Este trabalho foi apoiado pelo Will e Jeanne Caldwell Doowed Research Fund do Centro de Câncer Integral da Universidade de Michigan e, em parte, pela nih grant CA82529 e pela Gillson Longenbaugh Foundation.

1. Coloração ALDEFLUOR
<ol><li>Preparação de amostras celulares: Preparar suspensão celular única de células tumorais, seja de células tumorais cultivadas ou de amostras de tumores recém-colhidas. Conte as células e ajuste a suspensão celular a uma concentração de 1 x 106 células/ml no tampão de ensaio ALDEFLUOR.</li>
	<li>Rotular quatro tubos de teste de poliestireno de 12 mm x 75 mm como tubos de controle da seguinte forma: #1: Não manchado, #2: ALDEFLUOR, #3: ALDEFLUOR mais DEAB, e #4: 7AAD.</li>
	<li>Para estes tubos de controle, coloque a suspensão celular de 1ml em #1 e #4, mas 2 ml em tubos #2. Adicione 5 μl DEAB (inibidor de ALDH) no tubo #3 e mantenha no gelo. Em seguida, adicione 10 μl ativado substrato ALDEFLUOR ao tubo #2, misture e transfira imediatamente 1 ml da mistura para #3 do tubo.</li>
	<li>Ao mesmo tempo, adicione 2 μl ativado substrato ALDEFLUOR por milhão de células ao resto das células (tubos de amostra) também em 1 x 106 células/ml no Tampão de Ensaio ALDEFLUOR.</li>
	<li>Incubar tanto os 4 tubos de controle quanto os tubos de amostra por 30 minutos em banho-maria de 37 °C.</li>
	<li>Após a incubação, adicione 5 μl 7AAD no tubo de controle #4 e 1 μl 7AAD 1 x 106 células ao tubo de amostra. Incubar 10 min a 4 °C.</li>
	<li>Centrifugar todos os tubos por 5 min a 250 x g e remover supernaspe.</li>
	<li>Células resuspend em ALDEFLUOR Assay Buffer.</li>
	<li>Realize a classificação baseada em citometria de fluxo.</li>
	<li>Defina os portões de triagem usando células manchadas de ALDEFLUOR tratadas com DEAB como controle negativo e as células manchadas de propidium (PI) 7AAD para controle de viabilidade. Com base nesses controles, foi estabelecido um portão para distinguir as células ALDEFLUOR+/ALDHalta,D5 e SCC7. Esses portões serão então usados para classificar todas as células amostrais preparadas acima para as células ALDEFLUOR+/ALDHhigh D5 e SCC7.</li>
</ol>2. Preparação da vacina célula dendrítica de células-tronco-tronco do câncer (CSC-TPDC)
<ol><li>Eutanize camundongos (síngênicos B6 e C3H, respectivamente) usando CO2 e isolam ossos de fêmur e tíbia.</li>
	<li>Coloque os ossos em 75% de etanol por 1 min a temperatura ambiente. Em seguida, lave os ossos usando HBSS.</li>
	<li>Corte a cabeça dos ossos e use uma agulha de 21 G e uma seringa de 10 ml para empurrar as células em um prato.</li>
	<li>Aspire e assopre as células usando a seringa para fazer a suspensão de célula única.</li>
	<li>Após centrifugação em 1.500 rpm por 5 min, descarte o supernasce. Em seguida, adicione 5 ml de tampão de lise RBC à lise do RBC por 1 min em banho de água de 37 °C.</li>
	<li>Conte o número da célula e ajuste a suspensão celular para uma concentração de 1 milhão de células/ml em meio completo (CM) contendo 10 ng/ml GM-CSF e 10 ng/ml IL-4.</li>
	<li>Cultume as células e compense CM mais IL-4 e GM-CSF 3 dias depois.</li>
	<li>No5º dia, colhe as células dendríticas imaturas (DCs) e prepare o meio de isolamento DC contendo 4,2 ml de solução C mais 1 ml de gradiente de densidade OptiPrep.</li>
	<li>Suspenda as pelotas de célula em 5 ml CM. Adicione a suspensão da célula ao meio de isolamento lentamente. Centrífuga a 2.000 rpm à temperatura ambiente.</li>
	<li>Colete os DCs entre o CM e o meio de isolamento. Conte o número celular e ajuste a suspensão celular para uma concentração de 1 milhão de células/ml em meio de cultura contendo 10 ng/ml GM-CSF e 10 ng/ml IL-4.</li>
	<li>Prepare as células de tumores suspendendo as células D5 ou SCC7altas ou não sortidas de ALDH em meio de cultura e sujeitas a rápidas exposições de congelamento quatro vezes seguidas de giro em ∼100 x g por 5 min para coletar a porção de membrana dos lises.</li>
	<li>Para preparar csc-tpdc, pulso DCs com o lysate de célulasaltas ALDH autólogas. Para preparar h-TPDC, pulso DCs com linsato heterogêneo de células tumorais não variadas. A razão entre DC e células tumorais é a mesma.</li>
</ol>3. Vacinação e Avaliação da Eficácia
<ol><li>Vacinar camundongos B6 normais, respectivamente, com 2.500 células tumorais D5 CSC-TPDC ou D5 H-TPDC subcutânea(s.c). Vacinar animais C3H normais.c. com 5.000 células tumorais CSC-TPDC ou SCC7 H-TPDC, respectivamente.</li>
	<li>Após a vacinação, desafie os camundongos B6 com as células tumorais heterogêneas D5 i.v. Eutanize os camundongos usando CO2 20 dias depois. Colher os pulmões e enumerar metástases pulmonares.</li>
	<li>No modelo SCC7, desafie os camundongos C3H com células tumorais SCC7 não sortidas.c do lado oposto da vacina DC. Monitore o tamanho do tumor.</li>
	<li>No final dos experimentos eutanize os camundongos B6 e C3H usando CO2. No dia 34 após a primeira vacina, eutanize camundongos com CO2, e ao mesmo tempo colete os baços de cada grupo com um procedimento asséptico.</li>
	<li>Ative o baço células T e/ou B com anti-CD3 imobilizado mais anti-CD28 mAbs em CM contendo hrIL-2 ou LPS mais mAb anti-CD40 (FGK45).</li>
	<li>Após a ativação, colete supernacantes e use para ensaio ELISA.</li>
	<li>Para ensaio ELISA, cubra placas com anticorpos para IFNγ, GM-CSF e IgG a 4 °C O/N.</li>
	<li>Após a aplicação do buffer de bloqueio, adicione amostras e padrões e incubar à temperatura ambiente.</li>
	<li>Lave a placa e adicione anticorpo de detecção HRP e substrato TMB para incubação.</li>
	<li>Meça a absorvância em um leitor de placa ELISA em 450 nm dentro de 30 minutos após parar a reação.</li>
</ol>

