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According to the cancer stem cell (CSC) theory only a small subset of cancer cells are capable of forming tumors. We previously reported that CD44 isolates tumorigenic cells from HNSCC. Recent studies indicate that aldehyde dehydrogenase (ALDH) activity may represent a more specific marker of CSCs.
Six primary HNSCC were collected. Cells with high and low ALDH activity (ALDHhigh/ALDHlow) were isolated. ALDHhigh and ALDHlow populations were implanted into NOD/SCID mice and monitored for tumor development.
ALDHhigh cells represented a small percentage of the tumor cells (1-7.8%). ALDHhigh cells formed tumors from as few as 500 cells in 24/45 implantations while only 3/37 implantations of ALDHlow cells formed tumors.
ALDHhigh cells comprise a subpopulation cells in HNSCC that are tumorigenic and capable of producing tumors at very low numbers. This finding indicates that ALDH activity on its own is a highly selective marker for CSCs in HNSCC.
Head and neck cancer is a common malignancy that affects approximately 40,000 new patients in the United States each year1. Despite advances in therapy, which have improved quality of life, survival rates have remained static for many years. Mortality from this disease remains high due to the development of distant metastasis and the emergence of treatment-resistant local and regional recurrences. To develop more effective therapies for HNSCC it is essential that we gain a deeper understanding of the biology of this disease and the cells that are responsible for recurrent and persistent cancer.
The cancer stem cell theory of carcinogenesis postulates that tissue stem cells or progenitor cells are a target for genetic changes that lead to malignant transformation. Based on their similarity to normal stem cells cancer stem cells are also likely to be more resistant to therapy and may be responsible for tumor persistence and recurrence. Evidence has been accumulating that supports the validity of this theory in a number of malignant diseases2-8. Epithelial cancers, including head and neck squamous cell carcinoma, contain heterogeneous populations of cells, some of which are tumorigenic and many others that are not. Accumulated evidence indicates that cancer stem cells retain phenotypic features of their cell of origin whether that is a stem cell or early progenitor cell9,10. Stem cell markers or molecular processes that are also expressed on cancer stem cells should provide the means to identify and study cancer stem cells. Thus, conserved stem cell molecular pathways may possibly be used in the search for cancer stem cells.
In recent studies in breast and central nervous system cancers, tumorigenic subpopulations of “cancer stem cells” have been isolated based on expression of cell surface markers11-16. Using a modification of a method employed to identify cancer stem cells in breast cancer we isolated a subpopulation of HNSCC cells marked by the cell surface marker CD44 (CD44+ cells) that produce tumors in the NOD/SCID mouse model12,17. In contrast, even ten fold higher numbers of CD44 negative (CD44-) tumor cells were unable to form tumors. Although this was the first demonstration of cancer stem cells in HNSCC, the high percentage of HNSCC cancer cells found to be CD44+, and the number of such cells necessary (5 × 103) to develop a tumor suggested that although the CD44+ cell population contained cancer stem cells it was unlikely to be a pure population of cancer stem cells.
Aldehyde dehydrogenase (ALDH) expression has been suggested as a potential functional marker for stem cells and cancer stem cells. ALDH detoxifies intracellular aldehydes through oxidation and may have a role in the differentiation of stem cells through the oxidation of retinoic acid18-20. High ALDH activity has been used to isolate normal hematopoietic and central nervous system stem cells21-24. ALDH activity is also found in subsets of multiple myeloma and acute myeloid leukemia24,25. Wicha et al. (2007) used ALDH activity to isolate breast cancer stem cells26. ALDH 1 expression has recently been reported to be a putative marker for cancer stem cells in head and neck squamous cell cancer and colon cancer27,28. These findings indicate that ALDH expression may be an important new marker for the isolation of cancer stem cells. In this study we investigated the ALDH active population of HNSCC cells for tumorigenic activity.
Approval for the collection of the cancer specimens and for the use of the animal model was obtained through the appropriate review boards. The University of Michigan's guide for the care and use of laboratory animals was followed. Samples of HNSCC cancer were obtained from subjects undergoing surgical resection or biopsy of their tumor. Specimens were immediately transported to the lab in DMEM containing 10% Fetal Bovine Serum on ice. Single cell suspensions were created.
