Este protocolo foi testado e otimizado inicialmente no laboratório do Dr. Richard Hynes (MIT). Financiamento é assegurado pelo Prêmio IDEA NYSTEM (para LX) e Ruth L. Kirschstein Prêmio Nacional de Serviço de Investigação (para LX).

1. Preparação da Amostra <ol><li> Cultivar células confluentes para ~ 70% em seus meios específicos, com fatores de crescimento ou FBS (por exemplo, DMEM com SFB 10% para MC-1 células). Aspirar a mídia da placa e lavar cuidadosamente várias vezes com um x PBS (8 g / L de NaCl, 0,2 g / L de KCl, 1,15 g / L de Na 2 HPO 4 .7 H 2 O, 0,2 g / L de KH 2 PO 4, pH 7,3). </li><li> Aspirar PBS e adicionar 2 ml de tripsina 0,05% em versene (0,014 g / L de vermelho de fenol e 0,2 g / L de EDTA-Na em 1 x PBS, pH 7,2). Agite levemente placa para facilitar o desapego célula de prato. Observe as células sob um microscópio. Ele normalmente leva 2-5 minutos para as células de separar. </li><li> Adicionar um volume amplo de mídia contendo FBS (ou inibidor de tripsina de soja) para extinguir a atividade de tripsina e recolher as células em um tubo falcon 50ml. Contagem de células utilizando um hemocitômetro. Carga 10μL de suspensão de células em um hemocitômetro limpo. O número de células por ml é igual ao número médio de células em cada uma das cinco praças multiplicado por 10 4. </li><li> Centrifugar as células a 1000 RPM (ou ~ 200 xg) em uma centrífuga de bancada por 3 minutos. </li><li> Remova cuidadosamente os meios de comunicação, sem perturbar o pellet celular. Ressuspender as células em um volume adequado de solução tampão de Hanks Balanced (HBSS) para alcançar uma concentração final de ~ 5 x 10 6 células / mL. </li><li> Células do filtro através de um filtro de células Falcon 70 mM para excluir agregados de grandes células. Coloque a ponta da pipeta directamente para o filtro ao longo de um tubo de cultura rotulados 5mL Falcon. Rapidamente ejetar a suspensão através do filtro dentro do tubo de cultura. </li><li> Contagem de células novamente usando um hemocitômetro e diluí-los para uma concentração final de 2,5 x 10 6 / mL. Manter as células no gelo. </li><li> Determinar a viabilidade das células. Mix algumas células com azul de tripano e medir a percentagem de mortos (azul), as células sobre as células totais utilizando um hemocitômetro. A viabilidade das células deve ser ≥ 90% antes da injeção. </li></ol> 2. Injeção intravenosa <ol><li> Mudar de luvas. Agarram delicadamente a cauda de um rato imunodeficientes (nude, NOD SCID, ou NSG, Laboratório de Jackson) e puxe-o para o limitador mouse, com suas costas contra a fenda e sua cauda de fora da pequena abertura na parte de trás do limitador . </li><li> Lentamente, deslize o anel para dentro ao longo da fenda e fixá-la no lugar uma vez o anel de trava a boca do mouse. O rato não deve ser capaz de mover-se livremente, mas deve ter ritmo normal da respiração. </li><li> Encontrar as veias da cauda grande. Quatro vasos sanguíneos principais estão presentes em um rabo de rato. Vasos sangüíneos nos lados dorsal e ventral da cauda são artérias. Veias são nas laterais da cauda. </li><li> Desenhe mais de 200 mL das células preparadas para uma seringa de 1ml. Coloque a agulha 30 G1 / 2 polegada e empurrar para fora as bolhas de ar que possam existir. O volume final de células na seringa deve ser de 200 mL (ou seja, 5 x 10 5 células no total). </li><li> Injetar células dentro da veia. <ol class="lalpha"><li> Começar a partir da extremidade distal da cauda, ​​por isso, se o primeiro teste falhar, uma região mais proximal da cauda poderia ser usado para uma segunda tentativa. </li><li> Limpe o rabo com etanol 70%. Puxar o rabo reto. Segure a ponta da cauda com o polegar e apoiar o ponto de injeção com o dedo indicador. </li><li> Inserir a agulha na veia e injetar células. Certifique-se a agulha ea seringa são paralelas à veia durante a injeção, caso contrário, a agulha vai picar através da parede do vaso e injetar as células nos tecidos adjacentes cauda. </li><li> Retirar a agulha após a injecção. Sangue deve profusa no local da injecção, se a injeção foi com sucesso. Pressione uma