<ol>
	<li>Shafee, N., Smith, C. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cells+contribute+to+cisplatin+resistance+in+Brca1/p53-mediated+mouse+mammary+tumors.">Cancer stem cells contribute to cisplatin resistance in Brca1/p53-mediated mouse mammary tumors.</a> Cancer Res. 68, 3243-3250 (2008).</li><li>Nandi, S., Ulasov, I. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-dose+radiation+enhances+survivin-mediated+virotherapy+against+malignant+glioma+stem+cells.">Low-dose radiation enhances survivin-mediated virotherapy against malignant glioma stem cells.</a> Cancer Res. 68, 5778-5784 (2008).</li><li>Dallas, N. A., Xia, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemoresistant+colorectal+cancer+cells,+the+cancer+stem+cell+phenotype,+and+increased+sensitivity+to+insulin-like+growth+factor-I+receptor+inhibition.">Chemoresistant colorectal cancer cells, the cancer stem cell phenotype, and increased sensitivity to insulin-like growth factor-I receptor inhibition.</a> Cancer Res. 69, 1951-1957 (2009).</li><li>Yamauchi, K., Yang, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+cancer+metastasis+by+cyclophosphamide+pretreatment+of+host+mice:+an+opposite+effect+of+chemotherapy.">Induction of cancer metastasis by cyclophosphamide pretreatment of host mice: an opposite effect of chemotherapy.</a> Cancer Res. 68, 516-520 (2008).</li><li>Chang, A. E., Redman, B. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phase+I+trial+of+tumor+lysate-pulsed+dendritic+cells+in+the+treatment+of+advanced+cancer.">A phase I trial of tumor lysate-pulsed dendritic cells in the treatment of advanced cancer.</a> Clin. Cancer Res. 8, 1021-1032 (2002).</li><li>Ito, F., Li, Q., Shreiner, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-CD137+monoclonal+antibody+administration+augments+the+antitumor+efficacy+of+dendritic+cell-based+vaccines.">Anti-CD137 monoclonal antibody administration augments the antitumor efficacy of dendritic cell-based vaccines.</a> Cancer Res. 64, 8411-8419 (2004).</li><li>Kirk, C. J., Hartigan-O'Connor, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell-dependent+antitumor+immunity+mediated+by+secondary+lymphoid+tissue+chemokine:+augmentation+of+dendritic+cell-based+immunotherapy.">T cell-dependent antitumor immunity mediated by secondary lymphoid tissue chemokine: augmentation of dendritic cell-based immunotherapy.</a> Cancer Res. 61, 2062-2070 (2001).</li><li>Ning, N., Pan, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cell+vaccination+confers+significant+antitumor+immunity.">Cancer stem cell vaccination confers significant antitumor immunity.</a> Cancer Res. 72, 1853-1864 (2012).</li><li>Ginestier, C., Liu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CXCR1+blockade+selectively+targets+human+breast+cancer+stem+cells+in+vitro+and+in+xenografts.">CXCR1 blockade selectively targets human breast cancer stem cells in vitro and in xenografts.</a> J. Clin. Invest. 120, 485-497 (2010).</li><li>Carpentino, J. E., Hynes, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aldehyde+dehydrogenase-expressing+colon+stem+cells+contribute+to+tumorigenesis+in+the+transition+from+colitis+to+cancer.">Aldehyde dehydrogenase-expressing colon stem cells contribute to tumorigenesis in the transition from colitis to cancer.</a> Cancer Res. 69, 8208-8215 (2009).</li><li>Charafe-Jauffret, E., Ginestier, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+cancer+cell+lines+contain+functional+cancer+stem+cells+with+metastatic+capacity+and+a+distinct+molecular+signature.">Breast cancer cell lines contain functional cancer stem cells with metastatic capacity and a distinct molecular signature.</a> Cancer Res. 69, 1302-1313 (2009).</li></ol>

