Этот протокол был протестирован и оптимизирован первоначально в лаборатории д-р Ричард Хайнс (MIT). Финансирование предоставляется AWARD NYSTEM IDEA (для LX) и Рут Л. Kirschstein Национальный исследовательский Service Award (для LX).

1. Подготовка образцов <ol><li> Расти клетки ~ 70% сливающийся в их конкретных средств массовой информации с факторами роста или FBS (например, DMEM с 10% FBS для МС-1 клеток). Аспирируйте средств массовой информации из пластины и аккуратно мыть несколько раз с 1 х PBS (8 г / л NaCl, 0,2 г / л KCl, 1,15 г / л Na 2 HPO 4 .7 H 2 O, 0,2 г / л KH 2 PO 4, рН 7,3). </li><li> Аспирируйте PBS и добавьте 2 мл 0,05% трипсина в версена (0,014 г / л фенола красного и 0,2 г / л ЭДТА-Na в 1 х PBS, рН 7,2). Аккуратно каменную плиту, чтобы облегчить ячейки отрыв от пластины. Соблюдайте клетки под микроскопом. Обычно это занимает 2-5 минут для клеток необходимо отсоединить. </li><li> Добавить достаточным объемом сред, содержащих FBS (или ингибитором трипсина сои), чтобы утолить трипсин активности и сбора клеток в 50 мл трубки сокола. Граф клеток с использованием hemacytometer. Нагрузка 10 мкл суспензии клеток на чистую hemacytometer. Число клеток в мл равна средней # клеток в каждом из пяти квадратов умножается на 10 4. </li><li> Центрифуга клетки при 1000 оборотов в минуту (или ~ 200 мкг) в настольных центрифуг в течение 3 минут. </li><li> Осторожно снимите СМИ, не нарушая клеточный осадок. Ресуспендируют клеток в соответствующий объем Хэнкс Сбалансированный буферного раствора (HBSS), чтобы достичь конечной концентрации ~ 5 х 10 6 клеток / мл. </li><li> Фильтры клеток через Сокол 70 мкм сито ячейки, чтобы исключить крупные агрегаты клеток. Место кончика пипетки непосредственно на фильтре более помечены 5 мл трубку культуры Falcon. Быстро извлечь суспензии через фильтр в пробирку. </li><li> Граф еще раз, используя клетки hemacytometer и разбавить их конечной концентрации 2,5 х 10 6 / мл. Держите клетки на льду. </li><li> Определение жизнеспособности клеток. Смешайте несколько клеток с трипанового синего и измерить процентное содержание мертвых (синий) клетки более общий клеток с использованием hemacytometer. Жизнеспособность клеток должна быть ≥ 90% перед инъекцией. </li></ol> 2. Внутривенных инъекций <ol><li> Изменение перчатки. Аккуратно захватите хвост иммунодефицитных мышей (ню, NOD-SCID, или ГЯП, Лаборатория Джексона) и потяните ее на мыши фиксатор, с его спиной к щели и хвост, торчащие из маленького отверстия в задней части фиксатор . </li><li> Медленно слайд кольцо внутрь вдоль щели и зафиксировать ее на месте когда-то кольцо бросается в устье мыши. Мышь не должны иметь возможность свободно передвигаться, но должны иметь нормальный темп дыхания. </li><li> Найти основные вены хвоста. Четыре крупных кровеносных сосудов присутствуют в мышь хвоста. Кровеносные сосуды на спинной и брюшной сторонах хвоста артерий. Вены находятся на боковых сторонах хвоста. </li><li> Нарисуйте более 200 мкл подготовленных клеток в 1 мл шприца. Прикрепить 30 G1 / 2 дюйма иглы и вытолкнуть пузырьков воздуха, которые могут существовать. Конечный объем клеток в шприце должно быть 200 мкл (например, 5 х 10 5 клеток в общей сложности). </li><li> Inject клетки в хвостовую вену. <ol class="lalpha"><li> Начните с дистального конца хвоста, так что если первый процесс не удается, более проксимальной области хвоста могут быть использованы для второй попытки. </li><li> Протрите хвост с 70% этанола. Потяните хвост прямой. Держите кончик хвоста с большим и поддержку точки для инъекций с указательным пальцем. </li><li> Вставьте иглу в вену и ввести клетки. Убедитесь в том, игл и шприцев параллельны вены во время инъекции, иначе игла будет тыкать через стенку сосуда и придать клеток в окружающие ткани хвоста. </li><li> Снять иглу после инъекции. Кровь следует обильно из места инъекции, если инъекции прошел