Name: isolation and expansion of cytotoxic cytokine-induced killer t cells for cancer treatment
Uploaded: 2020-01-24T14:00:00
Description: Watch this Scientific Journal Video about Isolation and Expansion of Cytotoxic Cytokine-induced Killer T Cells for Cancer Treatment at JoVE.com

This study was supported by China Medical University Hospital (DMR-Cell-1809).

The clinical protocol was performed and approved in accordance with the guidelines of the Institutional Review Board of the China Medical University and Hospital Research Ethics Committee. Peripheral blood specimens were harvested from healthy volunteers with their informed consent.
1. Preparation of materials
<ol>
	<li>Store reagents, antibodies, and chemicals as shown in the Material Safety Data Sheet (MSDS). Dissolve the drugs or cytokines in solvents as stock solutions and then aliquot f...

<ol>
	<li>Cappuzzello, E., 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=Cytokines+for+the+induction+of+antitumor+effectors:+The+paradigm+of+Cytokine-Induced+Killer+(CIK)+cells.">Cytokines for the induction of antitumor effectors: The paradigm of Cytokine-Induced Killer (CIK) cells.</a> Cytokine &amp; Growth Factor Reviews. 36, 99-105 (2017).</li><li>Schmidt-Wolf, R. 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=Propagation+of+large+numbers+of+T+cells+with+natural+killer+cell+markers.">Propagation of large numbers of T cells with natural killer cell markers.</a> British Journal of Haematology. 87 (3), 453-458 (1994).</li><li>Grainger, 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=Wnt+Signaling+in+Hematological+Malignancies.">Wnt Signaling in Hematological Malignancies.</a> Progress in Molecular Biology and Translational Science. 153, 321-341 (2018).</li><li>Dai, 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=Implication+of+combined+PD-L1/PD-1+blockade+with+cytokine-induced+killer+cells+as+a+synergistic+immunotherapy+for+gastrointestinal+cancer.">Implication of combined PD-L1/PD-1 blockade with cytokine-induced killer cells as a synergistic immunotherapy for gastrointestinal cancer.</a> Oncotarget. 7 (9), 10332-10344 (2016).</li><li>Schmidt-Wolf, I. 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=Use+of+a+SCID+mouse/human+lymphoma+model+to+evaluate+cytokine-induced+killer+cells+with+potent+antitumor+cell+activity.">Use of a SCID mouse/human lymphoma model to evaluate cytokine-induced killer cells with potent antitumor cell activity.</a> TheJournal of Experimental Medicine. 174 (1), 139-149 (1991).</li><li>Introna, 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=Rapid+and+massive+expansion+of+cord+blood-derived+cytokine-induced+killer+cells:+an+innovative+proposal+for+the+treatment+of+leukemia+relapse+after+cord+blood+transplantation.">Rapid and massive expansion of cord blood-derived cytokine-induced killer cells: an innovative proposal for the treatment of leukemia relapse after cord blood transplantation.</a> Bone Marrow Transplantation. 38 (9), 621-627 (2006).</li><li>Schmeel, L. 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=Cytokine-induced+killer+(CIK)+cells+in+cancer+immunotherapy:+report+of+the+international+registry+on+CIK+cells+(IRCC).">Cytokine-induced killer (CIK) cells in cancer immunotherapy: report of the international registry on CIK cells (IRCC).</a> Journal of Cancer Research and Clinical Oncology. 141 (5), 839-849 (2015).</li><li>Rutella, 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=Adoptive+immunotherapy+with+cytokine-induced+killer+cells+generated+with+a+new+good+manufacturing+practice-grade+protocol.">Adoptive immunotherapy with cytokine-induced killer cells generated with a new good manufacturing practice-grade protocol.</a> Cytotherapy. 14 (7), 841-850 (2012).</li><li>Nausch, N., 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=NKG2D+ligands+in+tumor+immunity.">NKG2D ligands in tumor immunity.</a> Oncogene. 27 (45), 5944-5958 (2008).