A STEMCELL Technologies tem suportado as taxas de publicação do Open Access.

Os hospedeiros imunocomprometidos, como os camundongos SCID, impedem avaliações imunológicas de CSCs devido à falta de imunidade adaptativa dentro dos hospedeiros. Neste estudo, avaliamos a imunogenicidade dos CSCs em hospedeiros imunocompetntes, o que poderia imitar mais de perto as configurações dos pacientes. Os CSCs enriquecidos são imunogênicos e podem induzir imunidade protetora de tumores mais eficaz quando seus lisosatos são carregados para DCs como uma vacina em comparação com DCs com pulso de células tumorais não eleitos.  Mecanicamente, a proteção foi conferida por indução seletiva de anticorpos CSC-reativos e células T8, bem como a produção de citocina tipo 1, por exemplo, IFNγ e GM-CSF.
A maioria das imunoterapias atuais, incluindo vacinas baseadas em células dendríticas e a transferência de células T adotivas, são projetadas para atingir antígenos diferenciados por tumores. CSCs, que podem não expressar esses antígenos diferenciados, podem, portanto, escapar desses alvos imunológicos. Em contraste, a vacina CSC projetada para atingir especificamente células-tronco cancerígenas pode destruir essa população especial das células cancerosas e, assim, melhorar a eficácia terapêutica da vacina prevenindo a recaída tumoral e a metástase.
Em ambas as células tumorais cultivadas e tumores recém-colhidos, identificamos a população enriquecida com CSC por citometria de fluxo baseada na atividade desidrogenase de alto aldeído. Tais célulasaltas de ALDH poderiam ser isoladas pela triagem de fluxo para serem usadas como fonte de antígeno para pulsar DC para gerar CSC-TPDCs. A comparação com célulasaltas aldh isoladas de células tumorais cultivadas versus de tumores recém-colhidos não demonstrou diferença significativa em termos de indução da imunidade anti-CSC8. Esses resultados revelaram o potencial de uso de CSCs isolados de células tumorais cultivadas ou de tumores recém-colhidos para aplicação clínica.
Para ser clinicamente relevante, a vacina precisa ser examinada em um ambiente terapêutico. Esses experimentos estão sendo realizados em nosso laboratório.

As células-tronco cancerígenas são relativamente resistentes à quimioterapia convencional e radioterapia1,2. Por outro lado, essa população de células pode ser as células responsáveis pela recaída e progressão dos cânceres após as terapias tradicionais de câncer1-4. Devido à falta de expressão de antígenos tumorais diferenciados em células-tronco cancerosas, as células-tronco cancerígenas podem escapar das intervenções imunológicas atuais da terapia para o câncer, que são principalmente projetadas para atingir os antígenos nas células tumorais diferenciadas. Portanto, o desenvolvimento de novas estratégias especificamente voltadas e destruindo as células-tronco cancerígenas pode manter promessas de aumentar a eficácia terapêutica do tratamento atual do câncer. Para isso, isolamos populações de células-tronco cancerígenas (CSC) enriquecidas a partir de dois tumores animais (melanoma D5 e câncer de células escamosas SCC7), e as usamos como fonte de antígeno para pulsar células presentes de antígenos (células dendríticas, DC) para preparar a vacina CSC-TPDC. Em seguida, avaliamos a imunidade antitumor induzida pela vacina CSC-TPDC nos hospedeiros de imunocompetente sintérico, camundongos B6 e camundongos C3H, respectivamente. A eficácia do antitumor induzido pelo CSC-TPDC foi comparada com a vacina tradicional dc pulsada com lise de células tumorais heterogêneas não sortidas (H-TPDC), que já foi usada pelo nosso grupo5,6, bem como por outros pesquisadores7 tanto em estudos pré-clínicos quanto em ensaios clínicos.