Specimens were cut into small fragments with sterile scissors and minced with a sterile scalpel, rinsed with Hanks' Balanced Salt Solution (HBSS) containing 2% heat inactivated calf serum (HICS) and centrifuged for 5 min at 1000 rpm. The resulting tissue specimen was placed in a solution of DMEM F-12 containing 300u/mL collagenase and 100u/mL hyaluronidase (Stem Cell Technologies). The mixture was incubated at 37°C mixing to dissociate cells. The digestion was arrested with the addition of Fetal Bovine Serum and the cells were filtered through 40-μm nylon sieve. The cells were washed twice with HBSS/2% HICS and stained for flow cytometry as described below.
Aldehyde dehydrogenase activity was identified in the cancer cells using the ALDEFLUOR® substrate per the manufacturer's protocol (STEMCO Biomedical). Specimens that were analyzed for ALDH activity were counter stained with anti-CD44 (allophycocyanin (APC) conjugated: BD Pharmingen) at the appropriate dilution. Cells of other lineages were identified and removed using markers anti-CD2, CD3, CD10, CD16, CD18, (CyChrome (Cy) conjugated: BD Pharmingen) that are not expressed on the tumor cells. Non-viable cells were eliminated using DAPI (BD Pharmingen). During flow cytometry analysis other lineage cells and the DAPI stained dead cells were eliminated by gating. The specific flow gates for ALDH positive cells were set using a control sample of the isolated tumor cells in which ALDH activity was inhibited with diethylamino-benzaldehyde (DEAB). Subsequent flow cytometry runs were used to identify populations of cells with high aldehyde dehydrogenase activity (ALDHhigh) and those that express the surface marker CD44 (CD44+). When cell numbers allowed measurements assessed the percent of each population present; ALDHhigh, ALDHlow, CD44+ and CD44-. Overlap between these populations was assessed to identify populations of cells with the characteristics CD44+ ALDHhigh, CD44+ALDHlow, CD44-ALDHhigh and CD44- ALDHlow in HNSCC.
HNSCC subpopulations of interest were collected based on their ALDH expression. Subpopulations of ALDHhigh and ALDHlow cells were injected subcutaneously into NOD/SCID mice and evaluated for their tumorigenic potential. When sufficient numbers were available the cells were serially diluted prior to injection. The cells were mixed with Matrigel Basement Membrane Matrix (BD Pharmingen) solution to form a final volume of 200 μL. Injection sites were sealed with a liquid skin adhesive. The animals were assessed for tumor growth.
The tumorgenicity of the injected cell populations was evaluated by evidence of tumor growth in NOD/SCID mice and by histology. When the number of cells allowed for serial dilutions to be injected we determined the minimum number of cells required for each population to produce a tumor in the NOD/SCID mouse. The resultant tumors were assessed by flow cytometric and histologic analysis for tumor heterogeneity and proportion of cells with ALDH activity.
Six primary tumors were collected from subjects with head and neck squamous cell cancer. Two tumors originated in the oral cavity (floor of mouth, tongue), three originated in the oropharynx (two in the tonsil, one base of tongue) and one originated in the larynx. The subjects ranged in age from 45 to 74 years old. There were three moderately differentiated, two poorly differentiated and one well differentiated tumor (Table 1). Although most of the tumor samples were small, when sufficient numbers of cells were available co-stained flow cytometry sorts and injections of serially diluted cells were performed.
HNSCC can be separated into two subpopulations using the ALDEFLUOR® substrate to determine ALDH activity and flow cytometry to sort and collect the cells. As expected the majority of HNSCC cells had low ALDH activity. In these tumors, the proportion of ALDHhigh cells had a mean of 3.5% ± 2.8% (1.0 – 7.8 %).
The ALDHhigh subpopulation overlaps significantly with the CD44+ population of cells. When the ALDHhigh population was sorted for CD44, the majority of cells also expressed CD44, 50.6% - 74.4% (Figure 1 and Table 2). Conversely when CD44+ cells were sorted for ALDH activity, only 9.8% - 23.6% of the CD44+ cells had high ALDH activity. Indicating that a large proportion of ALDHhigh cells are CD44+, but a much smaller proportion of CD44+ cells are ALDHhigh.
