Os autores reconhecem com gratidão a concessão de equipamentos da ciência Natural e pesquisa de engenharia (NSERC) Conselho de Canadá para HPLC, a subvenção de funcionamento do Instituto canadense de pesquisa de saúde (CIHR) e pesquisa de câncer de mama canadense (CBCR) Aliança para X.Y. Wu e a bolsa da Universidade de Toronto para R.X. Zhang e T. Zhang.

todos os experimentos com animais foram aprovados pelo Animal conta Comissão de University Health Network no Ontario Cancer Institute e conduzidos de acordo com o Conselho canadense sobre as orientações de cuidado Animal.
 1. preparação da amostra biológica <ol><li>coletar o sangue total, os principais órgãos e tumor de mama nos pontos de tempo predeterminados após administração intravenosa (IV) de contendo fármaco formulações (por exemplo, DMPLN, DOX lipossomal) <ol><li>injetar um peito tumor-rolamento do mouse IV com uma formulação contendo fármaco preparada.</li>
		<li>Anestesiar o mouse nos pontos tempo designados (por exemplo, 15 min), dando inalável isoflurano de 2% em uma câmara selada.</li>
		<li>Colocar o mouse anestesiado em suas costas e colocar seu nariz através de um inalador nasal que constantemente fornece 2% isoflurano. 
		Nota: Para garantir que o mouse passa por anestesia profunda, suavemente belisca os membros dianteiros do mouse e procure qualquer movimento contrair.</li>
		<li>Limpe muito bem os peito e abdome regiões usando etanol a 70% e, em seguida, executar um procedimento terminal da punção cardíaca os ratos anestesiados profundos, usando uma seringa heparinizada 1 mL e uma agulha 23G.</li>
		Heparina <li>coletar o sangue total em um rotulado de sódio pulverizado tubo plástico e agite suavemente o tubo para garantir coletado sangue entra em contato com a heparina revestida da parede do tubo. Recolha um mínimo de 50 µ l de sangue total. Mantenha sempre as amostras no gelo</li>
		<li>Fita todos os quatro membros do rato para fixá-lo e abrir a cavidade abdominal e torácica do mouse usando um par de tesouras e pinças. Deslocar o intestino para o lado e empurre o fígado para cima para expor suficientemente a veia porta. Cortar a veia porta para a drenagem de sangue.</li>
		<li>Perfundir o corpo todo rato com 50 mL de soro fisiológico 0,9% gelado através do coração utilizando uma seringa de 10 mL com uma agulha de 25 G. 
		Nota: Dobrar a agulha a 90° para guiar a seringa na veia portal.</li>
		<li>Órgãos de impostos especiais de consumo no seguinte ordem: coração, pulmão, fígado, baço, rins. Em seguida, separe o tumor de mama os tecidos conectivos circundantes, usando um par de tesouras de incisão para a almofada de gordura mamária direita do mouse. Coletar todos os órgãos individualmente em tubos de polipropileno de 1,5 mL e rapidamente congelá-los em azoto líquido. 
		&#8203; Nota: separar a vesícula do fígado.</li>
		<li>Armazenar o sangue de toda a 4 ° C e tecidos extirpados em congelador a-80 ° C até posterior análise HPLC.</li>
	</ol></li> <li>Extrair DOX, MMC e DOXol de matrizes biológicas.
	<ol><li>Pesar todos os tecidos dissecados congelados rapidamente e transferi-los para um tubo cônico de fundo arredondado de 13 mL. Para evitar o metabolismo das drogas possível ou degradação, manter as amostras no gelo</li>
		<li>Adicionar 15 mL de tampão de lise celular gelada no tubo. 
		Nota: O volume de reserva para usar depende do peso de tecido com base na relação tecido-tampão de 1 g: 5 mL (p/v); para pequenos órgãos, tais como o coração e o baço, a proporção é de 1 g: 2 mL.</li>
		<li>Usar um movimento cima-baixo curso para homogeneizar as amostras no gelo à velocidade de 18.000 rpm usando um homogeneizador de mão elétrico. 
		Nota: Homogeneização completa requer cerca de 3 a 5 iterações de um processo de homogeneização curta de menos de 15 s, seguido de resfriamento com gelo entre cada curta homogeneização de tecidos.</li>
		<li>Lavar a sonda de gerador dente de serra 10 mm do homogenizador com água destilada deionizada (DDI) H 2 O etanol a 70% e em seguida DDI H 2 O entre cada amostra de tecido para evitar a contaminação cruzada.</li>
		<li>Transferir 50 µ l de homogeneizado de tecido ou sangue total em um tubo de centrífuga micro polipropileno 1,5 mL e spike com 5 µ l de uma interna padrão (I.S.) 4-methylumbelliferone (4-MU) (2000 ng/mL) no tubo. 
		Nota: solução de 4-MU foi preparada em metanol aqui.</li>
		<li>Adicionar 250 µ l de um solvente de extração gelada no tubo contendo sangue ou tecido homogeneizado. 
		Nota: O solvente de extração consiste em 60% acetonitrila (ACN) e acetato de amônio 40% (5 mM) com pH ajustado para pH = 3.5 usando 0,05% de ácido fórmico. Use uma amostra de (v/v) de 1:5: solvente de extração para relação volume.</li>
		<li>Vigorosamente a mistura por 2 min, centrifugar 3.000 x g força no 4 o C por 10 min e pipetar 200 µ l sobrenadante para outro tubo de microcentrífuga fresco pre-refrigerado de vórtice.</li>
		<li>Sobrenadante de evaporar a 60 ° C, sob uma corrente lenta de gás nitrogênio com proteção contra luz.</li>
		<li>Reconstituir o resíduo seco com 100 µ l de metanol gelado, vórtice vigorosamente por 30 s e centrifugar a 3000 x g a 4 ° C, mais 5 min.</li>
		<li>Transferir o sobrenadante para uma inserção do frasco HPLC e colocar os frascos de amostra em uma bandeja de mostruário para injeção.</li>
	</ol></li></ol> 2. HPLC Instrumentação e parâmetros de operação <ol><li>preparar HPLC fase móvel com reprodutibilidade consistente <ol><li>medir de HPLC-classe H 2 O usando um cilindro graduado de 500 mL.</li>
		<li>Medir 500 mL de acetonitrila grau CLAE (ACN) usando um cilindro graduado separado.</li>
		<li>, Cuidadosamente, adicionar 0,5 mL de ácido trifluoroacético (TFA) (cuidado) em cada um dos 500 mL de H 2 O e ACN para obter a fase móvel de H 2 O e ACN contendo 0,1% TFA, respectivamente. 
