequencing. The device allows ex vivo isolation from artificial rare cell samples (i.e. 500 target cells spiked into 5 mL of peripheral blood). Whereas detachment rates of cells are acceptable (50 - 90%), the recovery rate of detached cells onto slides spans a wide range dependent on the cell line used (&#60;10 - &#62;50%) and needs some further attention. This device is not cleared for the use in patients.

This protocol is to recover and prepare rare target cells from a mixture with non-target background cells for molecular genetic characterization at the single-cell level. DNA quality is equal to non-treated single cells and allows for single-cell application (both screening based and targeted analysis).

Amplificazione dell'obiettivo delle cellule pre-arricchimento e intero genoma per singola cella a valle caratterizzazione

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently ...

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by CTCs critically contributes to early lung metastatic processes, has been vigorously investigated. As such, animal models are the only approach that captures the full systemic process of metastasis. Given that problems occur in previous experimental designs for examining the contributions of CTCs to blood vessel extravasation, we established an in vivo lung colonization assay in which a long-term-fluorescence cell-tracer, carboxyfluorescein succinimidyl ester (CFSE), was used to label suspended tumor cells and lung perfusion was performed to clear non-specifically trapped CTCs prior to lung removal, confocal imaging, and quantification. Polymeric fibronectin (polyFN) assembled on CTC surfaces has been found to mediate lung colonization in the final establishment of metastatic tumor tissues. Here, to specifically test the requirement of polyFN assembly on CTCs for lung colonization and extravasation, we performed short term lung colonization assays in which suspended Lewis lung carcinoma cells (LLCs) stably expressing FN-shRNA (shFN) or scramble-shRNA (shScr) and pre-labeled with 20 &#956;M of CFSE were intravenously inoculated into C57BL/6 mice. We successfully demonstrated that the abilities of shFN LLC cells to colonize the mouse lungs were significantly diminished in comparison to shScr LLC cells. Therefore, this short-term methodology may be widely applied to specifically demonstrate the ability of CTCs within the circulation to colonize the lungs.

An animal model is needed to decipher the role of circulating tumor cells (CTCs) in promoting lung colonization during cancer metastasis. Here, we established and successfully performed an in vivo assay to specifically test the requirement of polymeric fibronectin (polyFN) assembly on CTCs for lung colonization.

L'istituzione di un'analisi di colonizzazione del polmone per la visualizzazione di cellule del tumore nei tessuti polmonari di circolazione

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by ...

Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer

A hallmark of advanced tumors is a switch to aerobic glycolysis that is readily measured by [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography (18F-FDG PET) imaging. Co-mutations in the KRAS proto-oncogene and the LKB1 tumor suppressor gene are frequent events in lung cancer that drive hypermetabolic, glycolytic tumor growth. A critical pathway regulating the growth and metabolism of these tumors is the mechanistic target of the rapamycin (mTOR) pathway, which can be effectively targeted using selective catalytic mTOR kinase inhibitors. The mTOR inhibitor MLN0128 suppresses glycolysis in mice bearing tumors with Kras and Lkb1 co-mutations, referred to as KL mice. The therapy response in KL mice is first measured by 18F-FDG PET and computed tomography (CT) imaging before and after the delivery of MLN0128. By utilizing 18F-FDG PET/CT, researchers are able to measure dynamic changes in the glucose metabolism in genetically engineered mouse models (GEMMs) of lung cancer following a therapeutic intervention with targeted therapies. This is followed by ex vivo autoradiography and a quantitative immunohistochemical (qIHC) analysis using morphometric software. The use of qIHC enables the detection and quantification of distinct changes in the biomarker profiles following treatment as well as the characterization of distinct tumor pathologies. The coupling of PET imaging to quantitative histology is an effective strategy to identify metabolic and therapeutic responses in vivo in mouse models of disease.

Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer

In this protocol, we describe how to utilize [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography (18F-FDG PET/CT) imaging to measure the tumor metabolic response to the targeted therapy MLN0128 in a Kras/Lkb1 mutant mouse model of lung cancer and coupled imaging with high resolution ex vivo autoradiography and quantitative histology.

Utilizzando 18F-FDG PET/CT Imaging e l'istologia quantitativa per misurare i cambiamenti dinamici nel metabolismo del glucosio in modelli murini di cancro ai polmoni

A hallmark of advanced tumors is a switch to aerobic glycolysis that is readily measured by [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography ...

Acellular and Cellular Lung Model to Study Tumor Metastasis

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor cells with a natural matrix and continual flow of nutrients, as well as a model that shows the interaction of tumor cells with normal cellular components and a natural matrix. The acellular ex vivo lung model is created by isolating a rat heart-lung block and removing all the cells using the decellularization process. The right main bronchus is tied off and tumor cells are placed in the trachea by a syringe. The cells move and populate the left lung. The lung is then placed in a bioreactor where the pulmonary artery receives a continual flow of media in a closed circuit. The tumor grown on the left lung is the primary tumor. The tumor cells that are isolated in the circulating media are circulating tumor cells and the tumor cells in the right lung are metastatic lesions. The cellular ex vivo lung model is created by skipping the decellularization process. Each model can be used to answer different research questions.

