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.