		Nota: O TFA é corrosivo e tóxico e deve ser tratado sob uma coifa de laboratório. Todas as misturas de solventes são preparadas à temperatura ambiente.</li>
		<li>Filtro fases móveis através de um filtro de membrana de nylon com um 0,45 µm reduz o tamanho dos poros e transferi-lo para limpas garrafas de reservatório HPLC.</li>
	</ol></li> <li>Instrumentação de HPLC de set-up para a deteção simultânea de DOX, MMC e DOXol e I.S. 4-MU.
	<ol><li>Ligue a bomba de gradiente, de gasser, auto-sampler, detetor de fotodiodo matriz e multi detector de fluorescência λ.</li>
		<li>Entrada as condições iniciais da composição da fase móvel para 16,5% H 2 O (0,1% TFA) e 83,5% ACN (0,1% TFA) (v/v).</li>
		<li>Definir o detector UV em dois canais, um em 310 nm para 4-MU (I.S.) e o outro a 360 nm para MMC.</li>
		<li>Definir o detector de fluorescência em dois canais, um de cada λ ex / λ em = 365/445 nm para 4-MU e o outro no λ ex / λ em = 480 nm/560 nm para DOX e DOXol, respectivamente.</li>
		<li>Definir uma taxa de fluxo de isocrática de 1,0 mL/min.</li>
		<li>Equilibrar uma coluna de 18 fase reversa pré-instalado C (4,6 mm x 250 mm, 5 µm) em temperatura ambiente por 10 min. para o estabelecimento da linha de base.</li>
	</ol></li> <li>Separar drogas (DOX, MMC, DOXol e 4-MU) usando condição gradiente de fase móvel.
	<ol><li>Injetar 15 µ l de amostras re-concentradas e extraídas usando o auto-sampler.</li>
		<li>Gradualmente mudar a condição inicial da fase móvel (consulte a etapa protocolo 2.2.2) para 100% ACN (0,1% TFA) mais 18 min usando a bomba gradiente automatizada. 
		Nota: Durante o processo de separação, quatro canais (dois absorventes UV e duas fluorescentes) aparecem simultaneamente com cada canal exibindo um medicamento composto (consulte a etapa de protocolo 2.2.3 e 2.2.4).</li>
		<li>Manter 100% de ACN (0,1% TFA) por 1 min e em seguida, retornar à condição inicial fase móvel dentro de 1 min.</li>
		<li>Re-condicionar a coluna com a fase móvel inicial com caudal de 1,5 mL/min durante 4 min para a próxima injeção de amostra.</li>
	</ol></li></ol> 3. Validação de HPLC <ol><li>preparar normas de trabalho de DOX, MMC e DOXol e 4-MU (I.S.).
	<ol><li>Pesar separadamente 1 mg de pó de drogas DOX e MMC (cuidado) e 4-MU em uma nova pesagem pequeno papel (3 x 3 polegadas 2). 
		Observe que todas as drogas anticâncer são consideradas um perigo para a saúde que pode causar mutagenicidade de toxicidade e células germinativas aguda na inalação ou ingestão. Devem ser manuseadas com cuidado com luvas e máscaras.</li>
		<li>Transferir o pesado DOX, MMC e 4-MU em Nova individual 1,5 mL polipropileno microtubo de centrífuga.</li>
		<li>Adicionar 1 mL de metanfetaminaadilia e vórtice brevemente para obter a concentração de 1 mg/mL de DOX e MMC.</li>
		<li>Adicionar 1 mL de metanol em um frasco contendo pré-pesados 1 mg de DOXol (cuidado) e vórtice brevemente para obter a concentração de 1 mg/mL de DOXol. 
		Nota: DOXol é um metabólito tóxico cardio e deve ser manuseado com cuidado.</li>
		<li>Pipeta 20 µ l de soluções estoque preparadas de DOX, MMC, DOXol e 4-MU em um novo separar o tubo de microcentrífuga polipropileno de 1,5 mL e adicionar 980 µ l de metanol para obter um padrão de trabalho de 20 µ g/mL de cada medicamento.</li>
		<li>Diluir 20 µ g/mL de DOX, MMC e DOXol usando metanol para obter as normas de trabalho de 50 ng - 20 µ g /mL para DOX, MMC e DOXol e 2000 ng/mL para I.S. 4-MU.</li>
		<li>Fechar a tampa do tubo de soluções de trabalho com um estreito pedaço de película coberta de parafina para evitar a evaporação do metanol, enrole o tubo inteiro com papel de alumínio para evitar a exposição à luz direta e loja em -20 ° C.</li>
	</ol></li> <li>Determinar a linearidade, precisão e exatidão de DOX, MMC e DOXol em matrizes biológicas (ou seja, o sangue total e tumor homogenate).
	<ol><li>Simultaneamente espigão 5 µ l de normas de trabalho de DOX e DOXol (50 ng/mL - 20 µ g/mL), MMC (1.000 ng/mL - 16 µ g/mL) e 4-MU (2 µ g/mL) em 50 µ l de sangue em branco ou tecido homogeneizado em polipropileno tubos de centrífuga de micro obter a curva padrão de concentração que variam de 5-2000 ng/mL para compostos de drogas e 200 ng/mL para o 4-MU (I.S.).</li>
		<li>Realizar o ensaio de extração de drogas descrito no protocolo 1.2.</li>
		<li>Usar concentrações baixas, média e altas de DOX e DOXol (50, 500 e 2.000 ng/mL) e MMC (100, 1000, 2.000 ng/mL) para precisão intra e inter dia. 
		Nota: Preparar concentrações padrão frescas no dia da análise.</li>
	</ol></li> <li>Análise de amostras <ol><li>injetar 15 µ l de amostra usando o auto-sampler.</li>
		<li>Gradualmente mudar a fase móvel mais de 0 a 18 min, aumentando a composição do Grupo ACN no intervalo.</li>
		<li>Depois de 18 min, segure a condição de fase móvel de 1 min.</li>
		<li>Retornar à condição inicial sobre a próxima 2 min e, em seguida, re-equilibrar para 4 min antes da próxima injeção.</li>
		<li>Após cada amostra executar, note que os picos de compostos de drogas com o seu tempo de retenção são mostrados como segue: MMC, DOXol, 4-MU (I.S.) e DOX.</li>
		<li>Integrar a área do pico sob a curva (AUC) de compostos de drogas usando HPLC software.</li>
		<li>Calcular a relação AUC entre compostos individuais sobre drogas e I.S. (equação 1) e use as curvas padrão elaboradas sob os mesmos procedimentos de extração para determinar as concentrações de droga de DOX, MMC e DOXol na formulação de DMPLN. 
		<img alt="Equation 1" src="/files/ftp_upload/56159/56159eq1.jpg" /></li> <li>calcular a percentagem de recuperação de drogas (equação 2), comparando as concentrações de droga reconstituídas utilizando metanol de extractos de amostras biológicas cravados àquele da norma (&#34; pura &#34;) solução em metanol de drogas. 
		<img alt="Equation 2" src="/files/ftp_upload/56159/56159eq2.jpg" /></li></ol></li></ol>

Os autores não têm interesses financeiros concorrentes e conflitos de interesse.