Here, we present a protocol for an ex vivo lung cancer model that mimics the steps of tumor progression and helps to isolate a primary tumor, circulating tumor cells, and metastatic lesions.

Modello del polmone acellulare e cellulari per lo studio di metastasi del tumore

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as tumors. Because digital polymerase chain reactions (PCR) enable the precise quantification of DNA copy number variants, they are becoming an essential tool for detecting rare genetic variations, such as drug-resistant mutations. It is expected that molecular diagnoses using digital PCR (dPCR) will be available in clinical practice in the near future; thus, how to efficiently conduct dPCR with human genetic material is a hot topic. Here, we introduce a method to detect Adenomatous polyposis coli (APC) somatic mosaicism using dPCR with the chip-in-a-tube format, which allows eight dPCR reactions to be simultaneously conducted. Care should be taken when filling and sealing the reaction mixture on the chips. This article demonstrates how to avoid the over- and underestimation of positive partitions. Furthermore, we present a simple procedure for collecting the dPCR product from the partitions on the chips, which can then be used to confirm the specific amplification. We hope that this methods report will help promote the dPCR with the chip-in-a-tube method in genetic research.

Digital polymerase chain reaction (PCR) is a useful tool for the high-sensitivity detection of single nucleotide variants and DNA copy number variants. Here, we demonstrate key considerations for measuring rare variants in the human genome using digital PCR with the chip-in-a-tube format.

Reazione a catena della polimerasi digitale per la variazione genetica in un paziente di poliposi adenomatosa familiare sporadica utilizzando il formato di Chip-in-a-tubo

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

Detection of Lung Tumor Progression in Mice by Ultrasound Imaging

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic alterations in oncogenes such as the KRAS oncogene, which constitutes ~25% of lung cancer cases. The difficulty in therapeutically targeting KRAS-driven lung cancer partly stems from having poor models that can mimic the progression of the disease in the lab. We describe a method that permits the relative quantification of primary KRAS lung tumors in a Cre-inducible LSL-KRAS G12D mouse model via ultrasound imaging. This method relies on brightness (B)-mode acquisition of the lung parenchyma. Tumors that are initially formed in this model are visualized as B-lines and can be quantified by counting the number of B-lines present in the acquired images. These would represent the relative tumor number formed on the surface of the mouse lung. As the formed tumors develop with time, they are perceived as deep clefts within the lung parenchyma. Since the circumference of the formed tumor is well-defined, calculating the relative tumor volume is achieved by measuring the length and width of the tumor and applying them in the formula used for tumor caliper measurements. Ultrasound imaging is a non-invasive, fast and user-friendly technique that is often used for tumor quantifications in mice. Although artifacts may appear when obtaining ultrasound images, it has been shown that this imaging technique is more advantageous for tumor quantifications in mice compared to other imaging techniques such as computed tomography (CT) imaging and bioluminescence imaging (BLI). Researchers can investigate novel therapeutic targets using this technique by comparing lung tumor initiation and progression between different groups of mice.

This protocol describes the steps taken to induce KRAS lung tumors in mice as well as the quantification of formed tumors by ultrasound imaging. Small tumors are visualized in early timepoints as B-lines. At later timepoints, relative tumor volume measurements are achieved by the measurement tool in the ultrasound software.

Rilevamento della progressione del tumore del polmone nei topi mediante Ultrasound Imaging

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic ...

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.

Intravenous injection of cancer cells is often used in metastasis research, but the metastatic tumor burden can be difficult to analyze. Herein, we demonstrate a tail-vein injection model of metastasis and include a novel approach to analyze the resulting metastatic lung tumor burden.

Analisi patologica delle metastasi polmonari a seguito di iniezione laterale della vena della coda di cellule tumorali

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous ...

Generation of Zebrafish Larval Xenografts and Tumor Behavior Analysis

Zebrafish larval xenografts are being widely used for cancer research to perform in vivo and real-time studies of human cancer. The possibility of rapidly visualizing the response to anti-cancer therapies (chemo, radiotherapy, and biologicals), angiogenesis and metastasis with single cell resolution, places the zebrafish xenograft model as a top choice to develop preclinical studies.

The zebrafish larval xenograft assay presents several experimental advantages compared to other models, but probably the most striking is the reduction of size scale and consequently time. This reduction of scale allows single cell imaging, the use of a relatively low number of human cells (compatible with biopsies), medium-high-throughput drug screenings, but most importantly enables a significant reduction of the time of the assay. All these advantages make the zebrafish xenograft assay extremely attractive for future personalized medicine applications.

Many zebrafish xenograft protocols have been developed with a wide diversity of human tumors; however, a general and standardized protocol to efficiently generate zebrafish larval xenografts is still lacking. Here we provide a step-by-step protocol, with tips to generate xenografts and guidelines for tumor behavior analysis, whole-mount immunofluorescence, and confocal imaging quantification.

Here, we provide a step-by-step protocol, with tips to generate xenografts and guidelines for tumor behavior analysis, whole-mount immunofluorescence, and confocal imaging quantification.

Generazione di xenografti larvali zebrafish e analisi del comportamento tumorale

Zebrafish larval xenografts are being widely used for cancer research to perform in vivo and real-time studies of human cancer. The possibility of rapidly ...