Name: Generation of a Novel Dendritic-cell Vaccine Using Melanoma and Squamous Cancer Stem Cells (Video) | JoVE Journal
Uploaded: 2014-01-06T14:30:00.000+00:00
Duration: 12 min 43 s
Description: Learn to generate a novel dendritic cell vaccine using melanoma and squamous cancer stem cells. This method demonstrates stronger anti-tumor immunity than traditional DC vaccines in immunocompetent hosts.

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5 minutes

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/pubmed?term=((Cancer+stem+cells+contribute+to+cisplatin+resistance+in+Brca1%2Fp53-mediated+mouse+mammary+tumors%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">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/pubmed?term=((Low-dose+radiation+enhances+survivin-mediated+virotherapy+against+malignant+glioma+stem+cells%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">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/pubmed?term=((Chemoresistant+colorectal+cancer+cells%2C+the+cancer+stem+cell+phenotype%2C+and+increased+sensitivity+to+insulin-like+growth+factor-I+receptor+inhibition%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">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/pubmed?term=((Induction+of+cancer+metastasis+by+cyclophosphamide+pretreatment+of+host+mice%3A+an+opposite+effect+of+chemotherapy%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">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/pubmed?term=((A+phase+I+trial+of+tumor+lysate-pulsed+dendritic+cells+in+the+treatment+of+advanced+cancer%5BTitle%5D)+AND+%22Clin.+Cancer+Res%22%5BJournal%5D)">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/pubmed?term=((Anti-CD137+monoclonal+antibody+administration+augments+the+antitumor+efficacy+of+dendritic+cell-based+vaccines%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">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/pubmed?term=((T+cell-dependent+antitumor+immunity+mediated+by+secondary+lymphoid+tissue+chemokine%3A+augmentation+of+dendritic+cell-based+immunotherapy%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">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/pubmed?term=((Cancer+stem+cell+vaccination+confers+significant+antitumor+immunity%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">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/pubmed?term=((CXCR1+blockade+selectively+targets+human+breast+cancer+stem+cells+in+vitro+and+in+xenografts%5BTitle%5D)+AND+%22J.+Clin.+Invest%22%5BJournal%5D)">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/pubmed?term=((Aldehyde+dehydrogenase-expressing+colon+stem+cells+contribute+to+tumorigenesis+in+the+transition+from+colitis+to+cancer%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">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/pubmed?term=((Breast+cancer+cell+lines+contain+functional+cancer+stem+cells+with+metastatic+capacity+and+a+distinct+molecular+signature%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">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><tbody><tr><td>ALDHEFLOUR KIT</td><td>Stemcell Technologies</td><td>1700</td><td></td></tr><tr><td>Murine IL-4</td><td>Pepro Tech</td><td>214-14</td><td></td></tr><tr><td>Murine GM-CSF</td><td>Pepro Tech</td><td>315-03</td><td></td></tr><tr><td>Mouse GM-CSF ELISA KIT</td><td>BD Biosciences</td><td>555167</td><td></td></tr><tr><td>OptiPrep Density Gradient Medium</td><td>Sigma Aldrich</td><td>D1556</td><td></td></tr><tr><td>BD OptEIA TMB Substrated Reagent Set</td><td>BD Biosciences</td><td>555214</td><td></td></tr><tr><td>Equipment</td><td></td><td></td><td></td></tr><tr><td>BD FACS Aria Cell Sorter</td><td>BD Biosciences</td><td>336834</td><td></td></tr><tr><td>Kcjunior</td><td>Bio-Tek Instruments</td><td>176058</td><td></td></tr></tbody></table>

