Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

The overall goal of the following procedure is to expand cancer stem cells in vitro using an Anchorage independent culture technique, and then test the metabolic effects of novel cancer stem cell targeting therapies. This is accomplished by first dissociating a human pancreatic adenocarcinoma tissue sample into a single cell suspension. In the second step, the tumor tissue is further digested with enzymes and then seeded onto a gelatin plate To remove the fibroblast cell population, the isolated cancer cells are then seeded in an ultra low attachment multi-well plate, and treated with the drug of interest to assess the metabolic activity and formation of the tumor spheres.

Ultimately, the drug of interest can be used in immune compromised mice bearing patient derived xenografts to validate the effects of the drug in vivo. These method is a powerful tool for studying cancer stem cell biology and serves as a first step for developing new cancer stem cell targeting therapies. Demonstrating the procedure will be myself, a junior investigator at the Center for Stem Cells in Cancer and Aging at the Breast Cancer Institute and Tini Cruz, a PhD student from my laboratory To isolate cancer stem cells from a pancreatic ductal adenocarcinoma in a sterile biosafety cabinet.

Use a sterile scalpel and forceps to mince the human tumor in a 60 by 15 millimeter culture dish containing one milliliter of sterile PBS, adding another three to four milliliters of PBS as necessary until the tissue is completely dissociated. Transfer the tissue suspension into a sterile conical tube and add PBS to a final volume of five milliliters. Then mechanically homogenize the sample with a dissociation centrifuge and digest the homogenized tissue with collagenase at 37 degrees Celsius.

After an hour centrifuge, the cells resus, suspending the pellet in 10 milliliters of complete medium. Filter the cell suspension through a 40 micrometer strainer and spin down the cells again. This time, resus suspending the pellet in five milliliters of a CK After five minutes at room temperature, remove the red blood cell lysis buffer by centrifugation and resuspend the pellet in cancer stem cell medium.

Then incubate the cells on a gelatin coated dish at 37 degrees Celsius to remove most of the quickly attaching fibroblast cells. After one hour, recover the supernatant and quantify the number of viable pancreatic cancer cells to facilitate the tumor sphere formation for cancer. Stem cells enrichment dilute the cells in the appropriate volume of cancer stem cell medium to a final concentration of two times 10 to the three cells per milliliter, and chill them on ice for a few minutes.

Then after adding 500 microliters of PBS to the first and last rows of a 24 ultra low attachment cell plate, re suspend the cells by pipette and seed one milliliter of cells per well into the empty wells of the plate. Treat four wells of cells with the drug of interest and at least four negative control wells with vehicle. Then incubate the cells at 37 degrees Celsius with 5%carbon dioxide for one week.

Every other day, add fresh treatment to our vehicle to each of the wells. On day five or seven, assess the number of formed tumor spheres with an automated cell counter that allows the identification of larger structures for serial packaging of the tumor spheres. On day seven, use a 40 micrometer cell strainer to harvest the spheres.

Next, spin down the spheres and incubate them in one milliliter of trypsin for 20 minutes at 37 degrees Celsius to dissociate the pellet into a single cell suspension. Then expand the cells for another seven days as just demonstrated to prepare the spheres for measurement of their reactive oxygen species or ROS production after dissociation as just demonstrated, spin down the spheres and resuspend the pellet in HBSS containing the appropriate probe. After a 20 minute incubation in the dark at 37 degrees Celsius, place the cells on ice until analysis by flow cytometry to prepare the spheres for measurement of their oxygen consumption.

First coat a 96 well cell culture plate with cell and tissue adhesive for at least 20 minutes at room temperature. Next, dissociate the spheres and trypsin as demonstrated. Then wash the plate two to three times with water to remove the excess adhesive plate, the singular cells at three times 10 to the fifth cells per well.

In 150 microliters of oxygen consumption assay medium containing glucose and sodium pyruvate adhere the cells to the bottom of the wells by centrifugation until 100 times G has been reached. Letting the centrifuge stop with the break off. Then reverse the orientation of the plate and repeat the spin.

Now incubate the cells for 30 minutes at 37 degrees Celsius without carbon dioxide. Meanwhile, load the assay cartridge imports. A, B, and C.Inject 25 microliters of a three, six, and nine millimolar concentration of the drug of interest respectively, and import D at 25 microliters of a one micromolar concentration of the mitochondrial inhibitor rone.

When the cells are ready, calibrate the cartridge and load the cell culture plate. Finally run the assay with the standard protocol of mixing and measurement In these representative experiments, treatment of pancreatic cancer stem cells with metformin strongly decreased the size and number of the tumor spheres. Further, these reductions were exhibited by secondary and tertiary sphere cultures.

Even when only the primary sphere culture had been treated with metformin, Metformin reportedly acts as a mitochondrial inhibitor. Indeed, in this experiment, the drug was observed to induce a rapid and dose dependent decrease in oxygen consumption in the tumor cells that correlates with its capacity to induce ROS production. Importantly, metformin treated mice exhibit a short term but significant reduction in pancreatic adenocarcinoma progression compared to control animals.

After watching this video, you should have a good understanding of how to dissociate fresh human tumors. Isolate primary cancer cells, set up as sphere formation culture, and evaluate the metabolic activity of pancreatic cancer stem cells.