parte limpa da toalha de papel ou chumaço de algodão no local da injecção a fim de facilitar a coagulação e apalpar o rabo para cima para empurrar qualquer amostra residual na veia em circulação. </li><li> Solte o mouse do limitador e devolvê-lo para a jaula. Registrar o processo de injeção (por exemplo, quantos ensaios que levou para injetar as células e quanto células foram injetadas), em um caderno de anotações. </li><li> Determinar a viabilidade das células após a injeção, como descrito na Seção de Preparação da Amostra, a Etapa 9. Esta etapa fornece garantia de que as células se manter vivo durante todo o processo de injeção. No final de um experimento de injeção, a viabilidade das células irá diminuir, mas deve ser acima de 80%. </li><li> (Opcional) Gire para baixo as células sobras e lavar as pelotas com PBS uma vez. Congelar a pellets a -80 oC para análises futuras (por exemplo, western blots para confirmar a expressão do gene ou knockdowns). </li></ol></li><li> Geralmente após um ou dois meses, os ratos serão dissecados e os locais de metástases são grosseiramente determinados. De pulmão é o principal local de metástase, uma vez que contém a primeira cama que as células capilares encontro depois que elesentrar na circulação. </li><li> Após lavagem com PBS, cada lóbulo do pulmão (ou outros tecidos que contêm metástases) é observada sob um microscópio de dissecação (Figura 1). O número de metástases detectáveis ​​em ambos os lados do pulmão é contada e os números de metástases pulmonares em todos os quatro lobos são somados como o número total de metástases pulmonares 1. Metástases de pulmão são mais facilmente detectado se os pulmões estão fixados em formol durante a noite , uma vez que as metástases se como manchas esbranquiçadas apppear em contraste com os tecidos adjacentes pulmão marrom escuro. </li></ol> 3. A cultura de células de metástases pulmonares <ol><li> Metástases pulmonares são isolados de camundongos injetados. Cada um deve ser cultivado separadamente. </li><li> Cada metástase é picada até o final de uma ponta de agulha (estéril) em um filtro de células 70 mM. </li><li> Lavar o filtro de células com vários mls de médio e células coletadas que passam através do filtro em um prato de cultura. </li><li> Incubar as células a 37 ° C durante pelo menos quatro dias sem perturbação. </li><li> Lavar sangue ou restos de tecido sobre o prato com PBS. </li><li> Adicionar meio fresco. No início, tanto as células cancerosas e células de fibroblastos crescer nas placas, mas, gradualmente, as células fibroblasto vai morrer e ser substituído por células cancerosas. Se as células cancerosas transportar qualquer genes resistentes aos medicamentos, selecione as células com antibióticos correspondente. </li><li> A pureza das células derivada é avaliada por imunomarcação utilizando anticorpos humanos contra proteínas específicas. Usamos um anti-anticorpo humano vimentina (VIM-NCL-V9, Novocastra). As células que contêm derivados em geral mais de 99% de células humanas, mesmo na ausência de seleção de medicamentos. As células recém-derivados pode ser injetado em camundongos imunodeficientes novamente para testar suas habilidades metastático, como descrito acima. </li></ol> 4. Resultados representante <ol><li> No final deste ensaio, uma linhagem de células altamente metastáticas de câncer geralmente dá origem a metástases pulmonares muitos (Figura 1), 1 enquanto metástases pulmonares muito poucos virão de uma linha mal câncer metastático de células. </li><li> As células derivadas usando este método costuma dar origem a metástases mais do que a linha dos pais, quando são testados novamente com este ensaio. 1-3 </li></ol><img src="/files/ftp_upload/1942/1942fig1.jpg" alt="Figura 1" /> Figura 1. Imagens representativas dos pulmões do rato após injeções veia da cauda das células cancerosas. 5 x 10 5 da linha celular humana melanoma metastático, as células SM, 4 foram injetados na veia da cauda de camundongos imunodeficientes. Dois meses depois, os pulmões foram isolados e as metástases foram encontrados dispersos entre o tecido pulmonar normal.