<table border="0" cellpadding="0" cellspacing="0" width="1022">
	<colgroup>
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="27">
			<td height="27" style="height:27px;width:405px;">ALDHEFLOUR KIT</td>
			<td style="width:399px;">Stemcell Technologies</td>
			<td style="width:219px;">1700</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:405px;">Murine IL-4</td>
			<td style="width:399px;">Pepro Tech</td>
			<td style="width:219px;">214-14</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:405px;">Murine GM-CSF</td>
			<td style="width:399px;">Pepro Tech</td>
			<td style="width:219px;">315-03</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:405px;">Mouse IFN-&#611; ELISA KIT</td>
			<td style="width:399px;">BD Biosciences</td>
			<td style="width:219px;">555138</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:405px;">Mouse GM-CSF ELISA KIT</td>
			<td style="width:399px;">BD Biosciences</td>
			<td style="width:219px;">555167</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:405px;">OptiPrep Density Gradient Medium</td>
			<td style="width:399px;">Sigma Aldrich</td>
			<td style="width:219px;">D1556</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:405px;">BD OptEIA TMB Substrated Reagent Set</td>
			<td style="width:399px;">BD Biosciences</td>
			<td style="width:219px;">555214</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;"></td>
			<td style="width:399px;"></td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Equipment</td>
			<td style="width:399px;"></td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">BD FACS Aria Cell Sorter</td>
			<td>BD Biosciences</td>
			<td>336834</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:405px;">Kcjunior</td>
			<td style="width:399px;">Bio-Tek Instruments</td>
			<td style="width:219px;">176058</td>
		</tr>
	</tbody>
</table>

generation of a novel dendritic-cell vaccine using melanoma and squamous cancer stem cells

Identificamos populações de células-tronco cancerígenas (CSC) enriquecidas de melanoma murino D5 síngênico a C57BL/6 camundongos escamosos SCC7 scc7 scc7 sgênicas para camundongos C3H usando ALDEFLUOR/ALDH como marcador, e testamos sua imunogenicidade usando o lisato celular como fonte de antígenos para pulsar células dendríticas (DCs). Os DCs pulsados com lises de CSCelevados aldh induziam imunidade antitumor protetora significativamente maior do que os DCs pulsados com os lises de células tumorais inteiras não variadas em ambos os modelos e em um ajuste de metástase pulmonar e um s.c. ajuste de crescimento do tumor, respectivamente. Este fenômeno deveu-se às respostas humorais induzidas pela vacina CSC, bem como às respostas anti-CSC celulares. Em particular, esplenócitos isolados do hospedeiro submetido à vacina CSC-DC produziram uma quantidade significativamente maior de IFNγ e GM-CSF do que esplenócitos isolados do hospedeiro submetidos à vacina de linsatrato pulsado de células tumorais não sortidas. Esses resultados apoiam os esforços para desenvolver uma vacina terapêutica baseada em CSC autóloga para uso clínico em um ambiente adjuvante.