</li><li>Gammaitoni, 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=Effective+activity+of+cytokine-induced+killer+cells+against+autologous+metastatic+melanoma+including+cells+with+stemness+features.">Effective activity of cytokine-induced killer cells against autologous metastatic melanoma including cells with stemness features.</a> Clinical Cancer Research. 19 (16), 4347-4358 (2013).</li><li>Rettinger, E., 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=The+cytotoxic+potential+of+interleukin-15-stimulated+cytokine-induced+killer+cells+against+leukemia+cells.">The cytotoxic potential of interleukin-15-stimulated cytokine-induced killer cells against leukemia cells.</a> Cytotherapy. 14 (1), 91-103 (2012).</li><li>Narayan, 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=Donor-derived+cytokine-induced+killer+cell+infusion+as+consolidation+after+nonmyeloablative+allogeneic+transplantation+for+myeloid+neoplasms.">Donor-derived cytokine-induced killer cell infusion as consolidation after nonmyeloablative allogeneic transplantation for myeloid neoplasms.</a> Biology of Blood and Marrow Transplantation. 19, 1083 (2019).</li><li>Castiglia, 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=Cytokines+induced+killer+cells+produced+in+good+manufacturing+practices+conditions:+identification+of+the+most+advantageous+and+safest+expansion+method+in+terms+of+viability,+cellular+growth+and+identity.">Cytokines induced killer cells produced in good manufacturing practices conditions: identification of the most advantageous and safest expansion method in terms of viability, cellular growth and identity.</a> Journal of Translational Medicine. 16 (1), 237 (2018).</li><li>Bonanno, 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=Thymoglobulin,+interferon-&#947;+and+interleukin-2+efficiently+expand+cytokine-induced+killer+(CIK)+cells+in+clinical-grade+cultures.">Thymoglobulin, interferon-&#947; and interleukin-2 efficiently expand cytokine-induced killer (CIK) cells in clinical-grade cultures.</a> Journal of Translational Medicine. 8, 129 (2010).</li><li>Iudicone, P., 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=Interleukin-15+enhances+cytokine+induced+killer+(CIK)+cytotoxic+potential+against+epithelial+cancer+cell+lines+via+an+innate+pathway.">Interleukin-15 enhances cytokine induced killer (CIK) cytotoxic potential against epithelial cancer cell lines via an innate pathway.</a> Human Immunology. 77 (12), 1239-1247 (2016).</li><li>Liu, 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=Phenotypic+characterization+and+anticancer+capacity+of+CD8++cytokine-induced+killer+cells+after+antigen-induced+expansion.">Phenotypic characterization and anticancer capacity of CD8+ cytokine-induced killer cells after antigen-induced expansion.</a> PLoS One. 12 (4), 0175704 (2017).</li><li>Chen, 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=Cytokine-induced+killer+cells+as+a+feasible+adoptive+immunotherapy+for+the+treatment+of+lung+cancer.">Cytokine-induced killer cells as a feasible adoptive immunotherapy for the treatment of lung cancer.</a> Cell Death &amp; Disease. 9 (3), 366 (2018).</li><li>Tario, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+cell+proliferation+by+dye+dilution:+considerations+for+probe+selection.">Monitoring cell proliferation by dye dilution: considerations for probe selection.</a> Methods in Molecular Biology. 1678, 249-299 (2018).</li><li>Last'ovicka, 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=Assessment+of+lymphocyte+proliferation:+CFSE+kills+dividing+cells+and+modulates+expression+of+activation+markers.">Assessment of lymphocyte proliferation: CFSE kills dividing cells and modulates expression of activation markers.</a> Cellular Immunology. 256 (1-2), 79-85 (2009).</li><li>Yoshida, T., 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=Characterization+of+natural+killer+cells+in+tamarins:+a+technical+basis+for+studies+of+innate+immunity.">Characterization of natural killer cells in tamarins: a technical basis for studies of innate immunity.</a> Frontiers in Microbiology. 1, 1-9 (2010).</li></ol>