<ol>
	<li>Xu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+changes+in+an+animal+melanoma+model+correlate+with+aggressiveness+of+human+melanoma+metastases.">Gene expression changes in an animal melanoma model correlate with aggressiveness of human melanoma metastases.</a> Mol Cancer Res. 6 (5), 760-760 (2008).</li><li>Clark, E. A., Golub, T. R., Lander, E. S., Hynes, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+analysis+of+metastasis+reveals+an+essential+role+for+RhoC.">Genomic analysis of metastasis reveals an essential role for RhoC.</a> Nature. 406 (6795), 532-532 (2000).</li><li>Xu, L., Begum, S., Hearn, J. D., Hynes, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GPR56,+an+atypical+G+protein-coupled+receptor,+binds+tissue+transglutaminase,+TG2,+and+inhibits+melanoma+tumor+growth+and+metastasis.">GPR56, an atypical G protein-coupled receptor, binds tissue transglutaminase, TG2, and inhibits melanoma tumor growth and metastasis.</a> Proc Natl Acad Sci U S A. 103 (24), 9023-9023 (2006).</li><li>Kozlowski, J. M., Hart, I. R., Fidler, I. J., Hanna, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+human+melanoma+line+heterogeneous+with+respect+to+metastatic+capacity+in+athymic+nude+mice.">A human melanoma line heterogeneous with respect to metastatic capacity in athymic nude mice.</a> J Natl Cancer Inst. 72 (4), 913-913 (1984).</li><li>Kang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multigenic+program+mediating+breast+cancer+metastasis+to+bone.">A multigenic program mediating breast cancer metastasis to bone.</a> Cancer Cell. 3 (6), 537-537 (2003).</li></ol>

Metástase é a principal causa de morte em pacientes com câncer. Trata-se de quatro etapas principais: separação de células de câncer de seus loci primários, sua entrada em circulação (intravasation), sua saída de circulação (extravasamento), e sobrevivência e crescimento em um órgão distante. Metástase em humanos é considerada um processo lento e muitas vezes se manifesta após anos de latência. Para estudar a sua progressão em tempo hábil, o ensaio relativamente rápido acima experimental foi estab...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10313-021</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>SH30088.03</td>
<td>&nbsp;</td>
<tr>
<td>HBSS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14175-095</td>
<td>&nbsp;</td>
<tr>
<td>NaCl</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S9888</td>
<td>&nbsp;</td>
<tr>
<td>KCl</td>
<td>&nbsp;</td>
<td>Mallinckrodt Chemicals</td>
<td>6838-04</td>
<td>&nbsp;</td>
<tr>
<td>Na2 HPO4.7H2O</td>
<td>&nbsp;</td>
<td>Mallinckrodt Chemicals</td>
<td>7896-04</td>
<td>&nbsp;</td>
<tr>
<td>KH2PO4</td>
<td>&nbsp;</td>
<td>Mallinckrodt Chemicals</td>
<td>7100-12</td>
<td>&nbsp;</td>
<tr>
<td>Phenol red</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P3532</td>
<td>&nbsp;</td>
<tr>
<td>EDTA-Na</td>
<td>&nbsp;</td>
<td>EMD Biosciences</td>
<td>4010</td>
<td>&nbsp;</td>
<tr>
<td>Trypan Blue</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15250-061</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin 2.5%</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15090-046</td>
<td>&nbsp;</td>
<tr>
<td>Ethanol</td>
<td>&nbsp;</td>
<td>Ultrapure</td>
<td>200-CSGP</td>
<td>&nbsp;</td>
<tr>
<td>50 ml Falcon Tube</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>89039-656</td>
<td>&nbsp;</td>
<tr>
<td>Microcentrifuge Tubes</td>
<td>&nbsp;</td>
<td>Axygen Scientific</td>
<td>MCT-175-C</td>
<td>&nbsp;</td>
<tr>
<td>Falcon 70&#181;m Cell Strainer</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>352350</td>
<td>&nbsp;</td>
<tr>
<td>5ml Falcon Culture Tube</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>60818-576</td>
<td>&nbsp;</td>
<tr>
<td>Hemacytometer</td>
<td>&nbsp;</td>
<td>Hausser Scientific</td>
<td>15170-208</td>
<td>&nbsp;</td>
<tr>
<td>1 ml syringe</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>329650</td>
<td>&nbsp;</td>
<tr>
<td>30 &frac12; gauge needle</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>305106</td>
<td>&nbsp;</td>
<tr>
<td>Mouse restrainer</td>
<td>&nbsp;</td>
<td>Plas Labs Inc.</td>
<td>551-BSRR</td>
<td>&nbsp;</td>
<tr>
<td>Surgical scissors</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>08-940</td>
<td>&nbsp;</td>
<tr>
<td>Forceps</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>08-902</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting Microscope</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>SMZ1500</td>
<td>&nbsp;</td>
<tr>
<td>Benchtop Centrifuge</td>
<td>&nbsp;</td>
<td>Thermoscientific</td>
<td>75003491</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