ALDEFLUOR/ALDH tem sido usado como um único marcador para isolar células-tronco em múltiplas malignidades8-11. Identificamos populações cancerígenas enriquecidas com células-tronco em dois modelos tumorais D5 e SCC7 usando ALDEFLUOR como marcador. Detectamos células ALDEFLUOR+ no melanoma murino B16-D5 e câncer de células escângenas SCC7. Verificou-se que as células ALDEFLUOR+ contribuem com aproximadamente 0,5% e 5,2% nas linhas de células tumorais cultivadas D5 e SCC7, respectivamente (Figura 1). Células tumorais recém-colhidas de tumores estabelecidos foram analisadas para confirmar a existência de células ALDEFLUOR+. Como também mostrado na Figura 1,houve 2,5% e 4,2% das células ALDEFLUOR+ dos tumores In vivo estabelecidos D5 e SCC7, respectivamente. A tumorigenicidade e a capacidade de autoconexão dessaspopulações classificadas D5 e SCC7 ALDEFLUOR+/ALDH foram avaliadas no hospedeiro de imunocompetente síngênico, os camundongos C57BL/6 e C3H,respectivamente 8.
Utilizamos células tumorais heterogêneas para pulsar DCs (H-TPDC) tanto em estudos em animais quanto em ensaios clínicos5,6. Para examinar a imunogenicidade dos CSCs, isolamos o CSCalto da ALDH e pulsamos DC com o lisato dos CSCs para gerar CSC-TPDC(Figura 2) e usamos o H-TPDC como um controle convencional de vacina contra o câncer para testar se o CSC-TPDC tem algum efeito benéfico na prevenção do crescimento tumoral.
Avaliamos a imunogenicidade dos CSCs examinando a imunidade antitumoral protetora induzida pelo CSC-TPDC. No modelo D5, foram vacinados camundongos imunocompetntes ingênuos.c com CSC-TPDC ou H-TPDC (na mesma razão: razão DC). Os grupos de controle receberam soro fisiológico (PBS). Uma semana após a última vacina, os camundongos foram desafiados com células tumorais D5 não variadas por via intravenosa(i.v.),e os pulmões foram colhidos 3 semanas depois para enumerar metástases pulmonares. Como mostrado na Tabela 1, em comparação com o grupo PBS, os camundongos tratados com o H-TPDC desenvolveram menos metástases pulmonares. É importante ressaltar que os camundongos tratados com CSC-TPDC apresentaram significativamente menos metástases pulmonares do que o grupo de vacinas H-TPDC em ambos os experimentos realizados. No modelo SCC7, foram vacinados animais C3H normais.c com SCC7 CSC-TPDC ou H-TPDC, respectivamente, no flanco direito, seguidos por desafiadores com células tumorais SCC7 não sortidas.c no flanco esquerdo. Comparado com o grupo PBS, h-TPDC induziu imunidade anti-tumor modesta contra o crescimento do tumor. No entanto, houve inibição significativa do crescimento tumoral em camundongos que foram tratados com CSC-TPDC quando comparados tanto com o grupo controle quanto com o grupo H-TPDC (p<0,05, Figura 3). Esses resultados indicam que os CSCs poderiam ser usados como uma fonte de antígeno mais eficaz para carregar DCs do que as células tumorais tradicionais induzindo imunidade protetora contra o desafio das células tumorais.
Para entender melhor os mecanismos subjacentes à imunidade antitumor protetora induzida pelo CSC, colhemos os baços dos animais submetidos à vacinação de DC no final dos experimentos. As células do baço foram então ativadas por anti-CD3/CD28/IL-2 ou anti-CD3/CD28/IL-2 + LPS/anti-CD40. Em seguida, os supernacantes culturais foram coletados para detectar a expressão de citocinas e anticorpos.  Houve produções significativamente maiores de IFNγ e GM-CSF pelas células do baço dos animais vacinados com D5 CSC-TPDC ou SCC7 CSC-TPDC (Figura 4). Além disso, houve uma produção significativa (p<0,05) maior de IgG por células de baço ativadas LPS/anti-CD40 coletadas dos animais vacinados com D5 CSC-TPDC ou SCC7 CSC-TPDC em comparação com D5 H-TPDC ou SCC7 H-TPDC. Esses anticorpos foram encontrados para se ligarem aos CSCs D5 e SCC7, respectivamente, e tal vinculação poderia resultar na lise CSC na presença do complemento8.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/50561/50561fig1highres.jpg" src="/files/ftp_upload/50561/50561fig1.jpg"> Figura 1. Asaltas populações de ALDHFLUOR+/ALDH foram detectadas em tumores de células escângenas murinas recém-colhidas e tumores escânceres SCC7. As células tumorais tratadas com 50 mmol/L DEAB, inibidor específico da ALDH, foram utilizadas como controle negativo.
<a href="https://www.jove.com/files/ftp_upload/50561/50561fig1highres.jpg" target="_blank">Clique aqui para ver imagem maior.</a> 
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/50561/50561fig2highres.jpg" src="/files/ftp_upload/50561/50561fig2.jpg"> Figura 2. Geração de vacinas contra células-tronco baseadas em células dendríticas. Para a preparação de CSC-TPDC e H-TPDC, as células dendríticas derivadas da medula óssea foram pulsadas com células tumoraisaltas ou não sortidas de ALDH, respectivamente.
<a href="https://www.jove.com/files/ftp_upload/50561/50561fig2highres.jpg" target="_blank">Clique aqui para ver imagem maior.</a> 
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/50561/50561fig3highres.jpg" src="/files/ftp_upload/50561/50561fig3.jpg"> Figura 3. A vacina dc pulsada com csc (CSC-TPDC) poderia induzir imunidade de antitumor protetor protetor mais eficaz na .c. Modelo de tumor SCC7. <a href="https://www.jove.com/files/ftp_upload/50561/50561fig3highres.jpg" target="_blank">Clique aqui para ver imagem maior.</a> 