The described method is a fast, convenient, and reliable protocol for the isolation and expansion of cytotoxic cytokine-induced killer (CIK) T cells from whole blood samples of healthy donors. It also shows the cytotoxic effect of CIK against leukemia (K562) and ovarian cancer cells (OC-3) using a flow cytometry setup and tracking (CS &#38; T) system. CIK cells can be induced and expanded in good manufactory practices (GMP) conditions by using GMP-grade cytokines and serum-free medium for further clinical infusion<sup cl...

Cytotoxic T lymphocytes are a specific immune effector cell population that mediates immune responses against cancer. Several effector cell populations including lymphokine-activated killer (LAK) cells, tumor-infiltrating lymphocytes (TILs), natural killer (NK) cells, &#947;&#948; T cells, and cytokine-induced killer (CIK) cells have been developed for adoptive T cell therapy (ACT) purposes1. There is a growing interest in CIK cells, because they represent a mixture of cytokine-induced cytotoxic cell populations expanded from autologous peripheral blood mononuclear cells (PBMCs)2.
The uncontrolled growth of lymphoid progenitor cells, myeloblasts, and lymphoblasts leads to three main types of blood cancers (i.e., leukemia, lymphoma, and myeloma), solid tumors, including carcinomas (e.g., lung cancer, gastric cancer, cervical cancer), and sarcomas, among other cancers3. CIK cells are a mixture of cell populations that exhibit a wide range of major histocompatibility complex (MHC)-unrestricted antitumor activity and thus hold promise for the treatment of hematological and advanced tumors4,5,6,7. CIK cells comprise a combination of cells, including T cells (CD3+CD56&#8722;), NK-T cells (CD3+CD56+), and NK cells (CD3&#8211;CD56+). Optimization of the CIK induction protocol by use of a fixed schedule for the addition of IFN-&#947;, anti-CD3 antibody, and IL-2, results in the expansion of CIK cells8. The cytotoxic capability of CIK cells against cancer cells mainly depends on the engagement of NK group 2 member D (a member of the C-type lectin-like receptor family) NKG2D ligands on tumor cells, and on perforin-mediated pathways9. The results of a preclinical study revealed that IL-15-stimulated CIK cells induced potent cytotoxicity against primary and acute myeloid leukemia cell lines in vitro and exhibited a lower alloreactivity against normal PBMCs and fibroblasts9. Recently, the outcome of one-time healthy donor-derived CIK (1&#8239;x&#8239;108/kg CD3+ cells) infusion as consolidation following nonmyeloablative allogeneic transplantation for myeloid neoplasms treatment in a phase II clinical study was published10.
In the present study, we developed an optimized cell culture formula composed of IFN-&#947;, IL-1&#945;, anti-CD3 antibody, and IL-2 added to the hematopoietic cell medium to increase CIK production, and investigated the cytotoxic effect of CIK cells against human chronic myeloid leukemia (K562) cells and ovarian cancer (OC-3) cells.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>7-Amino Actinomycin D</td>
			<td>BD</td>
			<td>559925</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>APC Mouse Anti-Human CD56 antibody</td>
			<td>BD</td>
			<td>555518</td>
			<td>B159</td>
			<td></td>
		</tr>
		<tr>
			<td>APC Mouse IgG1, &#954; Isotype Control</td>
			<td>BD</td>
			<td>555751</td>
			<td>MOPC-21</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSCanto II Flow Cytometer</td>
			<td>BD</td>
			<td>338962</td>
			<td></td>
			<td>SN: R33896202856</td>
		</tr>
		<tr>
			<td>Carboxyfluorescein diacetate succinimidyl ester (CFSE)</td>
			<td>BD</td>
			<td>565082</td>
			<td></td>
			<td>Reconstritution of CFSE dye (500 mg) with 90 mL of DMSO</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose solution</td>
			<td>SIGMA</td>
			<td>G8644</td>
			<td></td>
			<td>For K562 cell culture. Add 12.5 mL to 500 mL of complete medium</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium/F12</td>
			<td>HyClone</td>
			<td>SH30023.02</td>
			<td></td>
			<td>Basal medium for OC-3 cell culture</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>HyClone</td>
			<td>SH30084.03</td>
			<td></td>
			<td>For K562 and OC-3 cell culture. Complete medium contains 10% of FBS</td>
		</tr>
		<tr>
			<td>Ficoll-Paque Plus</td>
			<td>GE Healthcare Life Sciences</td>
			<td>71101700-EK</td>
			<td></td>
			<td>Density gradient solution</td>
		</tr>
		<tr>
			<td>FITC Mouse Anti-Human CD3 antibody</td>
			<td>BD</td>
			<td>555332</td>
			<td>UCHT1</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Mouse IgG1, &#954; Isotype Control</td>
			<td>BD</td>
			<td>555748</td>
			<td>MOPC-21</td>
			<td></td>
		</tr>
		<tr>
			<td>Human anti-CD3 mAb</td>
			<td>TaKaRa</td>
			<td>T210</td>
			<td>OKT3</td>
			<td>Add 2.5 mL of stock (1 mg/1 mL) to 50 mL of Induction medium. Storage stock at -80 &#176;C</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
			<td>Add 5 mL of stock (10,000 U/mL) to 500 mL of complete medium. Storage stock at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Proleukin</td>
			<td>NOVARTIS</td>
			<td></td>
			<td></td>
			<td>Reconstitution of Proleukin Powder (22x106 IU) with 1.2 mL of sterile water and add 2.7 mL to 50 mL of Induction medium. Storage stock at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Recombinant Human Interferon-gamma</td>
			<td>CellGenix</td>
			<td>1425-050</td>
			<td></td>
			<td>Reconstitution of rh IFN-g (5x105 IU/50 &#181;g) with 200 &#181;L of sterile water and add 20 mL to 50 mL of Induction medium. Storage stock at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Recombinant Human Interleukin-1 alpha</td>
			<td>PEPROTECH</td>
			<td>200-01A</td>
			<td></td>
			<td>Reconstitution rh IL-1&#945; (10 &#181;g) with 1 mL of sterile water and add 5 mL to 50 mL of Induction medium. Storage stock at -20 &#176;C</td>
		</tr>
		<tr>
			<td>RPMI1640 medium</td>
			<td>Gibco</td>
			<td>11875-085</td>
			<td></td>
			<td>Basal medium for K562 cell culture. Storage stock at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Sigma 3-18K Centrifuge</td>
			<td>Sigma</td>
			<td>10295</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express Enzyme</td>
			<td>Gibco</td>
			<td>12605028</td>
			<td></td>
			<td>Cell dissociation enzyme; For deattachment of adheren cells. Storage at room temperature</td>
		</tr>
		<tr>
			<td>X-VIVO 15 medium</td>
			<td>Lonza</td>
			<td>04-418Q</td>
			<td></td>
			<td>Basal medium for PBMC and CIK cells. Storage at 4 &#176;C</td>
		</tr>
	</tbody>
</table>

isolation and expansion of cytotoxic cytokine-induced killer t cells for cancer treatment