Em comparação com outros métodos cromatográficos que permitem a detecção de uma espécie única droga de cada vez, o presente protocolo HPLC é capaz de dosar simultaneamente três compostos de drogas (DOX, MMC e DOXol) na mesma matriz biológica sem a necessidade de mudar a fase móvel. Esse método de preparação e análise tem sido aplicado com sucesso para determinar a farmacocinética e bio-distribuição de dois sistemas de entrega de drogas baseados em nanopartículas (i.e., lipossomal DOX e DMPLN)...

A quimioterapia é uma modalidade de tratamento primário para muitos tipos de câncer ainda é frequentemente associado com efeitos adversos graves e eficácia limitada devido a resistência às drogas e outros fatores1,2,3. Regimes de combinação de drogas para melhorar o resultado da quimioterapia, foram aplicados na clínica com base em considerações como toxicidades não-sobreposição, diferentes mecanismos de ação de drogas e drogas não-cruz resistência4,5 , 6. em ensaios clínicos, uma melhor taxa de resposta do tumor foi muitas vezes observada usando simultaneamente administrados combinações de drogas, em comparação com um regime de drogas sequencial entrega7,8. No entanto, devido à sub-ótimo bio-distribuição de formulários de droga livre, injeção simultânea de múltiplas drogas pode causar toxicidade proeminentes tecido normal que supera o efeito terapêutico9,10,11. Entrega de drogas baseadas em nanocarreador foi mostrada para alterar a farmacocinética e bio-distribuição de medicamentos encapsulados, aumentando a acumulação de tumor-alvo12,13,14. Como comentado em nossos artigos recentes, nanopartículas co carregadas com combinações sinérgicas de drogas têm demonstrado a capacidade de atenuar os problemas enfrentados por combinações de drogas livre, devido a sua controlada entrega co temporal e espacial de várias drogas ao tecido do tumor, permitir efeitos sinérgicos droga contra o câncer células4,15,16. Como resultado, superior eficácia terapêutica e baixa toxicidade foram demonstradas em ambos os estudos pré-clínicos e clínicos4,17,18.
Nossos anterior em vitro estudos encontraram que a combinação de duas drogas anticâncer, doxorrubicina (DOX) e mitomicina C (MMC), produziu um efeito sinérgico contra várias linhas de células de câncer de mama e, além disso, co carregando DOX e MMC dentro nanopartículas de lipídios-polímero híbrido (DMPLN) superaram vários resistente a múltiplas drogas associado efluxo bombas (por exemplo, P-glicoproteína e proteína resistente ao cancro da mama)19,20,21. In vivo, DMPLN habilitado entrega co espaço-temporal de DOX e MMC para localizações tumorais e maior biodisponibilidade de drogas dentro de células de câncer, conforme indicado pela moderação da formação do metabólito DOX doxorubicinol (DOXol)22. Como resultado, o DMPLN reforçada a apoptose de células do tumor, inibição de crescimento do tumor e sobrevivência de acolhimento prolongado em comparação a livre combinação de DOX e MMC ou um lipossomal DOX formulação22,23,24, 25.
Analisando a quantidade real de drogas fornecido em parceria pela nanocarreador é fundamental para a concepção de formulações de nanopartículas eficaz. Muitos métodos foram desenvolvidos para analisar o nível plasmático de único doses DOX ou MMC usando alta cromatografia líquida (HPLC) sozinha ou em combinação com a espectrometria de massa (MS)26,,27,28 , 29 , 30 , 31 , 32 , 33 , 34. no entanto, estes métodos são muitas vezes demorado e impraticável para a terapia de combinação como um grande número de amostras biológicas precisa ser preparados separadamente para análise de múltiplas drogas (às vezes incluindo metabolitos de drogas). Além da ligação às proteínas plasmáticas forte de DOX e MMC, glóbulos vermelhos também têm uma grande capacidade de vincular e concentrar muitas drogas anticâncer35,36. Assim, a análise de plasma para DOX ou MMC pode ofuscar concentrações de droga de sangue real. O presente trabalho (Figura 1) descreve uma maneira simples e robusto método de análise de drogas múltiplas usando fase reversa HPLC para extrair simultaneamente e dosar DOX, MMC e o DOX metabólito doxorubicinol (DOXol) de sangue total e diversos tecidos ( por exemplo, tumores). Foi aplicado com sucesso para determinar a farmacocinética e bio-distribuição de DOX e MMC, bem como a formação de DOXol após a entrega da droga através de soluções livres ou formas de nanopartículas (i.e., DMPLN e DOX lipossomal) em um orthotopically implantou o modelo de rato da mama-tumor murino após a intravenosa (i.v.) injeção22.