experimental metastasis assay

Metástase é a principal causa de morte em pacientes com câncer. Para entender o mecanismo de metástases, um ensaio de metástase experimental foi estabelecida usando camundongos imunodeficientes. Este artigo delineia os procedimentos envolvidos neste ensaio, incluindo a preparação da amostra, injeção intravenosa, e a cultura de células de metástases pulmonares. Resumidamente, um número pré-determinado de células humanas de câncer foram preparados<em&gt; In vitro</em&gt; E injetado diretamente na circulação de camundongos imunodeficientes através das veias sua cauda. Um pequeno número de células sobreviver à turbulência na circulação e crescer como metástases em órgãos internos, como pulmão. Os ratos injetados são dissecados após um certo período. A distribuição do tecido de metástases é determinada sob um microscópio de dissecação. O número de metástases em um determinado tecido é contado e que se correlaciona diretamente com a capacidade metastática das células cancerosas injetadas. As metástases surgido são isoladas e cultivadas<em&gt; In vitro</em&gt; Como linhas celulares, que freqüentemente mostram maior capacidade metastática do que a linha dos pais quando injetado novamente em camundongos imunodeficientes. Esses derivativos altamente metastáticas se tornar ferramentas úteis para a identificação de genes ou as vias moleculares que regulam a progressão metastática.

Watch this Scientific Journal Video about Experimental Metastasis Assay at JoVE.com

Experimental Metastasis Assay

Este artigo descreve os procedimentos de um ensaio de metástase experimental que é usado para determinar o potencial metastático de linhagens de células humanas de câncer.

Ensaio Metástase Experimental

Metastasis is the leading cause of death in cancer patients.  To understand the mechanism of metastasis, an experimental metastasis assay was established ...

Research

JoVE Journal

Medicine

20.1K visualizações.  University of Rochester Medical Center. Este artigo descreve os procedimentos de um ensaio de metástase experimental que é usado para determinar o potencial metastático de linhagens de células humanas de câncer.

Vídeo: Experimental Metastasis Assay

Quantitative Analysis of Cancer Metastasis using an Avian Embryo Model

During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex process that can involve many tissue types, span variable time periods, and often occur deep within organs, making it difficult to investigate and quantify. In addition, the efficacy of the metastatic process is influenced by multiple steps in the metastatic cascade making it difficult to evaluate the contribution of a single aspect of tumor cell behavior. As a consequence, metastasis assays are frequently performed in experimental animals to provide a necessarily realistic context in which to study metastasis. Unfortunately, these models are further complicated by their complex physiology. The chick embryo is a unique in vivo model that overcomes many limitations to studying metastasis, due to the accessibility of the chorioallantoic membrane (CAM), a well-vascularized extra-embryonic tissue located underneath the eggshell that is receptive to the xenografting of tumor cells (figure 1). Moreover, since the chick embryo is naturally immunodeficient, the CAM readily supports the engraftment of both normal and tumor tissues. Most importantly, the avian CAM successfully supports most cancer cell characteristics including growth, invasion, angiogenesis, and remodeling of the microenvironment. This makes the model exceptionally useful for the investigation of the pathways that lead to cancer metastasis and to predict the response of metastatic cancer to new potential therapeutics. The detection of disseminated cells by species-specific Alu PCR makes it possible to quantitatively assess metastasis in organs that are colonized by as few as 25 cells. Using the Human Epidermoid Carcinoma cell line (HEp3) we use this model to analyze spontaneous metastasis of cancer cells to distant organs, including the chick liver and lung. Furthermore, using the Alu-PCR protocol we demonstrate the sensitivity and reproducibility of the assay as a tool to analyze and quantitate intravasation, arrest, extravasation, and colonization as individual elements of metastasis.