Adoptive cellular immunotherapy focuses on restoring cancer recognition via the immune system and improves effective tumor cell killing. Cytokine-induced killer (CIK) T cell therapy has been reported to exert significant cytotoxic effects against cancer cells and to reduce the adverse effects of surgery, radiation, and chemotherapy in cancer treatments. CIK can be derived from peripheral blood mononuclear cells (PBMCs), bone marrow, and umbilical cord blood. CIK cells are a heterogeneous subpopulation of T cells with CD3+CD56+ and natural killer (NK) phenotypic characteristics that include major histocompatibility complex (MHC)-unrestricted antitumor activity. This study describes a qualified, clinically applicable, flow cytometry-based method for the quantification of the cytolytic capability of PBMC-derived CIK cells against hematological and solid cancer cells. In the cytolytic assay, CIK cells are co-incubated at different ratios with prestained target tumor cells. After the incubation period, the number of target cells are determined by a nucleic acid-binding stain to detect dead cells. This method is applicable to both research and diagnostic applications. CIK cells possess potent cytotoxicity that could be explored as an alternative strategy for cancer treatment upon its preclinical evaluation by a cytometer setup and tracking (CS &#38; T)-based flow cytometry system.

The purpose of the present protocol is to isolate and expand cytokine-induced killer (CIK) T cells from peripheral blood monocytes and evaluate the cytotoxic effect of CIK against hematological malignancy and solid cancer cells, respectively. The induction of CIK was identified by the CD3/CD56 recognition. Figure 1A shows the protocol for CIK induction and expansion. The representative results of the gating strategy for analyzing the subpopulation of CD3+CD56...

Watch this Scientific Journal Video about Isolation and Expansion of Cytotoxic Cytokine-induced Killer T Cells for Cancer Treatment at JoVE.com

Isolation and Expansion of Cytotoxic Cytokine-induced Killer T Cells for Cancer Treatment

Here, we present a protocol to perform the isolation and expansion of peripheral blood mononuclear cells-derived cytokine-induced CD3+CD56+ killer cells and illustrate their cytotoxicity effect against hematological and solid cancer cells by using an in vitro diagnosis flow cytometry system.

Adoptive cellular immunotherapy focuses on restoring cancer recognition via the immune system and improves effective tumor cell killing. Cytokine-induced ...