<img alt="Figure 4" fo:content-width="5in" fo:src="/files/ftp_upload/50561/50561fig4highres.jpg" src="/files/ftp_upload/50561/50561fig4.jpg"> Figura 4. Respostas celulares sistêmicas mais potentes no hospedeiro imunocompetente vacinados com CSC-TPDC.  Os esplenócitos foram colhidos dos animais submetidos à vacinação H-TPDC ou CSC-TPDC, e foram ativados conforme indicado. Os supernacantes culturais foram então coletados para detecção de citocinas usando ELISA.
<a href="https://www.jove.com/files/ftp_upload/50561/50561fig4highres.jpg" target="_blank">Clique aqui para ver imagem maior.</a>

Watch this Scientific Journal Video about Generation of a Novel Dendritic-cell Vaccine Using Melanoma and Squamous Cancer Stem Cells at JoVE.com

Generation of a Novel Dendritic-cell Vaccine Using Melanoma and Squamous Cancer Stem Cells

Avaliada em hospedeiros de imunocompetente sintônico, a vacina de células dendríticas baseadas em células dendríticas baseadas em câncer (CSC) demonstrou imunidade antitumoral significativamente maior do que as vacinas tradicionais de DC pulsadas com células tumorais a granel heterogêneas.

Geração de uma nova vacina de células dendríticas usando melanoma e células-tronco de câncer escântico

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H ...

generation-novel-dendritic-cell-vaccine-using-melanoma-squamous

Research

JoVE Journal

Immunology and Infection

11.2K visualizações.  University of Michigan. Avaliada em hospedeiros de imunocompetente sintônico, a vacina de células dendríticas baseadas em células dendríticas baseadas em câncer (CSC) demonstrou imunidade antitumoral significativamente maior do que as vacinas tradicionais de DC pulsadas com células tumorais a granel heterogêneas.

Vídeo: Generation of a Novel Dendritic-cell Vaccine Using Melanoma and Squamous Cancer Stem Cells

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in peripheral tissues in steady state; however in response to antigen stimulation, DCs take up the antigen and rapidly migrate to the draining lymph nodes where they initiate T cell response against the antigen1,2. Additionally, DCs also play a key role in initiating autoimmune as well as allergic immune response3.
DCs play an essential role in both initiation of immune response and induction of tolerance in the setting of lung environment4. Lung environment is largely tolerogenic, owing to the exposure to vast array of environmental antigens5. However, in some individuals there is a break in tolerance, which leads to induction of allergy and asthma. In this study, we describe a strategy, which can be used to monitor airway DC maturation and migration in response to the antigen used for sensitization. The measurement of airway DC maturation and migration allows for assessment of the kinetics of immune response during airway allergic inflammation and also assists in understanding the magnitude of the subsequent immune response along with the underlying mechanisms.
Our strategy is based on the use of ovalbumin as a sensitizing agent. Ovalbumin-induced allergic asthma is a widely used model to reproduce the airway eosinophilia, pulmonary inflammation and elevated IgE levels found during asthma6,7. After sensitization, mice are challenged by intranasal delivery of FITC labeled ovalbumin, which allows for specific labeling of airway DCs which uptake ovalbumin. Next, using several DC specific markers, we can assess the maturation of these DCs and can also assess their migration to the draining lymph nodes by employing flow cytometry.

We describe a strategy to monitor maturation and migration of pulmonary dendritic cells in response to ovalbumin in the setting of ovalbumin induced allergic airway inflammation. This strategy can be modified to assess migration of pulmonary dendritic cells in settings of infection.

Análise da maturação das células dendríticas e migração pulmonar durante a inflamação alérgica das vias aéreas

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in ...