Using quantitative PCR, we demonstrate how the well-established chick CAM model can be used to quantitatively analyze the metastasis of human tumor cells to distant organs.

Análise quantitativa de metástase de câncer utilizando um modelo de Embrião aviária

During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex ...

Medicina

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

Ensaio in vitro de Apuramento mesotelial que os modelos dos primeiros passos da metástase do câncer de ovário

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

Modelos de metástase óssea

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

Experimental Human Pneumococcal Carriage

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major source of transmission and the prerequisite of invasive disease. A model of carriage will allow accurate determination of the immunological correlates of protection, the immunizing effect of carriage and the effect of host pressure on the pathogen in the nasopharyngeal niche. Further, methods of carriage detection useful in epidemiologic studies, including vaccine studies, can be compared.
Aim
We aim to develop an EHPC platform that is a safe and useful reproducible method that could be used to down-select candidate novel pneumococcal vaccines with prevention of carriage as a surrogate of vaccine induced immunity. It will work towards testing of candidate vaccines and descriptions of the mechanisms underlying EHPC and vaccine protection from carriage1. Current conjugate vaccines against pneumococcus protect children from invasive disease although new vaccines are urgently needed as the current vaccine does not confer optimal protection against non-bacteraemic pneumonia and there has been evidence of serotype replacement with non-vaccine serotypes2-4.
Method
We inoculate with S. pneumoniae suspended in 100 &mu;l of saline. Safety is a major factor in the development of the EHPC model and is achieved through intensive volunteer screening and monitoring. A safety committee consisting of clinicians and scientists that are independent from the study provides objective feedback on a weekly basis.
The bacterial inoculum is standardized and requires that no animal products are inoculated into volunteers (vegetable-based media and saline). The doses required for colonization (104-105) are much lower than those used in animal models (107)5. Detecting pneumococcal carriage is enhanced by a high volume (ideally &gt;10 ml) nasal wash that is relatively mucus free. This protocol will deal with the most important parts of the protocol in turn. These are (a) volunteer selection, (b) pneumococcal inoculum preparation, (c) inoculation, (d) follow-up and (e) carriage detection.
Results
Our current protocol has been safe in over 100 volunteers at a range of doses using two different bacterial serotypes6. A dose ranging study using S. pneumoniae 6B and 23F is currently being conducted to determine the optimal inoculation dose for 50% carriage. A predicted 50% rate of carriage will allow the EHPC model to have high sensitivity for vaccine efficacy with small study numbers.

Experimental human pneumococcal carriage offers a natural model of carriage and a potential model for use in vaccine development. This technique is valuable yet complex and involves clinical risk by introducing a pathogen into a human. We have developed a detailed protocol.

Transporte pneumocócica experimental humana

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major ...

The In ovo CAM-assay as a Xenograft Model for Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions.
This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

The In ovo CAM-assay as a Xenograft Model for Sarcoma

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

O<em&gt; Em ovo</em&gt; CAM-ensaio como um Xenograftmodel para o Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

Um Modelo murino de próstata humana Metástase Ortotópico

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Primary Tumor and MEF Cell Isolation to Study Lung Metastasis

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered genotypes now possess the ability to invade and survive in other tissues. In this protocol, mouse mammary tumors are removed and primary cells are prepared from tumors. The cells isolated from this procedure are then available for gene profiling experiments. For successful metastasis, these cells must be able to intravasate, survive in circulation, extravasate to distant organs, and survive in that new organ system. The lungs are the typical target of breast cancer metastasis. A set of genes have been discovered that mediates the selectivity of metastasis to the lung. Here we describe a method of studying lung metastasis from a genetically engineered mouse model.. Furthermore, another protocol for analyzing mouse embryonic fibroblasts (MEFs) from the mouse embryo is included. MEF cells from the same animal type provide a clue of non-cancer cell gene expression. Together, these techniques are useful in studying mouse mammary tumorigenesis, its associated signaling mechanisms and pathways of the abnormalities in embryos.