We optimized a highly qualified and clinically applicable method for the expansion of PBMC-derived cytokine-induced killer T cells and for evaluation of their cytotoxicity against cancers. The advantage of this technique is to provide a standard operating procedure for technicians and clinicians to quantify the potency of cytokine-induced killer cells in both scientific research and clinical evaluation. Begin this procedure with a preparation of CIK cells as described in the text protocol, then wash the CIK cells with 10 milliliters of sterile PBS.Centrifuge for 10 minutes at 300 times G and 18 to 20 degrees Celsius. Aspirate the supernatant and resuspend the cells with five milliliters of PBS. Count the cell numbers and test cell viability using the trypan blue exclusion assay.Aliquot the CIK cells into six steril 1.5 milliliter tubes at a density of approximately 5 to one million cells per milliliter of PBS. Label and treat the cells as detailed in the text protocol. Gently mix the CIK cells with the antibodies by gently pipetting up and down at least three times with a one milliliter steril pipette.Incubate the cells for 15 minutes at room temperature in the dark. Centrifuge the tubes for 10 minute at 300 times G and 18 to 20 degrees Celsius. Following centrifugation, aspirate the supernatant and resuspend the cell pellet with one milliliter of PBS, then gently pipette the cells up and down at least three times with a one milliliter sterile pipette.Transfer the cell suspension to a sterile five milliliter polystyrene round bottom tube with a cell strainer cap by gently pipetting through the cap, then place the tubes on the carousel in order. Open the flow cytometry analysis software and create an experimental folder, then click the new specimen button to add a specimen and tube to the experiment. Name the tubes as listed in the text protocol.To create a scatter gating system for the CIK cell populations, first select tube one and click on the dot plot button to create an FSCA SSCA plot. Draw a rectangle gate over the entire cell population with an FSCA threshold greater than 5000 to exclude cell debris. Select the FSCA FSCH parameter for the new dot plot and draw a polygon gate around all single cells.Select the count versus FITC-conjugated CD3 and count versus APC-conjugated CD56 parameter for the new histogram plot. Select the FITC-conjugated CD3 versus APC-conjugated CD56 parameter for the new dot plot and draw a four quadrant gate to define the four subpopulations. Record the data from 20, 000 single cells in each specimen.Click the load sample button to analyze the blank control sample first. Identify the whole CIK cell population by using the CD56 and CD3 channel parameters. Open the files containing the statistical values of the individual specimen to analyze CIK cell populations and reprint them into analysis files.To perform the cytotoxic assay, coculture CIK and human chronic myeloid leukemia K562 cells by adding one milliliter of K562 cells to each well in a six-well plate at a density of 5 million cells per milliliter, then add one milliliter of basal medium with or without CIK cells to the six-well plate as listed in the text protocol. Mix the cell suspensions by gently pipetting up and down at least three times. Place the plate in the incubator for 24 hours.Now, coculture CIK and ovarian OC-3 cells by adding one milliliter of basal medium with or without CIK cells to the six-well plate as listed in the text protocol. Mix the cell suspensions by gently pipetting up and down at least three times. Put the plate in the incubator for 24 hours.Following incubation, harvest the CIK K562 cell suspension directly into a 15 milliliter sterile tube. To harvest both the suspension and adherence cells from the CIK OC-3 groups, first transfer the cell suspension to a 15 milliliter sterile tube. Wash the well with one milliliter of steril PBS, collect the PBS, and add it to the tube, then add 5 milliliters of cell dissociation enzyme solution and incubate for five minutes at 37 degrees Celsius.Add one milliliter of the solution from the same tube to the corresponding well, and gently mix the cells by pipetting up and down at least three times with a one milliliter sterile pipette. Collect all the cells in the same tube. Centrifuge at 300 times G for 10 minutes.Aspirate the supernatant and resuspend the cells in one milliliter of sterile PBS. Pellet the cells at 300 times G for 10 minutes, then aspirate the supernatant and resuspend the cells in 100 microliters of sterile PBS. Add five microliters of 7-AAD dye to the cell suspension.Gently mix the cells by pipetting up and down at least three times with a one milliliter sterile pipette. Incubate for 10 minutes and leave in the dark before analysis. To perform the cytolytic capability assay, mix the cell suspension and repeat the preparation for flow cytometry.Click the new specimen button to add a specimen and tube to the experiment. Name the tubes as listed in the text protocol. To create a scatter gating system for the cytolytic assay, first select tube one and click on the dot plot button to create an FSCA SSCA plot.Draw a rectangle gate over all of events with an FSCA threshold greater than 50, 000 to exclude cell debris. Select the SSCA CFSE parameter for the new dot plot, then select the 7-AAD CFSE parameter for the new dot plot and draw a four quadrant gate to define the four subpopulations. Click the load sample button to analyze the blank control sample first.Adjust the voltage of SSCA and FSCA. Identify the dead cell population by using the CFSE and 7-AAD channel parameters. Record the data from greater than 20, 000 CFSE positive cells in each specimen.Open the files containing the statistical values of each individual specimen to analyze the nonviable cell populations, and export the data into analysis files. The representative results of the gating strategy for analyzing the subpopulation of CD-3 positive CD56 positive T-cells from healthy donors are shown with the statistical analysis of the CIK proportion from three individuals. The CD-3 positive CD56 positive cell proportion significantly increased after 14 days of expansion.This is evident through the 65%for the original PBMC which increased to 27.4%for the CIK cells harvested on day 14. In this culture system, the CIK cells yielded about half a hundred fold changes compared to the original number of PBMCs. K562 cells were stained with a nonfluorescent dye CFSE, which is cleaved by intracellular esterases within viable cells and becomes a highly fluorescent dye.The size and granularity of the CIK and CFSE-positive cells are illustrated. The CFSE stained K562 cells were co-treated with CIK cells at a ratio of zero to one, five to one, and 10 to one. These cells were stained with 7-AAD dye as a viability probe for dead cell exclusion.The 7-AAD positive cells of CFSE positive K562 cells were all evaluated. The CFSE-stained OC-3 cells were co-treated with CIK cells at a ratio of 10 to one. The obvious cytotoxicity of CIK against OC-3 cells is demonstrated here following 24 hours of incubation.CIK T-cells has been reported to exert significant cytotoxic effects against cancer cells and reduce the adverse effects of surgery, radiation, and chemotherapy in cancer treatments. Immunotherapy is a promising treatment for a number of cancers. CIK can be efficiently expanded in vitro by incubation of patient-derived PMBCs in GMP-grade condition for further autologous infusion.Following this protocol, we could execute the immune cellular therapy in a GTP-or GMP-grade facility for further clinical cancer treatment.