успешно. Пресс чистый кусок бумажной салфеткой или ватным тампоном на месте инъекции для облегчения свертывания крови, и ощупывать хвостом вверх, чтобы подтолкнуть любой остаточной образца в вену в обращение. </li><li> Отпустите кнопку мыши, из фиксатор и вернуть его в клетку. Запись инъекции процесса (например, сколько попыток понадобилось вводить клетки и сколько клетки вводили) в лаборатории ноутбука. </li><li> Определить жизнеспособность клеток после инъекции, как описано в разделе подготовки проб, Шаг 9. Этот шаг дает уверенность в том, что клетки, остаться в живых протяжении процесса впрыска. В конце эксперимента инъекции, жизнеспособность клеток будет снижаться, но должна быть выше 80%. </li><li> (Опционально) спином вниз оставшиеся клетки и промыть гранулы с PBS один раз. Замораживание гранул при температуре -80 ° С для будущего анализа (например, западная пятна, чтобы подтвердить экспрессии генов или нокдаунов). </li></ol></li><li> Обычно после одного-двух месяцев, мышей будет расчленена и расположения метастазов грубо определена. Легких является основной сайт для метастазирования, так как он содержит первую капиллярного русла, что клетки встреча после того как онивведите обращении. </li><li> После промывки PBS, каждый доли легкого (или других тканей, содержащих метастазов) наблюдается при вскрытии микроскопа (рис. 1). Число обнаруживаемых метастазов по обе стороны легких считается и количество метастазов в легких на все четыре доли складываются как общее количество метастазов в легких. 1 метастазов легкого легче обнаружить, если легкие фиксировали в формалине ночь , так как метастазы будет apppear как белесые пятна, в отличие от соседней темной легкого коричневого тканей. </li></ol> 3. Культивирования клеток от метастазов в легких <ol><li> Метастазов в легких изолированы от вводили мышам. Каждый из них должен быть культурным отдельно. </li><li> Каждый метастазы измельчается до конца колпачок с иглы (стерильные) на 70 ячейки фильтра мкм. </li><li> Промойте ячейки фильтра с несколькими мл средних и собранных клеток, которые проходят через фильтр в культуре блюдо. </li><li> Инкубируйте клетки при 37 ° С в течение по крайней мере, четыре дня без нарушений. </li><li> Смыть крови или тканей мусора на блюдо с PBS. </li><li> Добавить свежую среду. В начале, как раковые клетки и фибробласты клетки растут на пластинках, но постепенно, клеток фибробластов вымрет и будет заменен раковых клеток. Если раковые клетки несут никакой гены лекарственной устойчивостью, выделите ячейки с соответствующими антибиотиками. </li><li> Чистотой клеток, полученных является методом иммуногистохимии с использованием антител против человека-специфических белков. Мы используем анти-человеческий виментин антител (NCL-ВИМ-V9, Novocastra). Клетки мы получили обычно содержат более 99% клеток человека, даже в случае отсутствия наркотика выбора. Вновь клеток, полученных могло быть введен снова в иммунодефицитных мышей, чтобы проверить их метастатической способности, как описано выше. </li></ol> 4. Представитель Результаты <ol><li> В конце этого теста, высокой метастатической линии раковых клеток обычно возникает много метастаз легких (рис. 1), 1 в то время как очень немногие метастазы легких будет поступать из плохо метастатическим линии клеток рака. </li><li> Клетки, полученные с помощью этого метода обычно приводит к более метастазов, чем родительские линии, когда они проверяются еще раз, используя результаты анализа. 1-3 </li></ol><img src="/files/ftp_upload/1942/1942fig1.jpg" alt="Рисунок 1" /> Рисунок 1. Представителю изображения мышью легких после хвостовую вену инъекции раковых клеток. 5 х 10 5 метастатических линии человеческих клеток меланомы, С. М. клетки, 4 вводили в хвостовую вену иммунодефицитных мышей. Два месяца спустя, легкие были изолированы и метастазы были обнаружены рассеянные среди нормальной ткани легкого.

