Cancer stem cells (CSCs) have been identified in a number of malignancies. In this protocol we describe a flow cytometric method utilizing aldehyde dehydrogenase activity and CD44 and CD24 expression to isolate CSCs from human pancreatic adenocarcinoma xenografts. These viable cells can then be used in functional and analytical studies.
Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny1. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44+CD24low/-)2. Since then, CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties, including aldehyde dehydrogenase (ALDH) activity, have also been used to isolate CSCs from malignant tissues3-5.
Recently, we and others identified CSCs from pancreatic adenocarcinoma based on ALDH activity and the expression of the cell surface antigens CD44 and CD24, and CD1336-8. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH+ and CD44+CD24+ pancreatic CSCs are similarly tumorigenic, but ALDH+ cells are relatively more invasive8. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human xenografts9. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent, a fluorescent substrate of ALDH10. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells.
1. Harvest Xenografts from Mice
2. Generate a Single-cell Suspension from the Xenografts
3. Deplete Cell Suspension of Tissue Debris and Dead Cells
4. Stain Cells for Fluorescence Activated Cell Sorting
5. Isolate Cancer Stem Cells by Fluorescence Activated Cell Sorting
6. Representative Results
This protocol will lead to the isolation of ALDH+ and CD44+CD24+ pancreatic CSCs. The percentage of mouse-derived cells is variable, but we have found that most human pancreatic cancer xenografts contain 20-60% mouse-derived cells using the mouse CD31, mouse lineage cocktail, and mouse H-2Kd biotin-conjugated antibodies (Figure 1A). Likewise the frequency of each CSC population from different xenografts is variable, but generally we have found that 1-4% of the total cell population is ALDH+ (Figure 1B) and 0.2-5% is CD44+CD24+ (Figure 1C).
We routinely manually count sorted cells using a hemocytometer since the FACSAria machine counts are often inaccurate due to non-cellular particles detected by the FACSAria.
Figure 1. Flow cytometry plot depicting specific populations from a pancreatic cancer
xenograft. (A) Cells staining positively in the FL-3 channel (propidium iodide+, mouse
CD31+ mouse lineage+, or mouse H-2Kd+) are excluded so as to isolate only viable and
non-mouse derived cells. (B) ALDH activity in a human pancreatic cancer xenograft
was measured by flow cytometry using the ALDEFLUOR reagent in the presence and
absence of the ALDH1 inhibitor diethylamino-benzaldehyde (DEAB). The frames
represent gates that depict ALDH+ cells that were created based on cells treated with
DEAB and then applied to untreated cells. The percentages of ALDH+ cells are shown
in the gate. (C) The CD44+CD24+ gate was created based on cells stained with
ALDEFLUOR, and the IgG2bκ-APC (isotypic control for CD44-APC) and IgG2aκ-PE
(isotypic control for CD24-PE) antibodies. The percentages of CD44+CD24+ cells are
shown in the gate.
This protocol describes the isolation of pancreatic CSCs from human xenografts. Several key steps in this procedure will enhance the yield of viable CSCs. The recovery of a pure population of pancreatic CSCs is dependent on the purity of viable cells present in the cell suspension before sorting on the FACSAria. Taking care to use xenografts that are less than 1-cm in greatest diameter helps to reduce the amount of necrotic tissue in the center of the tumor. Additionally, depleting the cell suspension of tissue debris and dead cells during Ficoll-Paque gradient centrifugation is critical and can be repeated if a large number of tissue debris or non-viable cells are detected when the cells are counted on the hemocytometer.
The staining protocol described here utilizes the ALDEFLUOR reagent system to detect ALDH activity as well as fluorophore-conjugated antibodies to detect specific cell surface antigens. ALDEFLUOR staining is performed at 37°C and, therefore, needs to be completed before antibody staining (on ice) to prevent the potential for antibody capping, which can happen at 37°C. Since cells can efflux the ALDEFLUOR reagent, it is important to maintain the cells in ALDEFLUOR buffer at 4°C after ALDEFLUOR staining has been completed. The ALDEFLUOR buffer contains a drug efflux inhibitor that helps maintain intracellular levels of ALDEFLUOR.
Since this method isolates viable pancreatic CSCs they can be applied in a number of experiments that measure the physiological function of these cells. We have used these cells for tumor-initiation assays utilizing immunocompromised mice and in vitro cell migration/invasion assays. Furthermore, RNA, DNA, and protein can be collected from these cells for analytical studies comparing CSC and non-CSC populations.
The authors have nothing to disclose.
This work was supported by grants from the National Institutes of Health (CA127574, CA107040 and CA09071) to W.M.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
DMEM | Invitrogen (Carlsbad, CA) | 11965-118 | ||
Fetal calf serum | Harlan (Indianapolis, IN) | BT-9501 | ||
Penicillin-streptomycin | Invitrogen | 15140-122 | ||
Collagenase type IV | Invitrogen | 17104-019 | ||
Dispase | Sigma (St. Louis, MO) | D4818 | ||
100 x 20 mm culture dish | BD Biosciences (San Jose, CA) | 353003 | ||
70-μm filter | BD Biosciences | 352350 | ||
50-mL conical tube | BD Biosciences | 352070 | ||
Ficoll-Paque Plus | GE Healthcare (Upsala, Sweden) | 17-1440 | ||
ALDEFLUOR reagent system | Stem Cell Technologies (Vancouver, BC, Canada) | 01700 | ||
Trypan blue | Invitrogen | 15250-061 | ||
12 x 75 mm polystyrene test tubes | BD Biosciences | 352054 | ||
CD44-APC (clone G44-26) | BD Biosciences | 559942 | ||
CD24-PE (clone ML5) | BD Biosciences | 555428 | ||
Mouse CD31-biotin | BD Biosciences | 553371 | ||
Mouse lineage-biotin | Miltenyi Biotec (Auburn, CA) | 130-092-613 | ||
Mouse H-2Kd-biotin | BD Biosciences | 553564 | ||
IgG2bκ-APC | BD Biosciences | 555745 | ||
IgG2aκ-PE | BD Biosciences | 555574 | ||
Streptavidin-PerCP protein | BD Biosciences | 554064 | ||
Propidium iodide | Sigma | P4170 | ||
12 x 75 mm polystyrene test tubes with strainer caps | BD Biosciences | 352235 |