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

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) 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, naïve 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 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. 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&lt;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.  There were significantly higher productions of IFNγ 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&lt;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="Cell sorting flow cytometry graph, cultured/fresh D5 SCC7 with/without DEAB, shows cell population." fo:content-width="5in" fo:src="/files/ftp_upload/50561/50561fig1highres.jpg" src="/files/ftp_upload/50561/50561fig1.jpg" data-altupdatedat="1787630408000" data-alt="Figure 1"> Figure 1. 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="Mouse tumor cell sorting and dendritic cell pulsing diagram for ALDH\(^\text{high}\) cancer research." fo:content-width="5in" fo:src="/files/ftp_upload/50561/50561fig2highres.jpg" src="/files/ftp_upload/50561/50561fig2.jpg" data-altupdatedat="1787630408000" data-alt="Figure 2"> 
Figure 2. 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, respectively.
<a href="https://www.jove.com/files/ftp_upload/50561/50561fig2highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Tumor size comparison graph; PBS, H-TPDC, CSC-TPDC treatment effects; statistical significance." fo:content-width="5in" fo:src="/files/ftp_upload/50561/50561fig3highres.jpg" src="/files/ftp_upload/50561/50561fig3.jpg" data-altupdatedat="1787630408000" data-alt="Figure 3"> 
Figure 3. 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="Cytokine production analysis in TPDC populations; bar charts of IFN-γ and GM-CSF levels by activation method." fo:content-width="5in" fo:src="/files/ftp_upload/50561/50561fig4highres.jpg" src="/files/ftp_upload/50561/50561fig4.jpg" data-altupdatedat="1787630408000" data-alt="Figure 4"> 
Figure 4. More potent systemic cellular responses in immunocompetent host vaccinated with CSC-TPDC.  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>

Evaluated in syngeneic immunocompetent hosts, cancer stem cell (CSC) based dendritic cell (DC) vaccine demonstrated significantly higher antitumor immunity than traditional DC vaccines pulsed with heterogeneous bulk tumor 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 ...

