This work was supported by The Will and Jeanne Caldwell Endowed Research Fund of the University of Michigan Comprehensive Cancer Center and in part by NIH grant CA82529 and the Gillson Longenbaugh Foundation.

1. ALDEFLUOR Staining
<ol>
	<li>Cell sample preparation: Prepare single cell suspension of tumor cells, either from cultured tumor cells or from freshly harvested tumor samples. Count the cells and adjust cell suspension to a concentration of 1 x 106 cells/ml in ALDEFLUOR Assay Buffer.</li>
	<li>Label four 12 mm x 75 mm polystyrene test tubes as control tubes as follows: #1: Unstained, #2: ALDEFLUOR, #3: ALDEFLUOR plus DEAB, and&#160; #4: 7AAD.</li>
	<li>For these control tubes, place 1ml cell suspension into #1 and #4, but 2 ml into tube #2. Add 5 &#181;l DEAB (ALDH inhibitor) into tube #3 and keep on ice. Then add 10 &#181;l activated ALDEFLUOR substrate to tube #2, mix and immediately transfer 1 ml of the mixture to tube #3.</li>
	<li>At the same time, add 2 &#181;l activated ALDEFLUOR substrate per million cells to the rest of the cells (sample tubes) also at 1 x 106&#160;cells/ml in ALDEFLUOR Assay Buffer.</li>
	<li>Incubate both the 4 control tubes and the sample tubes for 30 min in 37 &#176;C water bath.</li>
	<li>Following incubation, add 5 &#181;l 7AAD into control tube #4, and 1 &#181;l 7AAD 1 x 106 cells to the sample tube. Incubate 10 min at 4 &#176;C.</li>
	<li>Centrifuge all tubes for 5 min at 250 x g and remove supernatant.</li>
	<li>Resuspend cells in ALDEFLUOR Assay Buffer.</li>
	<li>Perform flow cytometry-based sorting.</li>
	<li>Set the sorting gates using ALDEFLUOR-stained cells treated with DEAB as negative control and the propidium iodide (PI) 7AAD- stained cells for viability control. Based on these controls, a gate was established to distinguish the ALDEFLUOR+/ALDHhigh, D5 and SCC7 cells. These gates will then be used to sort all the sample cells prepared above for the ALDEFLUOR+/ALDHhigh D5 and SCC7 cells.</li>
</ol>
2. Preparation of Cancer Stem Cell Lysate-pulse Dendritic Cell (CSC-TPDC) Vaccine
<ol>
	<li>Euthanize mice (syngeneic B6 and C3H, respectively) using CO2 and isolate femur and tibia bones.</li>
	<li>Put the bones in 75% ethanol for 1 min at room temperature. Then wash the bones using HBSS.</li>
	<li>Cut head off bones and use a 21 G needle and 10 ml syringe to push cells into a dish.</li>
	<li>Aspirate and blow the cells using the syringe to make the single cell suspension.</li>
	<li>After centrifugation in 1,500 rpm for 5 min, discard the supernatant. Then add 5 ml RBC lysis buffer to lysis the RBC for 1 min in 37 &#176;C water bath.</li>
	<li>Count cell number and adjust cell suspension to a concentration of 1 million cells/ml in complete&#160;medium (CM) containing 10 ng/ml GM-CSF and 10 ng/ml IL-4.</li>
	<li>Culture the cells and compensate CM plus IL-4 and GM-CSF 3 days later.</li>
	<li>At the 5th day, harvest the immature dendritic cells (DCs) and prepare the DC isolation medium containing 4.2 ml solution C plus 1 ml OptiPrep density gradient medium.</li>
	<li>Suspend the cell pellets in 5 ml CM. Add the cell suspension onto the isolation medium slowly. Centrifuge at&#160;2,000 rpm at room temperature.</li>
	<li>Collect the DCs between the CM and isolation medium. Count cell number and adjust cell suspension to a concentration of 1 million cells/ml in culture medium containing 10 ng/ml GM-CSF and 10 ng/ml IL-4.</li>
	<li>Prepare tumor lysates by suspending ALDHhigh or unsorted D5 or SCC7 cells in culture medium and subject to rapid freeze-thaw exposures four times followed by spin at &#8764;100 x&#160;g for 5 min to collect the membrane portion of the lysates.</li>
	<li>To prepare CSC-TPDC, pulse DCs with the lysate of autologous ALDHhigh&#160;cells. To prepare H-TPDC, pulse DCs with unsorted heterogeneous tumor cell lysate. The ratio of DC to tumor cell lysate is the same.</li>
</ol>
3. Vaccination and Evaluation of the Efficacy
<ol>
	<li>Vaccinate normal B6 mice respectively with 2,500&#160;D5 CSC-TPDC or D5 H-TPDC tumor cells subcutaneously (s.c.). Vaccinate normal C3H animals s.c. with &#160;5,000 SCC7 CSC-TPDC or SCC7 H-TPDC tumor cells, respectively.&#160;</li>
	<li>After vaccination, challenge the B6 mice with the heterogeneous D5 tumor cells i.v.&#160;Euthanize the mice using CO2&#160; 20 days later. Harvest the lungs and enumerate lung metastases.</li>
	<li>In SCC7 model, challenge the C3H mice with unsorted SCC7 tumor cells s.c on the opposite side of the DC vaccine. Monitor the tumor size.</li>
	<li>At the end of the experiments euthanize the B6 and C3H mice using CO2.&#160;At Day 34 after first vaccine,&#160;euthanize&#160;mice with CO2, and at the same time collect the&#160;spleens of each group with an aseptic procedure.&#160;</li>
	<li>Activate spleen T and/or B cells with immobilized anti-CD3 plus anti-CD28 mAbs in CM containing hrIL-2 or LPS plus anti-CD40 (FGK45) mAb ascites.</li>
	<li>After activation, collect supernatants and use for ELISA assay.</li>
	<li>For ELISA assay, coat plates with antibodies for IFN&#947;, GM-CSF and IgG at 4 &#176;C O/N.</li>
	<li>After the application of blocking buffer, add samples and standards and incubate at room temperature.</li>
	<li>Wash the plate and add HRP detection antibody and TMB substrate for incubation.</li>
	<li>Measure the absorbance on an ELISA plate reader at 450 nm within 30 min after stopping the reaction.</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>