ALDHhigh HNSCC cells are highly tumorigenic in comparison to ALDHlow cells (Table 3). HNSCC cells with high ALDH activity produced tumors in the NOD/SCID mouse model in 24/45 injections (53%), while HNSCC cells with low ALDH activity resulted in tumors in only 3/37 injections (p<.00001 Chi squared test). Tumors developed in 7/15 (47%) mice injected with as few as 50-100 ALDHhigh HNSCC cells whereas no tumors occurred in 0/14 injections of the same number of ALDHlow cells.
ALDHhigh cells produce tumors that have similar histology to the original tumor (Figure 2). The ALDHhigh cells can be passaged in the mouse model and reproduce the original tumor heterogeneity for ALDH activity, that is the proportion of cells that are ALDHhigh is similar to the proportion in the original tumor (Figure 3).
The identification of highly tumorigenic subpopulations of cells, the cancer stem cells, in solid tumors, has significant implications regarding cancer biology, response to therapy and the development of new cancer treatments. Therapies based upon tumor regression may produce treatments effective against the majority of more differentiated cancer cells while sparing the cancer stem cell subpopulation8. The development of more effective cancer therapeutics will require the cancer stem cells to be selectively targeted and eliminated. For this strategy to be successful the cancer stem cell cells must be reliably identified and isolated so their characteristics can be studied.
CD44 is a cell surface marker that identifies a subpopulation of cancer cells from HNSCC that are highly tumorigenic. However, the CD44 subpopulation of HNSCC likely contains both non cancer stem cells and cancer stem cells as shown by the need to inject in the order of 5×103 cells to produce a tumor in the animal model17. In other cancers combinations of cell surface markers have been used successfully to isolate cancer stem cells, and smaller numbers of the isolated cells have been shown to produce tumors in an animal model suggesting this methodology is capable of isolating the cancer stem cells more selectively. These findings indicated a need to identify a more selective single marker or combination of markers for cancer stem cells in HNSCC.
Wicha recently demonstrated that ALDH expression can be used to identify breast cancer stem cells, and that injections of small numbers of these cells produce tumors in an animal model26. Interestingly the normal breast stem/progenitor cells also have high expression of ALDH strongly supporting the concept that stem and progenitor cells are the targets of malignant transformation in breast cancer26.
We have shown here that ALDH expression isolates a subpopulation of cancer cells from HNSCC that are highly tumorigenic. The ALDH positive cells are able to produce tumors at a 10 fold reduction over that which was possible with CD44+ cells alone17. The tumors grown from ALDHhigh cells recreate the original tumor heterogeneity and histology and the ALDHhigh cells can be passaged in the animal model fulfilling the requirements for CSC phenotype. This data along with the recent report by Chen et. al. strongly supports the use of ALDH expression as a method to select cancer stem cells in HNSCC27.
As expected the ALDH subpopulation of HNSCC cells mainly comprise a small subpopulation of the CD44+ cells, suggesting ALDH expression isolates a subset of CD44+ cells that contain the actual tumorigenic cancer stem cells (Figure 4). Thus we conclude that the tumorigenic cells have both phenotypic markers. The explanation for why CD44 and ALDH are expressed by the stem cell population is not known, however, one could speculate that the ability to oxidize retinoic acid, a proposed function of ALDH, is a requirement for stem cell activity. It is also not certain that both markers are always expressed on the tumorigenic stem cells since there appear to be a small fraction of ALDH positive cells that are CD44-. However, the limited number of cells precluded a separate selection of these cells for testing, and our prior experiments had shown that even large numbers of CD44-negative cells are not tumorigenic. No doubt there are other phenotypic markers that are expressed by cancer stem cells. For example in breast cancer CD24 is also a stem cell marker. In HNSCC this does not appear to be the case, thus there seem to be tissue specific stem cell markers as well as general stem cell markers. ALDH expression may represent a CSC that is generally applicable to all cancer stem cells although this is yet to be proven.
The small numbers of cancer stem cells obtained from HNSCC limit the critical experiments that must be carried out to understand the role of these cells in cancer persistence, recurrence and resistance to therapy. Repeated re-cultivation in the NOD/SCID mouse or in vitro culture methods would allow for repeated experiments and our future work will target these mechanisms.
Supported by the James Selleck Bower Endowed Research Fund, the NIH through a Career Development Award from the University of Michigan Head and Neck SPORE P50 CA097248 and The American Academy of Otolaryngology Head and Neck Surgery Foundation Percy Memorial Research Award.