The overall goal of this procedure is to evaluate the anti-tumor immunity of a cancer stem cell or CSC based dendritic cell or DC vaccine in an immunocompetent host. This is accomplished by first isolating alor positive aldehyde dehydrogenase or A LDH high CSCs from heterogeneous tumor cells. In the second step, CSC dcs are isolated and then pulsed with lysate for the vaccine.Next, the CSC lysate Pulses DC or TP DC vaccine is administered to normal syngeneic Immunocompetent hosts. Then in the final step, the protective anti-tumor efficacy of the C-S-C-T-P-D-C vaccine is assessed. Ultimately, ELI SA can be performed to evaluate the C-S-C-T-P-D-C vaccine-induced anti CSC immunity by host t-cells and antibodies.This project brings together two of the most exciting areas of cancer research, the area of cancer stem cells, and the area of tumor immunotherapy. Our lab was among the first to discover cancer stem cells in solid tumors. These are the seeds of the cancer that drive its growth as well as metastasis.What you'll see today is a new approach using immunotherapy to specifically target these cancer stem cells. We utilize a particular marker called aldehyde dehydrogenase, which can be detected by the Alde fluoro assay. You will see doctors Lin Liu and Huon Tau working along with Dr.Chow Lee.We hope that by targeting these cancer stem cells, we will improve the outcome for patients with many kinds of cancer. Begin by suspending the tumor cells of interest in alor assay buffer at a one times 10 to the six cells per milliliter concentration. Then transfer all but three milliliters of the tumor cells to each of the sample tubes and label four 12 by 75 millimeter polystyrene control test tubes as follows, unstained alor ALOR plus DEAB and seven A A D dispense one milliliter of the cell suspension into the unstained and seven A A D tubes and two milliliters into the alde floor tube.Then add five microliters of DEAB into the ALOR plus DEAB tube. Place the tubes on ice and then mix 10 microliters of activated alor substrate into the alor tube. Immediately transfer one milliliter of the substrate solution to the ALOR plus DEAB tube, and then add two microliters of activated alor substrate per 1 million cells to each of the sample tubes.Incubate all the tubes in a 37 degree Celsius water bath for 30 minutes, and then add five microliters of seven a a d to the seven A a D tube and one microliter of seven A a d per 1 million cells to each sample tube. Hold these tubes at four degrees Celsius for 10 minutes. Centrifuge all of the tubes for five minutes at 250 times G at room temperature and resuspend the pellets in Alde Fluor assay buffer for fluorescence activated cell sorting.To set the sorting gates, use the alde Fluor plus DEAB cells as the negative control and the seven A a D stained cells as the viability control. Based on these controls, establish a gait to distinguish the alor positive A LDH, high D five and SCC seven cells. Then use this to sort the sample cells into alor positive A LDH, high D five and SCC seven populations.To generate the dendritic cells first isolate the femur and tibia bones from a C 57 black six and a C3 H mouse. Sterilize the bones in 75%ethanol for one minute at room temperature and then wash them in HBSS. Next, cut the ends off the bones and then use a 10 milliliter syringe equipped with a 21 gauge needle to flush the bone marrow into a Petri dish, aspirate and dispense the cells a few times to create a single cell suspension and then spin down the cells.Incubate the pellet in five milliliters of red blood cell lysis buffer for one minute in a 37 degree Celsius water bath. After counting the cells, adjust the volume to one times 10 to the six cells per milliliter in culture medium supplemented with G-M-C-S-F and IL four. Then culture the cells at 37 degrees Celsius and 5%CO2, refreshing the media and cytokines after three days.On day five, harvest the immature dendritic cells. After spinning down the cells, resuspend the pellets in five milliliters of culture media, and then slowly layer the cell suspension onto freshly prepared isolation media. After centrifuging the cells, again, collect the dcs between the culture medium and isolation medium layers.Then after counting the cells, adjust the volume to a one times 10 to the six cell per milliliter concentration in culture medium, supplemented with G-M-C-S-F and IL four now create the tumor lysates by subjecting the sorted cells to rapid freeze thaw exposures four times followed by centrifugation to collect the membrane portion of the lysates. Then pulse the DCS with the lysate from the sorted autologous A LDH high cells. To prepare the CSC TPCs.To prepare the H-T-P-D-C pulse, the dcs with unsorted heterogeneous tumor cell lysate to vaccinate the experimental recipient animals subcutaneously inject one times 10 to the sixth of the pulsed cells, suspended in 100 microliters of PBS into the left flank of each mouse after vaccination intravenously, administer heterogeneous one times 10 to the fifth D five tumor cells in 100 microliters of PBS to challenge the C 57 black six recipient mice. For the SCC seven model subcutaneously. Challenge the C3 H recipient mice with one times 10 to the sixth unsorted SCC seven tumor cells in 100 microliters of PBS on the opposite side of the DC vaccine.Create a single cell suspension from the spleens of the recipient C3 H mice. Then activate the splenic t and B cells with immobilized anti CD three and anti CD 28 monoclonal antibodies in culture medium supplemented with the appropriate stimulus after four days, collect the supernatants and then coat Eliza plates with antibodies against interferon gamma G-M-C-S-F or IgG at four degrees Celsius overnight. The next day block for non-specific binding plate, the standards and the just collected supernatants, and then incubate the plates at room temperature after washing, add HRP detection antibody for one hour and then add the TMB substrate.Finally measure the absorbance on an ELISA plate reader at 450 nanometers within 30 minutes of stopping the reaction. Alor has been used as a marker to isolate stem cells in multiple malignancies, including the two cancer stem cell enriched populations D five and SCC seven specifically as demonstrated in these dot plots, alor positive cells contribute approximately 0.5%and 5.2%of cultured D five and SCC seven tumor cell lines respectively. Freshly harvested tumor cells also have been determined to contain 2.5%and 4.2%of the alor positive cells from in vivo established D five and SCC seven tumors respectively.Tumor cells treated with D-E-A-B-A specific A LDH inhibitor were used as a negative control. The immunogenicity of cancer stem cells was evaluated by examining the protective anti-tumor immunity induced by CSC TPCs using the just demonstrated vaccination model. As shown in this table, mice treated with the H TPCs developed fewer lung metastases than mice treated with PBS alone.Importantly, mice treated with CSCT PCs had still fewer lung metastases than animals treated with the H-T-P-D-C or PBS alone In the SCC seven model, normal C3 H animals were vaccinated as just demonstrated compared with the control group. H TPCs induced modest anti-tumor immunity while CSC TPCs induced significant tumor growth inhibition. These results indicate that CSCs may be able to be used as a more effective antigen source for loading dcs than traditional unsorted tumor cells for inducing protective immunity against the challenge of tumor cells.To further understand the mechanisms underlying the observed CSC induced protective anti-tumor immunity, cytes from DC vaccinated animals were activated and their cytokine production was measured by EISA as just demonstrated there was significantly higher cytokine production by the cytes harvested from animals vaccinated with D five CSC TPCs, or S CCC seven CSC PCs. Then those harvested from animals vaccinated by D five H TPCs or SCC seven HTCs. Today you've seen how to use the Alor assay to isolate cancer stem cells.These cancer stem cells can then be used to develop immunotherapies utilizing dendritic cells. It is our hope that these immunotherapies either used alone or in combination with existing therapies will improve the outlook for patients with many types of cancer.