STEMCELL Technologies has supported Open Access publication fees.

The immunocompromised hosts, such as SCID mice, preclude immunological assessments of CSCs due to the lack of adaptive immunity within the hosts. In this study, we evaluated the immunogenicity of CSCs in immunocompetent hosts, which could more closely mimic patient settings. Enriched CSCs are immunogenic and could induce more effective tumor protective immunity when their lysates are loaded to DCs as a vaccine compared with unselected tumor cell lysate-pulsed DCs.&#160; Mechanistically, the protection was conferred by selective induction of CSC-reactive antibodies and T cells8 as well as the production of type 1 cytokine, e.g. IFN&#947; and GM-CSF.
Most of the current immunotherapies, including dendritic cell-based vaccines and the adoptive T cell transfer, are designed to target tumor- differentiated antigens. CSCs, which may not express these differentiated antigens may therefore escape these immunological targeting. In contrast, CSC vaccine designed to specifically target cancer stem cells may destroy this special population of the cancer cells, and thus improve the therapeutic efficacy of the vaccine by preventing tumor relapse and metastasis.
In both cultured tumor cells and freshly harvested tumors, we identified CSC-enriched population by flow cytometry based on high aldehyde dehydrogenase activity. Such ALDHhigh cells could be isolated by flow sorting to be used as an antigen source to pulse DC to generate CSC-TPDCs. Comparison using ALDHhigh cells isolated from cultured tumor cells vs&#160; from freshly harvested tumors demonstrated no significantly difference in term of the induction of anti-CSC immunity8. These results revealed the potential to use CSCs isolated either from cultured tumor cells or from freshly harvested tumors for clinical application.
To be clinically relevant, a vaccine needs to be examined in a therapeutic setting. These experiments are now being performed in our laboratory.