<table style="border-collapse:collapse;width:635pt" width="846">
	<colgroup>
		<col style="width:188pt" width="250" />
		<col style="width:122pt" width="162" />
		<col style="width:89pt" width="119" />
		<col style="width:236pt" width="315" />
	</colgroup>
	<tbody>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">Doxorubicin&#160;</td>
			<td class="xl20" style="width:122pt" width="162">Polymed Theraeutics</td>
			<td class="xl20" style="width:89pt" width="119">111023</td>
			<td class="xl20" style="width:236pt" width="315">Anticancer drug</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">Mitomycin C</td>
			<td class="xl20" style="width:122pt" width="162">Polymed Theraeutics</td>
			<td class="xl20" style="width:89pt" width="119">060814</td>
			<td class="xl20" style="width:236pt" width="315">Anticancer drug</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl20" height="42" style="width:188pt;height:31.5pt" width="250">Doxorubicinol (DOXol)</td>
			<td class="xl20" style="width:122pt" width="162">Toronto Research Chemicals</td>
			<td class="xl20" style="width:89pt" width="119">D558020</td>
			<td class="xl21">Metabolite of DOX</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">4-Methylumbelliferone sodium salt&#160;</td>
			<td class="xl20" style="width:122pt" width="162">Sigma-Aldrich</td>
			<td class="xl20" style="width:89pt" width="119">M1508</td>
			<td class="xl21">Internal standard</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">Myristic Acid</td>
			<td class="xl20" style="width:122pt" width="162">Sigma-Aldrich</td>
			<td class="xl20" style="width:89pt" width="119">544-63-8&#160;&#160;</td>
			<td class="xl21">Materials for poly-lipid hybrid nanoparticles</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">Polyoxyethylene (100) Stearate</td>
			<td class="xl20" style="width:122pt" width="162">Spectrum</td>
			<td class="xl20" style="width:89pt" width="119">M1402</td>
			<td class="xl21">Materials for poly-lipid hybrid nanoparticles</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">Polyoxyethylene (40) Stearate</td>
			<td class="xl20" style="width:122pt" width="162">Sigma-Aldrich</td>
			<td class="xl20" style="width:89pt" width="119">P3440</td>
			<td class="xl21">Materials for poly-lipid hybrid nanoparticles</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">Pluronic F68 (PF68)</td>
			<td class="xl20" style="width:122pt" width="162">BASF Corp.</td>
			<td class="xl20" style="width:89pt" width="119">9003-11-6</td>
			<td class="xl21">Materials for poly-lipid hybrid nanoparticles</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl20" height="42" style="width:188pt;height:31.5pt" width="250">Ultrasonication (UP100H)</td>
			<td class="xl20" style="width:122pt" width="162">Hielscher, Ultrasound Technology</td>
			<td class="xl20" style="width:89pt" width="119">NA</td>
			<td class="xl21">Nanoparticle preparation</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">Water Bath (ISOTEMP 3016HS)</td>
			<td class="xl20" style="width:122pt" width="162">Fisher Scientific</td>
			<td class="xl20" style="width:89pt" width="119">NA</td>
			<td class="xl21">Nanoparticle preparation</td>
		</tr>
		<tr height="105" style="height:78.75pt">
			<td class="xl20" height="105" style="width:188pt;height:78.75pt" width="250">Liposomal Doxorubicin&#160; (Caelyx)</td>
			<td class="xl20" style="width:122pt" width="162">Janssen</td>
			<td class="xl20" style="width:89pt" width="119">Purchased from the pharmacy Princess Margaret Hospital</td>
			<td class="xl21">Clinically-approved nanoparticle formulation&#160;</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">HPLC-graded Methanol</td>
			<td class="xl21">Caledon Chemicals</td>
			<td class="xl20" style="width:89pt" width="119">6701-7-40</td>
			<td class="xl21">HPLC mobile phase composition</td>
		</tr>
		<tr height="25" style="height:18.75pt">
			<td class="xl20" height="25" style="width:188pt;height:18.75pt" width="250">HPLC-graded H2O</td>
			<td class="xl21">Caledon Chemicals</td>
			<td class="xl20" style="width:89pt" width="119">8801-7-40</td>
			<td class="xl21">HPLC mobile phase composition</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">HPLC-graded Acetonitrile&#160;</td>
			<td class="xl20" style="width:122pt" width="162">Caledon Chemicals</td>
			<td class="xl20" style="width:89pt" width="119">1401-7-40</td>
			<td class="xl21">HPLC mobile phase composition</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">Trifluoroacetic Acid</td>
			<td class="xl20" style="width:122pt" width="162">Sigma-Aldrich</td>
			<td class="xl20" style="width:89pt" width="119">302031</td>
			<td class="xl21">HPLC mobile phase composition</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl20" height="42" style="width:188pt;height:31.5pt" width="250">0.45 &#956;m Nylon Membrane Filter Paper</td>
			<td class="xl20" style="width:122pt" width="162">Whatman</td>
			<td class="xl20" style="width:89pt" width="119">WHA7404004</td>
			<td class="xl21">HPLC mobile phase preparation</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl29" height="42" style="height:31.5pt">1cc Plastic Syringes</td>
			<td class="xl20" style="width:122pt" width="162">Becton, Dickinson and Company</td>
			<td class="xl20" style="width:89pt" width="119">2606-309659</td>
			<td class="xl21">Treatment injection</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl29" height="42" style="height:31.5pt">5cc Plastic Syringes</td>
			<td class="xl20" style="width:122pt" width="162">Becton, Dickinson and Company</td>
			<td class="xl20" style="width:89pt" width="119">2608-309646</td>
			<td class="xl21">Tissue collections</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl20" height="42" style="width:188pt;height:31.5pt" width="250">30G 1/2 Needles</td>
			<td class="xl20" style="width:122pt" width="162">Becton, Dickinson and Company</td>
			<td class="xl20" style="width:89pt" width="119">305106</td>
			<td class="xl21">Treatment injection</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl20" height="42" style="width:188pt;height:31.5pt" width="250">25G 5/8 Needles</td>
			<td class="xl20" style="width:122pt" width="162">Becton, Dickinson and Company</td>
			<td class="xl20" style="width:89pt" width="119">305122</td>
			<td class="xl21">Tissue collections</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl20" height="42" style="width:188pt;height:31.5pt" width="250">Sterile 0.9% Saline</td>
			<td class="xl20" style="width:122pt" width="162">Univeristy of Toronto House Brand</td>
			<td class="xl20" style="width:89pt" width="119">1011</td>
			<td class="xl21">Tissue perfusion</td>
		</tr>
		<tr height="22" style="height:16.5pt">
