In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

The overall goal of the following experiment is to identify culture conditions that best enrich for ovarian cancer tumor initiating cells. This is achieved by first transferring the cells of interest to low attachment flasks in serum free stem cell media containing growth factors. As a second step flow cytometry is performed to assess the CD 1 33 expression and A LDH activity of the cells.

Next cells from the cultures that are most highly enriched for tumor initiating cell markers are subcutaneously injected into immunocompromised mice to verify the tumor genicity of the cultures. Ultimately flow cytometry and subcutaneous xenograft models can be used to determine the culture conditions that elicit the highest percentage of ovarian cancer tumor initiating cells. We first had the idea for this method when we noticed that some of our cell lines had unusual growth characteristics and appeared to thrive as free floating clusters, a phenotype that would likely resemble what occurs in vivo in ovarian cancer patients.

The implications of this technique extend towards the therapy of ovarian cancer as there is a high rate of recurrent chemo resistant disease in ovarian cancer and much evidence suggests that tumor initiating cells contribute to this phenomenon To generate a floater tumor initiating cell culture first transfer the floating cells of an 80%cofluent cell culture into a 50 milliliter polypropylene centrifuge tube while the cells are spinning down. Add 10 milliliters of stem cell media supplemented with growth factors to an ultra-low attachment surface polystyrene 75 squared centimeter tissue culture flask labeled with the name, date, and passage number of the cell line. Then aspirate the supernatant from the centrifuge tube and resuspend the pellet in six milliliters of stem cell media.

Transfer the cells to the flask and place them in a humidified cell culture incubator set to 37 degrees Celsius and 5%carbon dioxide monitoring the cells every two to three days to observe the steroid formation to create a traditional tumor initiating cell culture. Discard the floating cells from an 80%confluence cell culture and wash the adherent cells with warm PBS. Next, detach the cells with 1.5 milliliters of a trypsin EDTA solution at room temperature.

After three to five minutes, stop the reaction with six milliliters of stem cell media and tritrate the cell solution a few times to loosen the cells from the flask surface. Transfer the dissociated cells to an ultra low attachment surface polystyrene 75 squared centimeter tissue culture flask containing 10 milliliters of stem cell media, supplemented with growth factors. Then place the flask in the cell culture incubator and supplement the cells every 48 to 72 hours with an additional two milliliters of stem cell media and fresh growth factors.

Monitor the traditional tumor initiating cell culture until 60 to 80%confluence is reached. Then split the culture by transferring equal volumes of the supernatant into two new ultra low attachment flasks and add enough fresh stem cell media to achieve a final volume of 18 milliliters to generate multicellular steroid cultures from chemo resistant patient ascites. Begin removing the cap from a one liter sterile vacuum bottle of ascites fluid and transfer 10 milliliters of the ascites to a polystyrene 75 square centimeter tissue culture flask containing 10 milliliters of traditional media.

Place the flask in the cell culture incubator for 72 to 96 hours and then perform the first complete media change once the cells reach 60 to 80%confluence. Split the culture one to two as just demonstrated for use as tumor initiating cultures to stay in the cells for flow Cyto metric analysis begin by labeling five 1.5 milliliter einor tubes for each of the culture conditions listed in the table. Next, distribute approximately 500, 000 cells each to tubes one to four.

Then spin down the cells, resuspend the pellets in 500 microliters of al the Fluor assay buffer and add 10 microliters of DEAB an A LDH inhibitor to tube number five. For the experimental negative control, add five microliters of activated al theor reagent to tubes tube and four flicking each tube to mix. Then immediately transfer half the volume of the cells from tube four into tube five, mix tube five by flicking as well, and then incubate all of the tubes for 30 to 45 minutes at 37 degrees Celsius protected from light.

At the end of the incubation period, spin down all the tubes and resuspend the pellets in 500 microliters of alor assay buffer. Next, add CD 1 33 A PC to tubes three and four and incubate the tubes on ice protected from light. After 15 minutes, spin down the cells twice and resuspend the pellets in 500 microliters of alor assay buffer.

Then filter all of the cell suspensions through individual polystyrene round bottom tubes with cell strainer caps. Ensure that the entire volume is filtered through the cap and then use tubes one to three to set the compensation controls on the flow cytometer gate the cells using the forward and side scatter parameters. Taking care to exclude the debris.

Use the FL one and FL four channels to establish the A LDH and CD 1 33 Double positive cells respectively then analyze the cells. The tumor initiating cell rich cultures will exhibit a higher level of staining for the A LDH and CD 1 33 cell surface markers compared to the non-tumor initiating cell cultures. To confirm the tumor genicity of the tumor initiating cells first collect the cells from the multicellular OID cultures as just demonstrated.

Resuspend each cell population in one milliliter of PBS and then determine the number of viable cells by trian blue exclusion. Next, add 1, 500, 000 cells diluted in two milliliters of PBS into each of three 15 milliliter conical tubes per tumor, initiating cell culture condition. Then using 27 gauge needles, inject 0.5 milliliters of cells from the traditional adherent cultures, traditional TIC cultures floater, TIC cultures or PBS alone subcutaneously into the right flank of each three mice per group return each mouse to the original cage post-injection.

Finally weigh the animals and measure any palpable tumors in two dimensions twice weekly by veer caliper to establish the length and width of the tumors as demonstrated in these dot plots. An increased percentage of CD 1 33 positive cells, coupled with a higher evidence of A LDH enzymatic activity and low attachment serum free conditions as compared to traditional adherent cultures indicates an enriched tumor initiating cell population in the multicellular steroid cultures. Although the traditional adherent cultures contain CD 1 33 positive cells, the percentage increases under conditions that enhance OID formation analysis of the pluripotency marker.

Draw one 60 further confirms that the tumor initiating cell culture conditions and rich for tumor initiating cells, indeed CD 1 33 A LDH double positive a CI 1 23 cells are largely limited to cultures grown under tumor initiating cell enhancing conditions, and a progressive increase in each of these different transcriptional factors is observed from traditional to floater tumor initiating cell conditions compared to the relatively low levels evident in the adherent culture. Finally, as expected a CI 1 25 and OV R five cells from traditional adherent cultures were observed to be less tumorogenic than those from the tumor initiating cell cultures. More the floater tumor initiating cell cultures produced larger tumors in a shorter period of time than the traditional adherent or the traditional tumor initiating cell cultures taken together.

These data suggest that even with a modest increase in ovarian tumor initiating cell markers, there is a dramatic phenotypic change in tumor initiating cell enriched cultures. While attempting this procedure, it's important to remember to monitor and replenish the cells with fresh media and growth factors. Every two to three days too short of a culture period will not provide sufficient time for enrichment and too long may result in the differentiation of the cells and a subsequent loss of their markers in tumor genicity.