Cancer stem cells are relatively resistant to conventional chemotherapy and radiotherapy1,2. On the other hand, this population of cells might be the cells responsible for the relapse and progression of cancers after traditional cancer therapies1-4. Due to the lack of expression of differentiated tumor antigens on cancer stem cells, cancer stem cells may escape current immunologic interventions of therapy for cancer, which are mostly designed to target the antigens on the differentiated tumor cells. Therefore, development of new strategies specifically targeting and destroying the cancer stem cells may hold promises to increase the therapeutic efficacy of current cancer treatment. To this end, we isolated cancer stem cell (CSC)-enriched populations from two animal tumors (melanoma D5 and squamous cell cancer SCC7), and used them as an antigen source to pulse antigen presenting cells (dendritic cells, DC) to prepare the CSC-TPDC vaccine. We then evaluated the antitumor immunity induced by the CSC-TPDC vaccine in the syngeneic immunocompetent hosts, B6 mice and C3H mice respectively. The CSC-TPDC-induced antitumor efficacy was compared with the traditional DC vaccine pulsed with lysate from unsorted heterogeneous tumor cells (H-TPDC), which has previously been used by our group5,6, as well as by other investigators7 both in preclinical studies and in clinical trials.

<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

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 mice using ALDEFLUOR/ALDH as a marker, and tested their immunogenicity using the cell lysate as a source of antigens to pulse dendritic cells (DCs). DCs pulsed with ALDHhigh CSC lysates induced significantly higher protective antitumor immunity than DCs pulsed with the lysates of unsorted whole tumor cell lysates in both models and in a lung metastasis setting and a s.c.&#160;tumor growth setting, respectively. This phenomenon was due to CSC vaccine-induced humoral as well as cellular anti-CSC responses. In particular, splenocytes isolated from the host subjected to CSC-DC vaccine produced significantly higher amount of IFN&#947; and GM-CSF than splenocytes isolated from the host subjected to unsorted tumor cell lysate pulsed-DC vaccine. These results support the efforts to develop an autologous CSC-based therapeutic vaccine for clinical use in an adjuvant setting.