			<td class="xl20" height="22" style="width:188pt;height:16.5pt" width="250">13 ml Rounded-bottom conical tube&#160;</td>
			<td class="xl20" style="width:122pt" width="162">SARSTEDT</td>
			<td class="xl20" style="width:89pt" width="119">62.515.006</td>
			<td class="xl21">Prolyprolene, tissue homogenization</td>
		</tr>
		<tr height="23" style="height:17.25pt">
			<td class="xl30" height="23" style="height:17.25pt">Alpha Minimum Essential Medium (MEM)&#160;</td>
			<td class="xl27">Gibco</td>
			<td class="xl28" style="width:89pt" width="119">12571063</td>
			<td class="xl21">Cell medium</td>
		</tr>
		<tr height="22" style="height:16.5pt">
			<td class="xl20" height="22" style="width:188pt;height:16.5pt" width="250">1 x Phosphate Buffer Saline</td>
			<td class="xl20" style="width:122pt" width="162">Gibco</td>
			<td class="xl20" style="width:89pt" width="119">10010023</td>
			<td class="xl21">Tissue homogenization</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">Triton X-100</td>
			<td class="xl20" style="width:122pt" width="162">Sigma-Aldrich</td>
			<td class="xl20" style="width:89pt" width="119">X100-100 ML</td>
			<td class="xl21">Tissue homogenization</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">Formic acid</td>
			<td class="xl20" style="width:122pt" width="162">Caledon Chemicals</td>
			<td class="xl20" style="width:89pt" width="119">1/5/3840</td>
			<td class="xl21">Adjust pH for extraction solvent</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl20" height="42" style="width:188pt;height:31.5pt" width="250">Sodium heparin sprayed plastic tubes</td>
			<td class="xl20" style="width:122pt" width="162">Becton, Dickinson and Company</td>
			<td class="xl20" style="width:89pt" width="119">367878</td>
			<td class="xl21">Blood collection</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">Analytical Weigh Balance&#160;</td>
			<td class="xl20" style="width:122pt" width="162">Sartorius&#160;</td>
			<td class="xl21">CPA225D</td>
			<td class="xl21">NA</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">pH meters&#160;</td>
			<td class="xl20" style="width:122pt" width="162">Fisher Scientific</td>
			<td class="xl26">13-637-671</td>
			<td class="xl21">accumet BASIC</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">Vortex Mixter</td>
			<td class="xl20" style="width:122pt" width="162">Fisher Scientific</td>
			<td class="xl20" style="width:89pt" width="119">02-215-365</td>
			<td class="xl21">Vortexing samples at desired speed</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">1.5 ml&#160; Microcentrifuge Tube</td>
			<td class="xl20" style="width:122pt" width="162">Fisherbrand</td>
			<td class="xl20" style="width:89pt" width="119">2043-05408129</td>
			<td class="xl21">Prolyprolene</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">Model 1000 homogenizer</td>
			<td class="xl22">Fisher Scientific</td>
			<td class="xl20" style="width:89pt" width="119">08-451-672</td>
			<td class="xl21">Tissue homogenization</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">Centrifuge 5702R</td>
			<td class="xl20" style="width:122pt" width="162">Eppendorf</td>
			<td class="xl20" style="width:89pt" width="119">5702R</td>
			<td class="xl21">Extraction preparation</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">Heated Evaporator System</td>
			<td class="xl20" style="width:122pt" width="162">Glas-Col</td>
			<td class="xl20" style="width:89pt" width="119">NA</td>
			<td class="xl21">Sample reconstitution</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">HPLC Screw Thread Vials</td>
			<td class="xl20" style="width:122pt" width="162">DIKMA</td>
			<td class="xl20" style="width:89pt" width="119">5320</td>
			<td class="xl21">HPLC sample injection</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl20" height="42" style="width:188pt;height:31.5pt" width="250">HPLC Screw Caps with PTFE White Silicone Septa</td>
			<td class="xl20" style="width:122pt" width="162">DIKMA</td>
			<td class="xl20" style="width:89pt" width="119">5325</td>
			<td class="xl21">HPLC sample injection</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">HPLC Polypropylene Insert&#160;&#160;</td>
			<td class="xl20" style="width:122pt" width="162">Agilent Technologies</td>
			<td class="xl20" style="width:89pt" width="119">5182-0549</td>
			<td class="xl21">Maximum volume 250 &#956;l, HPLC sample injection</td>
		</tr>
		<tr height="25" style="height:18.75pt">
			<td class="xl20" height="25" style="width:188pt;height:18.75pt" width="250">Xbridge C18 Column</td>
			<td class="xl23">Waters Corporation</td>
			<td class="xl20" style="width:89pt" width="119">186003117</td>
			<td class="xl21">Drug analysis</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">Gradient pump&#160;</td>
			<td class="xl23">Waters Corporation</td>
			<td class="xl20" style="width:89pt" width="119">W600</td>
			<td class="xl21">Drug analysis</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">Auto-sampler</td>
			<td class="xl23">Waters Corporation</td>
			<td class="xl20" style="width:89pt" width="119">W2707</td>
			<td class="xl21">Drug analysis</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">Photodiode array detector&#160;</td>
			<td class="xl23">Waters Corporation</td>
			<td class="xl20" style="width:89pt" width="119">W2998</td>
			<td class="xl21">Drug analysis</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">Multi &#955; fluoresence detector&#160;</td>
			<td class="xl23">Waters Corporation</td>
			<td class="xl20" style="width:89pt" width="119">W2475</td>
			<td class="xl21">Drug analysis</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">EMPOWER 2</td>
			<td class="xl23">Waters Corporation</td>
			<td class="xl20" style="width:89pt" width="119">NA</td>
			<td class="xl21">Data analysis software</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">Scientist</td>
			<td class="xl20" style="width:122pt" width="162">Micromath</td>
			<td class="xl20" style="width:89pt" width="119">NA</td>
			<td class="xl21">Pharmacokinetic analysis</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">Female Balb/c Mice</td>
			<td class="xl20" style="width:122pt" width="162">Jackson Laboratory</td>
			<td class="xl20" style="width:89pt" width="119">001026</td>
			<td class="xl21">In vivo</td>
		</tr>
		<tr height="63" style="height:47.25pt">
			<td class="xl20" height="63" style="width:188pt;height:47.25pt" width="250">EMT6/WT Breast Cancer Cells</td>
			<td class="xl20" style="width:122pt" width="162">Provided by Dr. Ian Tannock; Ontario Cancer Institute</td>
			<td class="xl20" style="width:89pt" width="119">NA</td>
			<td class="xl21">In vivo</td>
		</tr>
	</tbody>
</table>