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Cancer Research

Generation and Isolation of Cell Cycle-arrested Cells with Complex Karyotypes

Chromosome mis-segregation leads to aneuploidy, a condition in which cells harbor an imbalanced chromosome number. Several lines of evidence strongly indicate that aneuploidy triggers genome instability, ultimately generating cells with complex karyotypes that arrest their proliferation. Isolation and characterization of cells harboring complex karyotypes are crucial to study the impact of an imbalanced chromosome number on cell physiology. To date, no methods have been established to reliably isolate such aneuploid cells. This paper provides a protocol for the enrichment and analysis of aneuploid cells with complex karyotypes utilizing standard, inexpensive tissue culture techniques. This protocol can be used to analyze several features of aneuploid cells with complex karyotypes including their induced senescence-associated secretory phenotype, pro-inflammatory properties, and ability to interact with immune cells. Because cancer cells often harbor imbalances in chromosome number, it is crucial to decipher how aneuploidy impacts cell physiology in normal cells, with the ultimate goal of uncovering both its pro- and anti-tumorigenic effects.

Aneuploidy leads to genome instability, which eventually produces cell cycle-arrested cells with complex karyotypes. This paper provides a simple and convenient method to isolate aneuploid cells with complex karyotypes that cease to divide.

Chromosome mis-segregation leads to aneuploidy, a condition in which cells harbor an imbalanced chromosome number. Several lines of evidence strongly ...

Preparation of Exosomes for siRNA Delivery to Cancer Cells

Extracellular vesicles, in particular exosomes, have recently gained interest as novel drug delivery vectors due to their biological origin, abundance, and intrinsic capability in intercellular delivery of various biomolecules. This work establishes an isolation protocol to achieve high yield and high purity of exosomes for siRNA delivery. Human Embryonic Kidney cells (HEK-293 cells) are cultured in bioreactor flasks and the culture supernatant (hereon referred to as conditioned medium) is harvested on a weekly basis to allow for enrichment of HEK-293 exosomes. The conditioned medium (CM) is pre-cleared of dead cells and cellular debris by differential centrifugation and is subjected to ultracentrifugation onto a sucrose cushion followed by a washing step, to collect the exosomes. Isolated HEK-293 exosomes are characterized for yield, morphology and exosomal marker expression by nanoparticle tracking analysis, protein quantification, electron microscopy and flow cytometry, respectively. Small interfering RNA (siRNA), fluorescently labeled with Atto655, is loaded into exosomes by electroporation and excess siRNA is removed by gel filtration. Cell uptake in PANC-1 cancer cells, after 24 h incubation at 37 &#176;C, is confirmed by flow cytometry. HEK-293 exosomes are 107.0 &#177; 8.2 nm in diameter. The exosome yield and particle-to-protein ratio (P:P) ratio are 6.99 &#177; 0.22 &#215; 1012 particle/mL and 8.3 &#177; 1.7 &#215; 1010 particle/&#181;g, respectively. The encapsulation efficiency of siRNA in exosomes is ~ 10-20%. Forty percent of the cells show positive signals for Atto655 at 24 h post-incubation. In conclusion, exosome isolation by ultracentrifugation onto sucrose cushion offers a combination of good yield and purity. siRNA could be successfully loaded into exosomes by electroporation and subsequently delivered into cancer cells in vitro. This protocol offers a standard procedure for developing siRNA-loaded exosomes for efficient delivery to cancer cells.

An exosome is a new generation of drug delivery carriers. We established an exosome isolation protocol with high yield and purity for siRNA delivery. We also encapsulated fluorescently labelled non-specific siRNA into exosomes and investigated the cellular uptake of siRNA-loaded exosomes in cancer cells.

Extracellular vesicles, in particular exosomes, have recently gained interest as novel drug delivery vectors due to their biological origin, abundance, ...

Manufacturing Chimeric Antigen Receptor (CAR) T Cells for Adoptive Immunotherapy

Adoptive immunotherapy holds promise for the treatment of cancer and infectious disease. We describe a simple approach to transduce primary human T cells with chimeric antigen receptor (CAR) and expand their progeny ex vivo. We include assays to measure CAR expression as well as differentiation, proliferative capacity and cytolytic activity. We describe assays to measure effector cytokine production and inflammatory cytokine secretion in CAR T cells. Our approach provides a reliable and comprehensive method to culture CAR T cells for preclinical models of adoptive immunotherapy.

Manufacturing Chimeric Antigen Receptor CAR T Cells for Adoptive Immunotherapy

We describe an approach to reliably generate chimeric antigen receptor (CAR) T cells and test their differentiation and function in vitro and in vivo.

Adoptive immunotherapy holds promise for the treatment of cancer and infectious disease. We describe a simple approach to transduce primary human T cells ...