ALDEFLUOR/ALDH has been used as a single marker to isolate stem cells in multiple malignancies8-11. We identified cancer stem cell-enriched populations in two tumor models D5 and SCC7 by using ALDEFLUOR as a marker. We detected ALDEFLUOR+ cells in murine melanoma B16-D5 and squamous cell cancer SCC7. We found that ALDEFLUOR+ cells contribute approximately 0.5% and 5.2% in cultured D5 and SCC7 tumor cell lines, respectively (Figure 1). Freshly harvested tumor cells from established tumors have been analyzed to confirm the existence of ALDEFLUOR+ cells. As also shown in Figure 1, there were 2.5% and 4.2% of the ALDEFLUOR+ cells from in vivo established D5 and SCC7 tumors, respectively. The tumorigenicity and the self-renewal capacity of these sorted D5 and SCC7 ALDEFLUOR+/ALDHhigh populations were evaluated in the syngeneic immunocompetent host, the C57BL/6 and C3H mice, respectively8.
We have used heterogeneous unsorted tumor cell lysate to pulse DCs (H-TPDC) both in animal studies and in clinical trials5,6. To examine the immunogenicity of CSCs, we isolated ALDHhigh CSC and pulsed DC with the lysate of the CSCs to generated CSC-TPDC (Figure 2)&#160;and used H-TPDC as a conventional cancer vaccine control to test if CSC-TPDC has any beneficial effect in preventing tumor growth.
We evaluated the immunogenicity of CSCs by examining protective antitumor immunity induced by CSC-TPDC. In D5 model, na&#239;ve immunocompetent mice were vaccinated s.c with CSC-TPDC or H-TPDC (at the same lysate: DC ratio). Control groups received saline (PBS). One week after the last vaccine, the mice were challenged with unsorted D5 tumor cells intravenously (i.v.), and&#160;the lungs were harvested 3 weeks later to enumerate lung metastases. As shown in Table 1, compared with PBS group, mice treated with the H-TPDC developed less lung metastases. Importantly, mice treated with CSC-TPDC had significantly fewer lung metastases than H-TPDC vaccine group in both experiments performed.&#160;In SCC7 model, normal C3H animals were vaccinated s.c with SCC7 CSC-TPDC or H-TPDC respectively on the right flank, followed by challenging with unsorted SCC7 tumor cells s.c into the left flank. Compared with PBS group, H-TPDC induced modest anti-tumor immunity against tumor growth. However, there was significant inhibition of tumor growth in mice that were treated with CSC-TPDC when compared with both the control group and H-TPDC group (p&#60;0.05, Figure 3). These results indicate that CSCs could be used as an more effective antigen source to load DCs than traditional unsorted tumor cells in inducing protective immunity against the challenge of tumor cells.
To further understand the mechanisms underlying the observed CSC-induced protective antitumor immunity, we harvested the spleens from the animals subjected to DC vaccinations at the end of the experiments. The spleen cells were then activated by anti-CD3/CD28/IL-2 or anti-CD3/CD28/IL-2 + LPS/anti-CD40. Then the culture supernatants were collected to detect the expression of cytokines and antibody.&#160; There were significantly higher productions of IFN&#947; and GM-CSF by the spleen cells from the animals vaccinated with D5 CSC-TPDC or SCC7 CSC-TPDC (Figure 4). Furthermore, there was significantly (p&#60;0.05) higher IgG production by LPS/anti-CD40 activated spleen cells collected from the animals vaccinated with D5 CSC-TPDC or SCC7 CSC-TPDC compared with D5 H-TPDC or SCC7 H-TPDC. These antibodies were found to bind to D5 and SCC7 CSCs respectively, and such binding could result in the CSC lysis in the presence of complement8.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/50561/50561fig1highres.jpg" src="/files/ftp_upload/50561/50561fig1.jpg" /> Figure 1.&#160;ALDHFLUOR+/ALDHhigh populations were detected in cultured as well as newly harvested fresh murine D5 melanoma and SCC7 squamous cell tumors. Tumor cells treated with 50 mmol/L DEAB, a specific ALDH inhibitor, were used as the negative control.
<a href="https://www.jove.com/files/ftp_upload/50561/50561fig1highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/50561/50561fig2highres.jpg" src="/files/ftp_upload/50561/50561fig2.jpg" /> 
Figure 2.&#160;Generation of dendritic cell-based cancer stem cell vaccines. For the preparation of CSC-TPDC and H-TPDC, bone marrow-derived dendritic cells were pulsed with ALDHhigh or unsorted tumor cell lysates,&#160;respectively.
<a href="https://www.jove.com/files/ftp_upload/50561/50561fig2highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/50561/50561fig3highres.jpg" src="/files/ftp_upload/50561/50561fig3.jpg" /> 
Figure 3.&#160;CSC lysate pulsed DC (CSC-TPDC) vaccine could induce more effective protective antitumor immunity in the s.c. SCC7 tumor model.
<a href="https://www.jove.com/files/ftp_upload/50561/50561fig3highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Figure 4" fo:content-width="5in" fo:src="/files/ftp_upload/50561/50561fig4highres.jpg" src="/files/ftp_upload/50561/50561fig4.jpg" /> 
Figure 4.&#160;More potent systemic cellular responses in immunocompetent host vaccinated with CSC-TPDC.&#160; Splenocytes were harvested from the animals subjected to H-TPDC or CSC-TPDC vaccination, and were activated as indicated. The culture supernatants were then collected for cytokine detection using ELISA.
<a href="https://www.jove.com/files/ftp_upload/50561/50561fig4highres.jpg" target="_blank">Click here to view larger image.</a>

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

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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 ...