<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>

Метастазы являются основной причиной смерти у онкологических больных. Она включает в себя четыре основных этапа: отряд раковые клетки от их основных локусов, их вступление в обращении (intravasation), их выезда из кровообращения (кровоизлияние), а выживание и рост в далеком органа. Метастазы в...

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<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10313-021</td>
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<td>Hyclone</td>
<td>SH30088.03</td>
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<td>HBSS</td>
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<td>Invitrogen</td>
<td>14175-095</td>
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<td>NaCl</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S9888</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
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<td>6838-04</td>
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<td>Na2 HPO4.7H2O</td>
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<td>Sigma-Aldrich</td>
<td>P3532</td>
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<td>Invitrogen</td>
<td>15250-061</td>
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<td>Invitrogen</td>
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<td>Ethanol</td>
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<td>50 ml Falcon Tube</td>
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<td>VWR</td>
<td>89039-656</td>
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<td>305106</td>
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<td>Plas Labs Inc.</td>
<td>551-BSRR</td>
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<td>Fisher Scientific</td>
<td>08-940</td>
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<td>Forceps</td>
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<td>Fisher Scientific</td>
<td>08-902</td>
<td>&nbsp;</td>
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<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>
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experimental metastasis assay

Метастазы являются основной причиной смерти у онкологических больных. Чтобы понять механизм метастазирования, экспериментальные анализа метастазы был установлен с использованием иммунодефицитных мышей. Данная статья описывает процедуры, связанные в этом анализе, в том числе подготовка проб, внутривенные инъекции, и культивирования клеток от метастазов в легких. Короче говоря, заранее оговоренное число раковых клеток человека были подготовлены<em&gt; В пробирке</em&gt; И непосредственно вводится в обращение иммунодефицитных мышей в их жилах хвост. Небольшое число клеток выживать турбулентности в обращении и расти, как метастазы во внутренних органах, таких как рак легких. Вводили мышам расчленены по истечении определенного периода. Распределение в тканях метастазов определяется по рассекает микроскопом. Количество метастазов в конкретной ткани считается и прямо коррелирует с метастатическим способность вводили раковые клетки. Возникли метастазы изолированных и культурной<em&gt; В пробирке</em&gt; Как клеточных линий, которые часто показывают расширение метастатическим способностями, чем родительские линии, когда вводили опять в иммунодефицитных мышей. Эти высоко метастатической производных стать полезным инструментом для выявления генов или молекулярных путей, которые регулируют метастатической прогрессии.

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

Experimental Metastasis Assay

В этой статье описываются процедуры анализа экспериментальных метастазов, который используется для определения метастатического потенциала человеческих клеточных линий рака.

Экспериментальные Пробирной метастазы

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

20.2K Views.  University of Rochester Medical Center. В этой статье описываются процедуры анализа экспериментальных метастазов, который используется для определения метастатического потенциала человеческих клеточных линий рака.

Video: 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.

Количественный анализ рака метастазы использовании Птичий Модель эмбрионов

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

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.

В пробирке мезотелиальной Анализ просвет, что модели ранних стадиях рака яичников Метастазы

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.

Модели костных метастазов

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.

Экспериментальные человека перевозке пневмококковой

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.

<em&gt; В ово</em&gt; CAM-анализа, как Xenograftmodel саркомы

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.

Ортотопическая мышиной модели человеческого рака простаты Метастазы

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.

Первичная опухоль и MEF Выделение клеток для изучения метастазов в легких

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.

Чик Сердце Вторжение Пробирной для тестирования инвазивность раковых клеток и активность потенциально антиинвазивный соединений

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.

А &quot;Пациент-лайк&quot; Ортотопическая Сингенная мышь Модель гепатоцеллюлярной карциномы Метастазы

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.

Количественное внутрибрюшинным метастаз рака яичников

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 ...