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

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of patients with advanced cancer. Adoptive T cell transfer (ACT) of highly enriched tumor antigen-specific T cells has been shown to cause durable regression of metastatic cancer in some patients. However, during expansion, these cells may become exhausted or senescent, limiting their effector function and persistence in vivo. Induced pluripotent stem cell (iPSC) technology may overcome these obstacles by leading to in vitro generation of large numbers of less differentiated tumor antigen-specific T cells. Human iPSC (hiPSC) have the capacity to differentiate into any type of somatic cell, including lymphocytes, which retain the original T cell receptor (TCR) genomic rearrangement when a T cell is used as a starting cell. Therefore, reprogramming of human tumor antigen-specific T cells to hiPSC followed by redifferentiation to T cell lineage has the potential to produce rejuvenated tumor antigen-specific T cells. Described here is a method for generating tumor antigen-specific CD8&#945;&#946;+ single positive (SP) T cells from hiPSC using OP9/DLL1 co-culture system. This method is a powerful tool for in vitro T cell lineage generation and will facilitate the development of in vitro derived T cells for use in regenerative medicine and cell-based therapies.

This article describes a method to generate functional tumor antigen-specific induced pluripotent stem cell-derived CD8&#945;&#946;+ single positive T cells using OP9/DLL1 co-culture system.

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of ...

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response.
This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 &#181;L basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFN&#947; and TNF&#945;) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFN&#947;+, TNF&#945;+ and CD107a+ CD4+ and CD8+ T cells was quantified.
This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

Isolating Human Peripheral Blood Mononuclear Cells and CD4+ T cells from S&#233;zary Syndrome Patients for Transcriptomic Profiling

Cutaneous T-cell lymphomas (CTCL) are derived from the transformation and uncontrolled proliferation of mature skin-homing T cells, and mycosis fungoides (MF) and S&#233;zary syndrome (SS) represent the most common subtypes. Despite a number of studies on characterizing gene expression, genetic alterations, and epigenetic abnormalities of CTCL, the molecular pathogenesis of MF/SS remains unclear. MF refers to the more common CTCL with a skin-predominance, and is usually limited to skin, whereas&#160;SS is an aggressive leukemic variant of CTCL with widespread skin involvement and is characterized by neoplastic distribution mainly involving blood, skin, and lymph node. Focusing on clinical practice, the identification of gene expression biomarkers has&#160;enormous potential to improve diagnosis and treatment of MF/SS. Indeed, recent transcriptomic studies have identified potential diagnostic biomarkers from differences in gene expression between normal and malignant T cells, which may improve our understanding of SS biology, and reveal potential therapeutic targets. This manuscript describes a detailed reproducible protocol for the isolation of peripheral blood mononuclear cells from fresh whole blood from patients diagnosed with SS, selection of CD4+ memory T cells (CD4+CD45RO+ T cells), chemical stimulation, and preparation of RNA suitable for transcriptomic profiling to discover novel prognostic molecular markers to gain additional insight in disease etiology. The stimulation using chemical agonist to activate nuclear regulation provides more specific assessment for pathways important in the dynamic transcription regulation and gene expression and eliminates confounding defects that may arise from upstream signaling defects arising from TCR antigen loss at the cell membrane. The data obtained from comparison of transcriptome of unstimulated to stimulated SS T cells unmasks functional regulatory gene expression defects not evident from analysis of quiescent unstimulated cells. Furthermore, the method outlined from this approach can be adapted for studying T cell gene expression defects in other T cell immune diseases.

We present a simple protocol for the isolation of peripheral blood mononuclear cells from whole blood obtained from patients diagnosed with S&#233;zary Syndrome, followed by selection of CD4+ T cells, their stimulation with phorbol12-myristate13-acetate and A23187 ionophore, and preparation of RNA for transcriptomic profiling.

Cutaneous T-cell lymphomas (CTCL) are derived from the transformation and uncontrolled proliferation of mature skin-homing T cells, and mycosis fungoides ...

Natural Killer (NK) and CAR-NK Cell Expansion Method using Membrane Bound-IL-21-Modified B Cell Line

Chimeric antigen receptor (CAR)-modified immune cell therapy has become an emerging treatment for cancers and infectious diseases. NK-based immunotherapy, particularly CAR-NK cell, is one of the most promising 'off-the-shelf' development without severe life-threatening toxicity. However, the bottleneck for developing a successful CAR-NK therapy is achieving sufficient numbers of non-exhaustive, long-lived, 'off-the-shelf' CAR-NK cells from a third party. Here, we developed a new CAR-NK expansion method using an Epstein-Barr virus- (EBV) transformed B cell line expressing a genetically modified membrane form of interleukin-21 (IL-21). In this protocol, step-by-step procedures are provided to expand NK and CAR-NK cells from cord blood and peripheral blood, as well as solid organ tissues. This work will significantly enhance the clinical development of CAR-NK immunotherapy.