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Ultra-Fast Amplicon-Based Next-Generation Sequencing in Non-Squamous Non-Small Cell Lung Cancer

a vaccine-pulsed dendritic cell-based in vitro assay to induce lymphocyte proliferation

This video demonstrates a monocyte-derived dendritic cell, MoDC-based assay to measure the immunogenicity of commercially available vaccines. MoDC assays investigate dendritic cell responses to antigens, pathogens, or treatments and can provide insights into immune responses, vaccine development, and immunotherapy...

A Vaccine-Pulsed Dendritic Cell-Based In Vitro Assay to Induce Lymphocyte Proliferation

generation of monocyte-derived dendritic cells with differing sialylated phenotypes

Author Spotlight: Dendritic Cells Maturation Using Sialidases-Based Enzymatic Treatment of the Cell Surface 

A unique, comprehensive protocol to generate de-sialylated human monocyte-derived dendritic cells (mo-DCs) from isolated peripheral blood mononuclear cells (PBMCs) using a sialidase treatment is presented. Further, methods to assess the phenotypic and functional characterization of mo-DCs and evaluate how sialidase treatment improves the maturation level of mo-DCs are...

Generation of Monocyte-Derived Dendritic Cells with Differing Sialylated Phenotypes

generation of immature, mature and tolerogenic dendritic cells with differing metabolic phenotypes

Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

Immature dendritic cells can be selectively differentiated into tolerogenic or mature dendritic cells to regulate the balance between immunity and tolerance. This work presents a means to generate from immature monocyte derived dendritic cells (moDCs), in vitro tolerogenic and mature moDCs that differ in metabolic...

generation of immature, mature, and tolerogenic dendritic cells from monocytes

This video demonstrates the generation of immature, mature, and tolerogenic dendritic cells, or DCs, from human monocytes. The incubation of monocytes with growth factors and cytokines leads to the production of immature DCs. In response to immune regulatory molecules, immature DCs differentiate into tolerogenic and mature...

Generation of Immature, Mature, and Tolerogenic Dendritic Cells from Monocytes

generation of a humanized mouse liver using human hepatic stem cells

Generation of a Humanized Mouse Liver Using Human Hepatic Stem Cells

Here, we present a novel humanized mouse liver model generated in Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice following the transplantation of immature and expandable human hepatic stem...

induced pluripotent stem cell generation from blood cells using sendai virus and centrifugation

Induced Pluripotent Stem Cell Generation from Blood Cells Using Sendai Virus and Centrifugation

We propose a protocol for reprogramming peripheral blood mononuclear cells (PBMCs) into induced pluripotent stem cells (iPSCs). By plating the transduced blood cells onto matrix-coated plates with centrifugation, iPSCs are successfully induced from floating cells. This technique suggests a simple and effective reprogramming protocol for cells such as PBMCs and...

a robust discovery platform for the identification of novel mediators of melanoma metastasis

A Robust Discovery Platform for the Identification of Novel Mediators of Melanoma Metastasis

This article describes a workflow of techniques employed for testing novel candidate mediators of melanoma metastasis and their mechanism(s) of action.

bone marrow-derived dendritic cells generation: a method to generate dendritic cells from mouse bone marrow

This video describes a protocol for the generation of dendritic cells--key antigen-presenting cells that activate T cells--from the bone marrow of an adult...

Bone Marrow-Derived Dendritic Cells Generation: A Method to Generate Dendritic Cells from Mouse Bone Marrow

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.

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

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.

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.

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.

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.

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.

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.

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.

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

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

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.

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