The goal of this protocol is to study breast tumorigenesis. With this technique, mouse mammary tumors are removed and primary cells are prepared from tumors. A lung extraction protocol is included for studying lung metastasis. Furthermore, another protocol for analyzing mouse embryonic fibroblasts from the mouse embryo is included.

Tumor primário e isolamento de células MEF para o Estudo de metástases pulmonares

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered ...

Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish between invasive and non-invasive cells, and enables study of the effects of test compounds on tumor invasion. Cancer cells - either as aggregates or single cells - are confronted with fragments of embryonic chick heart. After organ culture in suspension for a few days or weeks the confronting cultures are fixed and embedded in paraffin for histological analysis. The three-dimensional interaction between the cancer cells and the normal tissue is then reconstructed from serial sections stained with hematoxylin-eosin or after immunohistochemical staining for epitopes in the heart tissue or the confronting cancer cells. The assay is consistent with the recent concept that cancer invasion is the result of molecular interactions between the cancer cells and their neighbouring stromal host elements (myofibroblasts, endothelial cells, extracellular matrix components, etc.). Here, this stromal environment is offered to the cancer cells as a living tissue fragment. Supporting aspects to the relevance of the assay are multiple. Invasion in the assay is in accordance with the criteria of cancer invasion: progressive occupation and replacement in time and space of the host tissue, and invasiveness and non-invasiveness in vivo of the confronting cells generally correlates with the outcome of the assay. Furthermore, the invasion pattern of cells in vivo, as defined by pathologists, is reflected in the histological images in the assay. Quantitative structure-activity relation (QSAR) analysis of the results obtained with numerous potentially anti-invasive organic congener compounds allowed the study of structure-activity relations for flavonoids and chalcones, and known anti-metastatic drugs used in the clinic (e.g., microtubule inhibitors) inhibit invasion in the assay as well. However, the assay does not take into account immunological contributions to cancer invasion.

Here, we present a protocol to study the invasion of tumor cells into living normal tissue fragments in three dimensions. This organ culture technique is mainly applied to test potentially anti-invasive drugs in vitro.

Pintainho Coração Invasion Ensaio para testar a invasão das células neoplásicas e da Actividade de compostos potencialmente anti-invasivos

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish ...

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

A &quot;Patient-Like&quot; Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

A &quot;Patient-Like&quot; Orthotopic Singênico Rato Modelo de carcinoma hepatocelular Metástase

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...

Quantitation of Intra-peritoneal Ovarian Cancer Metastasis

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States. Mortality is due to diagnosis of 75% of women with late stage disease, when metastasis is already present. EOC is characterized by diffuse and widely disseminated intra-peritoneal metastasis. Cells shed from the primary tumor anchor in the mesothelium that lines the peritoneal cavity as well as in the omentum, resulting in multi-focal metastasis, often in the presence of peritoneal ascites. Efforts in our laboratory are directed at a more detailed understanding of factors that regulate EOC metastatic success. However, quantifying metastatic tumor burden represents a significant technical challenge due to the large number, small size and broad distribution of lesions throughout the peritoneum. Herein we describe a method for analysis of EOC metastasis using cells labeled with red fluorescent protein (RFP) coupled with in vivo multispectral imaging. Following intra-peritoneal injection of RFP-labelled tumor cells, mice are imaged weekly until time of sacrifice. At this time, the peritoneal cavity is surgically exposed and organs are imaged in situ. Dissected organs are then placed on a labeled transparent template and imaged ex vivo. Removal of tissue auto-fluorescence during image processing using multispectral unmixing enables accurate quantitation of relative tumor burden. This method has utility in a variety of applications including therapeutic studies to evaluate compounds that may inhibit metastasis and thereby improve overall survival.

Ovarian cancer metastasis is characterized by numerous diffuse intra-peritoneal lesions, such that accurate visual quantitation of tumor burden is challenging. Herein we describe a method for in situ and ex vivo quantitation of metastatic tumor burden using red fluorescent protein (RFP)-labeled tumor cells and optical imaging.

A quantificação da Intra-peritoneal Ovarian Cancer Metástase

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States.  Mortality is due to diagnosis of 75% of ...