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Research

JoVE Journal

Immunology and Infection

This video demonstrates a cell-based tumor vaccination assay. A mouse, intradermally injected with B16-F10 melanoma cells for tumor formation, is vaccinated with B16-F10 cells secreting Flt3L cytokine near the developed tumor. The secreted Flt3L from the vaccine promotes the maturation and proliferation of dendritic cells that then present tumor antigens to T cells in lymph nodes, triggering a targeted anti-tumor immune response.

Tumor Implantation and Injection of a Tumor Vaccine in a Mouse Model

The most commonly used method for generating large numbers of autologous dendritic cells (DCs) for use in tumor immunotherapy is described.  The method uses IL-4 and GM-CSF to differentiate DCs from monocytes.  The immature DCs are stimulated to mature and then loaded with antigens before they are injected back into the patient.

Preparation of Tumor Antigen-loaded Mature Dendritic Cells for Immunotherapy

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

The goal of this protocol is to show the protocol for reprogramming melanoma tumor-infiltrating lymphocytes into induced pluripotent stem cells.

Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes

This article describes a novel method to generate tumor antigen-specific induced pluripotent stem cell-derived thymic emigrants (iTE) by a three-dimensional (3D) thymic culture system. iTE are a homogenous subset of T cells closely related to naïve T cells with the capacity for proliferation, memory formation, and tumor suppression.

A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants

The transimmunization (TI) device or plate and related protocols have been developed to replicate the key features of extracorporeal photochemotherapy (ECP), in an experimental setting, allowing for production of physiologically activated, tunable dendritic cells (DCs) for cancer immunotherapy.

Novel Protocol for Generating Physiologic Immunogenic Dendritic Cells

This article describes a method to generate functional tumor antigen-specific induced pluripotent stem cell-derived CD8αβ+ single positive T cells using OP9/DLL1 co-culture system.

Using Human Induced Pluripotent Stem Cells for the Generation of Tumor Antigen-specific T Cells

Presented here is a protocol for the effective suppression of hepatitis B virus (HBV) replication in mice by utilizing adoptive cell transfer (ACT) of stem cell-derived viral antigen (Ag)-specific T lymphocytes. This procedure may be adapted for potential ACT-based immunotherapy of HBV infection.

Stem Cell-Derived Viral Ag-Specific T Lymphocytes Suppress HBV Replication in Mice

The protocol presents a cancer immunotherapy model using cell-based tumor vaccination with Flt3L-expressing B16-F10 melanoma. This protocol demonstrates the procedures, including preparation of cultured tumor cells, tumor implantation, cell irradiation, measurement of tumor growth, isolation of intratumoral immune cells, and flow cytometry analysis.

Experimental Melanoma Immunotherapy Model Using Tumor Vaccination with a Hematopoietic Cytokine

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 described.

Generation of Monocyte-Derived Dendritic Cells with Differing Sialylated Phenotypes

This protocol describes the in vivo reprogramming of mouse cancer cells into type 1 dendritic-like cells within the tumor microenvironment through enforced expression of the transcription factors PU.1, IRF8, and BATF3.

Tractable In Vivo Reprogramming of Tumor Cells to Type 1 Conventional Dendritic Cell-like Cells

Learn to generate a novel dendritic cell vaccine using melanoma and squamous cancer stem cells. This method demonstrates stronger anti-tumor immunity than traditional DC vaccines in immunocompetent hosts.

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

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