Natural Killer NK and CAR-NK Cell Expansion Method using Membrane Bound-IL-21-Modified B Cell Line

Here, we present a method to expand peripheral blood natural killer (PBNK), NK cells from liver tissues, and chimeric antigen receptor (CAR)-NK cells derived from peripheral blood mononuclear cells (PBMCs) or cord blood (CB). This protocol demonstrates the expansion of NK and CAR-NK cells using 221-mIL-21 feeder cells in addition to the optimized purity of expanded NK cells.

Chimeric antigen receptor (CAR)-modified immune cell therapy has become an emerging treatment for cancers and infectious diseases. NK-based immunotherapy, ...

Expansion and Enrichment of Gamma-Delta (&#947;&#948;) T Cells from Apheresed Human Product

Although V&#947;9V&#948;2 T cells are a minor subset of T lymphocytes, this population is sought after for its ability to recognize antigens in a major histocompatibility complex (MHC)-independent manner and develop strong cytolytic effector function that makes it an ideal candidate for cancer immunotherapy. Due to the low frequency of Gamma-Delta (&#947;&#948;) T cells in the peripheral blood, we developed an effective protocol to greatly expand a highly pure &#947;&#948; T cells drug product for first-in-human use of allogeneic &#947;&#948; T cells in patients with acute myeloid leukemia (AML). Using healthy donor apheresis as an allogenic cell source, the lymphocytes are isolated using a validated device for a counterflow centrifugation method of separating cells by size and density. 
The lymphocyte-rich fraction is utilized, and the &#947;&#948; T cells are preferentially activated with zoledronic acid (FDA-approved) and interleukin (IL)-2 for 7 days. Following the preferential expansion of &#947;&#948; T cells, a clinical-grade magnetic cell-separation device and TCR&#945;&#946; beads are used to deplete contaminating T-cell receptor (TCR)&#945;&#946; T cells. The highly enriched &#947;&#948; T cells then undergo a second expansion using engineered artificial antigen-presenting cells (aAPCs) derived from K562 cells-genetically engineered to express single-chain variable fragment (scFv) for CD3 and CD28, 41BBL (CD137L) and IL15-RA-together with zoledronic acid and IL-2. Seeding all day-7 enriched &#947;&#948; T cells in co-culture with the aAPCs facilitates the manufacture of highly pure &#947;&#948; T cells with an average fold expansion of &gt;229,000-fold from healthy donor blood.

Expansion and Enrichment of Gamma-Delta &#947;&#948; T Cells from Apheresed Human Product

Presented is a protocol for the expansion of Gamma-Delta (&#947;&#948;) T cell drug product. Lymphocytes are isolated by elutriation and &#947;&#948;-enriched with zoledronic acid and interleukin-2. Alpha-beta T cells are depleted using a clinical-grade magnetic separation device. The &#947;&#948; cells are co-cultured with K562-derived, artificial antigen-presenting cells and expanded.

Although V&#947;9V&#948;2 T cells are a minor subset of T lymphocytes, this population is sought after for its ability to recognize antigens in a major ...

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

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

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

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

Orthotopic Implantation of Patient-Derived Cancer Cells in Mice Recapitulates Advanced Colorectal Cancer

Over the last decade, more sophisticated preclinical colorectal cancer (CRC) models have been established using patient-derived cancer cells and 3D tumoroids. Since patient derived tumor organoids can retain the characteristics of the original tumor, these reliable preclinical models enable cancer drug screening and the study of drug resistance mechanisms. However, CRC related death in patients is mostly associated with the presence of metastatic disease. It is therefore essential to evaluate the efficacy of anti-cancer therapies in relevant in vivo models that truly recapitulate the key molecular features of human cancer metastasis. We have established an orthotopic model based on the injection of CRC patient-derived cancer cells directly into the cecum wall of mice. These tumor cells develop primary tumors in the cecum that metastasize to the liver and lungs, which is frequently observed in patients with advanced CRC. This CRC mouse model can be used to evaluate drug responses monitored by microcomputed tomography (&#181;CT), a clinically relevant small-scale imaging method that can easily identify primary tumors or metastases in patients. Here, we describe the surgical procedure and the required methodology to implant patient-derived cancer cells in the cecum wall of immunodeficient mice.

This protocol describes the orthotopic implantation of patient-derived cancer cells in the cecum wall of immunodeficient mice. The model recapitulates advanced colorectal cancer metastatic disease and allows for the evaluation of new therapeutic drugs in a clinically relevant scenario of lung and liver metastases.

Over the last decade, more sophisticated preclinical colorectal cancer (CRC) models have been established using patient-derived cancer cells and 3D ...