sample extraction and simultaneous chromatographic quantitation of doxorubicin and mitomycin c following drug combination delivery in nanoparticles to tumor-bearing mice

Quimioterapia de combinação é frequentemente usada na clínica para tratamento de câncer; no entanto, os efeitos adversos associados ao tecido normal podem limitar seu benefício terapêutico. Combinação de drogas baseados em nanopartículas foi mostrada para atenuar os problemas encontrados pela terapia de combinação livre de drogas. Nossos estudos anteriores têm demonstrado que a combinação de duas drogas anticâncer, doxorrubicina (DOX) e mitomicina C (MMC), produziu um efeito sinérgico contra ambos murino e células de câncer de mama humano em vitro. Nanopartículas de híbrido de polímero carregado co-lipídico DOX e MMC (DMPLN) através de várias bombas de transportador de efluxo que conferem multirresistência e demonstrada eficácia reforçada em modelos de tumor de mama. Em comparação com formas de solução convencional, tal eficácia superior de DMPLN foi atribuída à farmacocinética sincronizada de DOX e MMC e biodisponibilidade de aumento intracelular de drogas dentro de células de tumor habilitado pelo nanocarreador PLN.
Para avaliar a farmacocinética e bio-distribuição de co administrado DOX e MMC em solução livre e formas de nanopartículas, um método de análise de múltiplas drogas simples e eficiente usando reverso-fase de alto desempenho foi de cromatografia líquida (HPLC) desenvolvido. Em contraste com os métodos anteriormente relatados que analisaram DOX ou MMC individualmente no plasma, esse novo método HPLC é capaz de dosar simultaneamente DOX, MMC e um metabolito principal de DOX cardio-tóxico, doxorubicinol (DOXol), em diferentes matrizes biológicas ( por exemplo, sangue total, tumor de mama e coração). Uma dupla sonda fluorescente e ultravioleta de absorvente 4-methylumbelliferone (4-MU) foi usado como um padrão interno (I.S.) para a deteção de uma etapa de análise de drogas múltiplas com comprimentos de onda diferentes da deteção. Este método foi aplicado com sucesso para determinar as concentrações de DOX e MMC entregado por abordagens tanto nanopartículas e solução no sangue e vários tecidos em modelo murino de tumor de mama um ortotópico. O método analítico apresentado é uma ferramenta útil para a análise pré-clínico de entrega baseados em nanopartículas de combinações de drogas.

Duas drogas anticâncer, DOX e MMC, bem como o metabólito DOX, DOXol, foram detectado simultaneamente sem qualquer interferência biológica sob a condição HPLC aplicada mesma gradiente usando 4-MU como o I.S. para os detectores de UV e fluorescência. DOX, MMC, DOXol e 4-MU foram bem separados uns dos outros com tempos de retenção de 5,7 min para MMC, 10,4 min para DOXol, 10,9 min para 4-MU e 11,1 min para DOX (Figura 2). Cada fármaco no sangue e vári...

Watch this Scientific Journal Video about Sample Extraction and Simultaneous Chromatographic Quantitation of Doxorubicin and Mitomycin C Following Drug Combination Delivery in Nanoparticles to Tumor-b

Sample Extraction and Simultaneous Chromatographic Quantitation of Doxorubicin and Mitomycin C Following Drug Combination Delivery in Nanoparticles to Tumor-bearing Mice

Este protocolo descreve um processo analítico eficiente e conveniente de extração de amostra e determinação simultânea de múltiplas drogas, doxorrubicina (DOX), mitomicina C (MMC) e um metabólito tóxico cardio DOX, doxorubicinol (DOXol), no biológico amostras de um modelo de tumor de mama pré-clínico tratadocom com formulações de nanopartículas de combinação sinérgica de drogas.

Extração de amostra e quantificação cromatográfica simultânea de doxorrubicina e mitomicina C após entrega de combinação de drogas em nanopartículas para ratos de Tumor-rolamento

Combination chemotherapy is frequently used in the clinic for cancer treatment; however, associated adverse effects to normal tissue may limit its ...

sample-extraction-simultaneous-chromatographic-quantitation

Research

Education

JoVE Core

JoVE Journal

Pharmacokinetics and Pharmacodynamics

Cancer Research

Unit 1

Pharmacokinetic Models

SCIENTISTS IN ACTION

10.8K visualizações.  University of Toronto. Este protocolo descreve um processo analítico eficiente e conveniente de extração de amostra e determinação simultânea de múltiplas drogas, doxorrubicina (DOX), mitomicina C (MMC) e um metabólito tóxico cardio DOX, doxorubicinol (DOXol), no biológico amostras de um modelo de tumor de mama pré-clínico tratadocom com formulações de nanopartículas de combinação sinérgica de drogas.

Vídeo: Sample Extraction and Simultaneous Chromatographic Quantitation of Doxorubicin and Mitomycin C Following Drug Combination Delivery in Nanoparticles to Tumor-bearing Mice

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance can be caused by a range of mechanisms, including increased drug elimination, decreased drug uptake, drug inactivation and alterations of drug targets. Recent data showed that other than by well-known genetic (mutation, amplification) and epigenetic (DNA hypermethylation, histone post-translational modification) modifications, drug resistance mechanisms might also be regulated by splicing aberrations. This is a rapidly growing field of investigation that deserves future attention in order to plan more effective therapeutic approaches. The protocol described in this paper is aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of several in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes. In particular, we evaluated the differential splicing of DDX5 and PKM transcripts. The aberrant splicing detected by the computational tool MATS was validated in leukemic cells, showing that different DDX5 splice variants are expressed in the parental vs. resistant cells. In these cells, we also observed a higher PKM2/PKM1 ratio, which was not detected in the Panc-1 gemcitabine-resistant counterpart compared to parental Panc-1 cells, suggesting a different mechanism of drug-resistance induced by gemcitabine exposure.

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Here we describe a protocol aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of parental and resistant in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes.

Usando RNA-sequenciação para detectar Novel Splice variantes relacionadas à resistência aos medicamentos em<em&gt; In Vitro</em&gt; modelos de cancro

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance ...

Pesquisa do câncer

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.

O Colon-26 Carcinoma Tumor-rolamento Mouse como um modelo para o estudo do câncer caquexia

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

Potentiation of Anticancer Antibody Efficacy by Antineoplastic Drugs: Detection of Antibody-drug Synergism Using the Combination Index Equation

Potentiation of hostile monoclonal antibodies (mAb) by chemotherapeutic agents constitutes a valuable strategy for designing effective and safer therapy against cancer. Here we provide a protocol to identify a rational combination at the preclinical step. First, we describe a cell-based assay to assess the synergism between anticancer mAb and cytotoxic drugs, that uses the combination index equation of Chou and Talalay1. This includes the measurement of tumor cell drug- and antibody-sensitivity using an MTT assay, followed by an automated computer analysis to calculate the combination index (CI) values. CI values of &lt;1 indicate synergism between tested mAbs and cytotoxic agents1. To corroborate the in vitro findings in vivo, we further describe a method to assess the combination regimen efficacy in a xenograft tumor model. In this model, the combined regimen significantly delays tumor growth, which results in a significant extended survival in comparison to single-agent controls. Importantly, the in vivo experimentation reveals that the combination regimen is well tolerated. This protocol allows the effective evaluation of anticancer drug combinations in preclinical models and the identification of rational combination to evaluate in clinical trials.

This protocol describes how to assess synergism between an anticancer antibody and antineoplastic drugs in preclinical models by using the combination index equation of Chou and Talalay.

Potenciação da eficácia de anticorpos anticâncer por fármacos antineoplásicos: deteção de sinergismo de anticorpo-droga usando a equação de índice de combinação

Potentiation of hostile monoclonal antibodies (mAb) by chemotherapeutic agents constitutes a valuable strategy for designing effective and safer therapy ...