Imunologia e Infecção

Mass Spectrometric Analysis of Glycosphingolipid Antigens

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes such as embryonic development, signal transduction, and immune receptor recognition1-2. They contain complex sugar moieties in the form of isomers, and lipid moieties with variations including fatty acyl chain length, unsaturation, and hydroxylation. Both carbohydrate and ceramide portions may be basis of biological significance. For example, tri-hexosylceramides include globotriaosylceramide (Gal&alpha;4Gal&beta;4Glc&beta;1Cer) and isoglobotriaosylceramide (Gal&alpha;3Gal&beta;4Glc&beta;1Cer), which have identical molecular masses but distinct sugar linkages of carbohydrate moiety, responsible for completely different biological functions3-4. In another example, it has been demonstrated that modification of the ceramide part of alpha-galactosylceramide, a potent agonist ligand for invariant NKT cells, changes their cytokine secretion profiles and function in animal models of cancer and auto-immune diseases5. The difficulty in performing a structural analysis of isomers in immune organs and cells serve as a barrier for determining many biological functions6.
Here, we present a visualized version of a method for relatively simple, rapid, and sensitive analysis of glycosphingolipid profiles in immune cells7-9. This method is based on extraction and chemical modification (permethylation, see below Figure 5A, all OH groups of hexose were replaced by MeO after permethylation reaction) of glycosphingolipids10-15, followed by subsequent analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and ion trap mass spectrometry. This method requires 50 million immune cells for a complete analysis. The experiments can be completed within a week. The relative abundance of the various glycosphingolipids can be delineated by comparison to synthetic standards. This method has a sensitivity of measuring 1% iGb3 among Gb3 isomers, when 2 fmol of total iGb3/Gb3 mixture is present9.
Ion trap mass spectrometry can be used to analyze isomers. For example, to analyze the presence of globotriaosylceramide and isoglobtriaosylceramide in the same sample, one can use the fragmentation of glycosphingolipid molecules to structurally discriminate between the two (see below Figure 5). Furthermore, chemical modification of the sugar moieties (through a permethylation reaction) improves the ionization and fragmentation efficiencies for higher sensitivity and specificity, and increases the stability of sialic acid residues. The extraction and chemical modification of glycosphingolipids can be performed in a classic certified chemical hood, and the mass spectrometry can be performed by core facilities with ion trap MS instruments.

A specific and sensitive method to gain insight into the expression profile of glycosphingolipid antigens in immune organs and cells is described. The method takes advantage of the ion trap mass spectrometry allowing step-wise fragmentation of glycosphingolipid molecules for structural analysis in comparison to chemically synthesized standards.

Análise por espectrometria de massa de Antígenos glicoesfingolipídeos

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes ...

Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many advantages for ease and standardization of immunological assessments, but detrimental effects of this process have been observed on some cell subsets, such as granulocytes, B cells, and dendritic cells 1-3. Assaying fresh leukocytes gives a more accurate picture of the in vivo state of the cells, but is often difficult to perform in the context of large clinical trials. Fresh cell assays are dependent upon volunteer commitments and timeframes and, if time-consuming, their application can be impractical due to the working hours required of laboratory personnel. In addition, when trials are conducted at multiple centers, laboratories with the resources and training necessary to perform the assays may not be located in sufficient proximity to clinical sites. To address these issues, we have developed an 11-color antibody staining panel that can be used with Trucount tubes (Becton Dickinson; San Jose, CA) to phenotype and enumerate the major leukocyte populations within the peripheral blood, yielding more robust cell-type specific information than assays such as a complete blood count (CBC) or assays with commercially-available panels designed for Trucount tubes that stain for only a few cell types. The staining procedure is simple, requires only 100 &mu;l of fresh whole blood, and takes approximately 45 minutes, making it feasible for standard blood-processing labs to perform. It is adapted from the BD Trucount tube technical data sheet (<a href="http://www.bdbiosciences.com/external_files/is/doc/tds/Package_Inserts_CE/live/web_enabled/23-3483-06_PI_Trucount tubes_CE_EN.pdf" target="_blank">version 8/2010</a>). The staining antibody cocktail can be prepared in advance in bulk at a central assay laboratory and shipped to the site processing labs. Stained tubes can be fixed and frozen for shipment to the central assay laboratory for multicolor flow cytometry analysis. The data generated from this staining panel can be used to track changes in leukocyte concentrations over time in relation to intervention and could easily be further developed to assess activation states of specific cell types of interest. In this report, we demonstrate the procedure used by blood-processing lab technicians to perform staining on fresh whole blood and the steps to analyze these stained samples at a central assay laboratory supporting a multicenter clinical trial. The video details the procedure as it is performed in the context of a clinical trial blood draw in the HIV Vaccine Trials Network (HVTN).

In this report, we demonstrate the staining and analysis steps of a phenotyping assay performed on fresh whole blood to enumerate major innate and adaptive leukocyte populations. We emphasize considerations for performing these procedures in the context of a multicenter clinical trial.

Enumeração dos principais Populações de Leucócitos do Sangue Periférico de ensaios clínicos multicêntricos usando um ensaio fenotipagem Sangue

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many ...

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

Monitoramento da Migração de Células dendríticas usando<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H Ressonância Magnética

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field 4. The use of cell-based arenavirus minigenome systems together with the ability to generate recombinant infectious arenaviruses with predetermined mutations in their genomes has facilitated the investigation of the contribution of viral determinants to the different steps of the arenavirus life cycle, as well as virus-host interactions and mechanisms of arenavirus pathogenesis 1, 3, 11 . In addition, the development of trisegmented arenaviruses has permitted the use of the arenavirus genome to express additional foreign genes of interest, thus opening the possibility of arenavirus-based vaccine vector applications 5 . Likewise, the development of single-cycle infectious arenaviruses capable of expressing reporter genes provides a new experimental tool to improve the safety of research involving highly pathogenic human arenaviruses 16 . The generation of recombinant arenaviruses using plasmid-based reverse genetics techniques has so far relied on the use of rodent cell lines 7,19 , which poses some barriers for the development of Food and Drug Administration (FDA)-licensed vaccine or vaccine vectors. To overcome this obstacle, we describe here the efficient generation of recombinant arenaviruses in FDA-approved Vero cells.

Rescue of recombinant arenaviruses from cloned cDNAs, an approach referred to as reverse genetics, allows researchers to investigate the role of specific viral gene products, as well as the contribution of their different specific domains and residues, to many different aspects of the biology of arenavirus. Likewise, reverse genetics techniques in FDA-approved cell lines (Vero) for vaccine development provides novel possibilities for the generation of effective and safe vaccines to combat human pathogenic arenaviruses.

Geração de Arenavirus recombinante de Desenvolvimento de Vacinas no FDA-approved células Vero

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field  4. The use of cell-based ...

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
LacI transgenic mice carry a recoverable &#955;&#160;phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of &#946;-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.
The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model.&#160;The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants&#160;spontaneously arisen or after exposure to genotoxins.

A identificação de DNA Mutações em Purificada-Tronco Hematopoéticas / células progenitoras

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

Usando Click Chemistry medir o efeito da infecção viral sobre a síntese de RNA do Host-Cell

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-&#947;). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.

The goal of this protocol is to manufacture pathogen-specific clinical-grade T cells using a bench-top, automated, second generation cell enrichment device that incorporates a closed cytokine capture system and does not require dedicated staff or use of a GMP facility. The cytomegalovirus pp65-specific-T cells generated can be directly administered to patients.

As células T de enriquecimento de células automatizado de citomegalovírus específico-para aplicações clínicas usando o Sistema de captura de citocinas

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

Na Vivo Ensaio para detecção de células T antígeno-específicas função citolítica usando um modelo de vacinação

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have recently been recognized to be more heterogeneous than previously appreciated. These cultures routinely contain moDC as well monocyte-derived macrophages (moMac), and even some less developed cells such as monocytes. The goal of this protocol is to provide a consistent method for identification and separation of the many cell types present in these cultures as they develop, so that their specific functions may be further investigated. The sorting strategy presented here separates cells first into four populations based on expression of Ly6C and CD115, both of which are expressed transiently by cells as they develop in GM-CSF-driven culture. These four populations include Common myeloid progenitors or CMP (Ly6C-, CD115-), granulocyte/macrophage progenitors or GMP (Ly6C+, CD115-), monocytes (Ly6C+, CD115+), and monocyte-derived macrophages or moMac (Ly6C-, CD115+). CD11c is also added to the sorting strategy to distinguish two populations within the Ly6C-, CD115- population: CMP (CD11c-) and moDC (CD11c+). Finally, two populations may be further distinguished within the Ly6C-, CD115+ population based on the level of MHC class II expression. MoMacs express lower levels of MHC class II, while a monocyte-derived DC precursor (moDP) expresses higher MHC class II. This method allows for the reliable isolation of several developmentally distinct populations in numbers sufficient for a variety of functional and developmental analyses. We highlight one such functional readout, the differential responses of these cell types to stimulation with Pathogen-Associated Molecular Patterns (PAMPs).

Here we provide a method for identifying and isolating large numbers of GM-CSF driven myeloid cells using high speed cell sorting. Five distinct populations (Common myeloid progenitors, granulocyte/macrophage progenitors, monocytes, monocyte-derived macrophages, and monocyte-derived DCs) can be identified based on Ly6C and CD115 expression.

Geração de um grande número de progenitores mieloides e células dendríticas precursores de murino medula óssea usando um celular romance classificação estratégia

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have ...