Transfer of Manipulated Tumor-associated Neutrophils into Tumor-Bearing Mice to Study their Angiogenic Potential In Vivo

The contribution of neutrophils to the regulation of tumorigenesis is getting increased attention. These cells are heterogeneous, and depending on the tumor milieu can possess pro- or anti-tumor capacity. One of the important cytokines regulating neutrophil functions in a tumor context are type I interferons. In the presence of interferons, neutrophils gain anti-tumor properties, including cytotoxicity or stimulation of the immune system. Conversely, the absence of an interferon signaling results in prominent pro-tumor activity, characterized with strong stimulation of tumor angiogenesis. Recently, we could demonstrate that pro-angiogenic properties of neutrophils depend on the activation of nicotinamide phosphoribosyltransferase (NAMPT) signaling pathway in these cells. Inhibition of this pathway in tumor-associated neutrophils leads to their potent anti-angiogenic phenotype. Here, we demonstrate our newly established model allowing in vivo evaluation of tumorigenic potential of manipulated tumor-associated neutrophils (TANs). Shortly, pro-angiogenic tumor-associated neutrophils can be isolated from tumor-bearing interferon-deficient mice and repolarized into anti-angiogenic phenotype by blocking of NAMPT signaling. The angiogenic activity of these cells can be subsequently evaluated using an aortic ring assay. Anti-angiogenic TANs can be transferred into tumor-bearing wild type recipients and tumor growth should be monitored for 14 days. At day 14 mice are sacrificed, tumors removed and cut with their vascularization assessed. Overall, our protocol provides a novel tool to in vivo evaluate angiogenic capacity of primary cells, such as tumor-associated neutrophils, without a need to use artificial neutrophil cell line models. vc

Here, we show therapeutic potential of anti-angiogenic tumor-associated neutrophils after their transfer into tumor-bearing mice. This protocol can be used to manipulate neutrophil activity ex vivo and to subsequently evaluate their functionality in vivo&#160;in developing tumors. It is an appropriate model for studying potential neutrophil-based immunotherapies.

Transferência de neutrophils tumor-associados manipulados em ratos do tumor-rolamento para estudar seu potencial angiogênico em vivo

The contribution of neutrophils to the regulation of tumorigenesis is getting increased attention. These cells are heterogeneous, and depending on the ...

In Vivo Imaging and Quantitation of the Host Angiogenic Response in Zebrafish Tumor Xenografts

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A zebrafish xenograft is created by implanting mammalian tumor cells into the perivitelline space of two days-post-fertilization zebrafish embryos, followed by measuring the extent of the angiogenic response observed at an experimental endpoint up to two days post-implantation. The key advantage to this method is the ability to accurately quantitate the zebrafish host angiogenic response to the graft. This enables detailed examination of the molecular mechanisms as well as the host vs tumor contribution to the angiogenic response. The xenografted embryos can be subjected to a variety of treatments, such as incubation with potential anti-angiogenesis drugs, in order to investigate strategies to inhibit tumor angiogenesis. The angiogenic response can also be live-imaged in order to examine more dynamic cellular processes. The relatively undemanding experimental technique, cheap maintenance costs of zebrafish and short experimental timeline make this model especially useful for the development of strategies to manipulate tumor angiogenesis.

The aim of this method is to generate an in vivo model of tumor angiogenesis by xenografting mammalian tumor cells into a zebrafish embryo that has fluorescently-labelled blood vessels. By imaging the xenograft and associated vessels, a quantitative measurement of the angiogenic response can be obtained.

In vivo Imaging e Quantitation da resposta angiogênica hospedeira em xenoenxertos de tumor de zebrafish

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A ...

Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1&#945; Nanoparticles

Cytokine therapy is a promising immunotherapeutic strategy that can produce robust antitumor immune responses in cancer patients. The proinflammatory cytokine interleukin-1 alpha (IL-1&#945;) has been evaluated as an anticancer agent in several preclinical and clinical studies. However, dose-limiting toxicities, including flu-like symptoms and hypotension, have dampened the enthusiasm for this therapeutic strategy. Polyanhydride nanoparticle (NP)-based delivery of IL-1&#945; would represent an effective approach in this context since this may allow for a slow and controlled release of IL-1&#945; systemically while reducing toxic side effects. Here an analysis of the antitumor activity of IL-1&#945;-loaded polyanhydride NPs in a head and neck squamous cell carcinoma (HNSCC) syngeneic mouse model is described. Murine oropharyngeal epithelial cells stably expressing HPV16 E6/E7 together with hRAS and luciferase (mEERL) cells were injected subcutaneously into the right flank of C57BL/6J mice. Once tumors reached 3-4 mm in any direction, a 1.5% IL-1a - loaded 20:80 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane:1,6-bis(p-carboxyphenoxy)hexane (CPTEG: CPH) nanoparticle (IL-1&#945;-NP) formulation was administered to mice intraperitoneally. Tumor size and body weight were continuously measured until tumor size or weight loss reached euthanasia criteria. Blood samples were taken to evaluate antitumor immune responses by submandibular venipuncture, and inflammatory cytokines were measured through cytokine multiplex assays. Tumor and inguinal lymph nodes were resected and homogenized into a single-cell suspension to analyze various immune cells through multicolor flow cytometry. These standard methods will allow investigators to study the antitumor immune response and potential mechanism of immunostimulatory NPs and other immunotherapy agents for cancer treatment.

Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1&amp;#945; Nanoparticles

A standard protocol is described to study the antitumor activity and associated toxicity of IL-1&#945; in a syngeneic mouse model of HNSCC.

Avaliação da Atividade Antitumoral In vivo de Nanopartículas de Polianidrido IL-1α

Cytokine therapy is a promising immunotherapeutic strategy that can produce robust antitumor immune responses in cancer patients. The proinflammatory ...

Exploring Mitochondrial Energy Metabolism of Single 3D Microtissue Spheroids Using Extracellular Flux Analysis

Three-dimensional (3D) cellular aggregates, termed spheroids, have become the forefront of in vitro cell culture in recent years. In contrast to culturing cells as two-dimensional, single-cell monolayers (2D culture), spheroid cell culture promotes, regulates, and supports physiological cellular architecture and characteristics that exist in vivo, including the expression of extracellular matrix proteins, cell signaling, gene expression, protein production, differentiation, and proliferation. The importance of 3D culture has been recognized in many research fields, including oncology, diabetes, stem cell biology, and tissue engineering. Over the last decade, improved methods have been developed to produce spheroids and assess their metabolic function and fate.
Extracellular flux (XF) analyzers have been used to explore mitochondrial function in 3D microtissues such as spheroids using either an XF24 islet capture plate or an XFe96 spheroid microplate. However, distinct protocols and the optimization of probing mitochondrial energy metabolism in spheroids using XF technology have not been described in detail. This paper provides detailed protocols for probing mitochondrial energy metabolism in single 3D spheroids using spheroid microplates with the XFe96 XF analyzer. Using different cancer cell lines, XF technology is demonstrated to be capable of distinguishing between cellular respiration in 3D spheroids of not only different sizes but also different volumes, cell numbers,&#160;DNA content and type.
The optimal mitochondrial effector compound concentrations of oligomycin, BAM15, rotenone, and antimycin A are used to probe specific parameters of mitochondrial energy metabolism in 3D spheroids. This paper also discusses methods to normalize data obtained from spheroids and addresses many considerations that should be considered when exploring spheroid metabolism using XF technology. This protocol will help drive research in advanced in vitro spheroid models.

These protocols will help users probe mitochondrial energy metabolism in 3D cancer cell-line-derived spheroids using Seahorse extracellular flux analysis.

Explorando o metabolismo de energia mitocondrial de spheróides microtissue 3D únicos usando análise de fluxo extracelular

Three-dimensional (3D) cellular aggregates, termed spheroids, have become the forefront of in vitro cell culture in recent years. In contrast to culturing ...

Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, senescence is a fundamentally different machinery restraining&#160;propagation of cancer cells. Decades of scientific studies have revealed the complex pathological effects of senescent cancer cells in tumors and microenvironments that modulate cancer cells and stromal cells. New evidence suggests that senescence is a potent prognostic factor during cancer treatment, and therefore rapid and accurate detection of senescent cells in cancer samples is essential. This paper presents a method to visualize and detect therapy-induced senescence (TIS) in cancer cells. Diffuse large B-cell lymphoma (DLBCL) cell lines were treated with mafosfamide (MAF) or daunorubicin (DN) and examined for the senescence marker, senescence-associated &#946;-galactosidase (SA-&#946;-gal), the DNA synthesis marker 5-ethynyl-2&#8242;-deoxyuridine (EdU), and the DNA damage marker gamma-H2AX (&#947;H2AX). Flow cytometer imaging can help generate high-resolution single-cell images in a short period of time to simultaneously visualize and quantify the three markers in cancer cells.

Here we present a flow cytometry-based method for visualization and quantification of multiple senescence-associated markers in single cells.

Detecção simultânea baseada em imagens e flutometria de marcadores de senescência fluorescentes múltiplas em células cancerígenas senescentes induzidas por terapia

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, ...

Non-Destructive Evaluation of Regional Cell Density Within Tumor Aggregates Following Drug Treatment

Multicellular tumor spheroid (MCTSs) models have demonstrated increasing utility for in vitro study of cancer progression and drug discovery. These relatively simple avascular constructs mimic key aspects of in vivo tumors, such as 3D structure and pathophysiological gradients. MCTSs models can provide insights into cancer cell behavior during spheroid development and in response to drugs; however, their requisite size drastically limits the tools used for non-destructive assessment. Optical Coherence Tomography structural imaging and Imaris 3D analysis software are explored for rapid, non-destructive, and label-free measurement of regional cell density within MCTSs. This approach is utilized to assess MCTSs over a 4-day maturation period and throughout an extended 5-day treatment with Trastuzumab, a clinically relevant anti-HER2 drug. Briefly, AU565 HER2+ breast cancer MCTSs were created via liquid overlay with or without the addition of Matrigel (a basement membrane matrix) to explore aggregates of different morphologies (thicker, disk-like 2.5D aggregates or flat 2D aggregates, respectively). Cell density within the outer region, transitional region, and inner core was characterized in matured MCTSs, revealing a cell-density gradient with higher cell densities in core regions compared to outer layers. The matrix addition redistributed cell density and enhanced this gradient, decreasing outer zone density and increasing cell compaction in the cores. Cell density was quantified following drug treatment (0 h, 24 h, 5 days) within progressively deeper 100 &#181;m zones to assess potential regional differences in drug response. By the final timepoint, nearly all cell death appeared to be constrained to the outer 200 &#181;m of each aggregate, while cells deeper in the aggregate appeared largely unaffected, illustrating regional differences in the drug response, possibly due to limitations in drug penetration. The current protocol provides a unique technique to non-destructively quantify regional cell density within dense cellular tissues and measure it longitudinally.

The present protocol develops an image-based technique for rapid, non-destructive, and label-free regional cell density and viability measurement within 3D tumor aggregates. Findings revealed a cell-density gradient, with higher cell densities in core regions than outer layers in developing aggregates and predominantly peripheral cell death in HER2+ aggregates treated with Trastuzumab.

Avaliação não destrutiva da densidade celular regional dentro dos agregados tumorais após o tratamento medicamentoso

Multicellular tumor spheroid (MCTSs) models have demonstrated increasing utility for in vitro study of cancer progression and drug discovery. These ...

Quantificando o comprimento dos telômeros usando detecção de quimioluminescência não radioativa

Optimization of Performance Parameters of the TAGGG Telomere Length Assay

Telomeres are repetitive sequences which are present at chromosomal ends; their shortening is a characteristic feature of human somatic cells. Shortening occurs due to a problem with end replication and the absence of the telomerase enzyme, which is responsible for maintaining telomere length. Interestingly, telomeres also shorten in response to various internal physiological processes, like oxidative stress and inflammation, which may be impacted due to extracellular agents like pollutants, infectious agents, nutrients, or radiation. Thus, telomere length serves as an excellent biomarker of aging and various physiological health parameters. The TAGGG telomere length assay kit is used to quantify average telomere lengths using the telomere restriction fragment (TRF) assay and is highly reproducible. However, it is an expensive method, and because of this, it is not employed routinely for large sample numbers. Here, we describe a detailed protocol for an optimized and cost-effective measurement of telomere length using Southern blots or TRF analysis and non-radioactive chemiluminescence-based detection.

Autor em destaque: Otimização dos parâmetros de desempenho do ensaio de comprimento dos telômeros TAGGG  

Here, we describe in detail the protocol for quantifying telomere length using non-radioactive chemiluminescent detection, with a focus on the optimization of various performance parameters of the TAGGG telomere length assay kit, such as buffer quantities and probe concentrations.

Otimização dos parâmetros de desempenho do ensaio de comprimento telomérico TAGGG

Telomeres are repetitive sequences which are present at chromosomal ends; their shortening is a characteristic feature of human somatic cells. Shortening ...