Aldehyde dehydrogenase activity as a functional marker for lung cancer

Abstract

Aldehyde dehydrogenase (ALDH) activity has been implicated in multiple biological and biochemical pathways and has been used to identify potential cancer stem cells. Our main hypothesis is that ALDH activity may be a lung cancer stem cell marker. Using flow cytometry, we sorted cells with bright (ALDH(br)) and dim (ALDH(lo)) ALDH activity found in H522 lung cancer cell line. We used in vitro proliferation and colony assays as well as a xenograft animal model to test our hypothesis. Cytogenetic analysis demonstrated that the ALDH(br) cells are indeed a different clone, but when left in normal culture conditions will give rise to ALDH(lo) cells. Furthermore, the ALDH(br) cells grow slower, have low clonal efficiency, and give rise to morphologically distinct colonies. The ability to form primary xenografts in NOD/SCID mice by ALDH(br) and ALDH(lo) cells was tested by injecting single cell suspension under the skin in each flank of same animal. Tumor size was calculated weekly. ALDH1A1 and ALDH3A1 immunohistochemistry (IHC) was performed on excised tumors. These tumors were also used to re-establish cell suspension, measure ALDH activity, and re-injection for secondary and tertiary transplants. The results indicate that both cell types can form tumors but the ones from ALDH(br) cells grew much slower in primary recipient mice. Histologically, there was no significant difference in the expression of ALDH in primary tumors originating from ALDH(br) or ALDH(lo) cells. Secondary and tertiary xenografts originating from ALDH(br) grew faster and bigger than those formed by ALDH(lo) cells. In conclusion, ALDH(br) cells may have some of the traditional features of stem cells in terms of being mostly dormant and slow to divide, but require support of other cells (ALDH(lo)) to sustain tumor growth. These observations and the known role of ALDH in drug resistance may have significant therapeutic implications in the treatment of lung cancer.

Figure 1

Aldefluor flow cytometry based assay to identify cells with high ALDH activity (ALDH^br) among the H522 lung cancer cells. The top panels show the histograms of Aldefluor fluorescence with and without the addition of DEAB, an ALDH activity inhibitor, which demonstrate shifting of cells with high ALDH activity to the right (right top panel). The same information is shown in the bottom panels, where the 1^st panel on the left shows the side scatter of the viable H522 cells (gate 1), and the other two panels show gate 2 that is defined by the addition of DEAB (middle panel) and within which the ALDH^br cells fall. In this analysis, the ALDH^br cells constitute about 29% of the parent cell line.

Figure 2

Comparison of the dominant cytogenetic karyotypes identified in H522 parent cell line as well as H522 cells sorted by flow cytometry with either high or low ALDH activity. Similar karyotypes were dominant in both the parent cell line and the cells with low ALDH activity, while the karyotype of the cells with high ALDH activity was clearly different. Interestingly, other less frequent karyotypes were identified in the parent cell line, including one similar to that found to be dominant among the cells exhibiting high ALDH activity.

Figure 3

The morphology of colony types identified during colony forming assay of H522 cells. A. Three different known types of colonies were identified when culturing the parent H522 cell lines and the sorted cells with low ALDH activity (ALDH^lo). B. Only one, completely different, type of colony was identified in a similar colony forming assay from sorted H522 cells with high ALDH activity (ALDH^br).

Figure 4

Semiquantitative RT-PCR for NANOG and OCT4, human embryonal stem cell genes, was performed on RNA obtained from 3 different groups of cells. GAPDH, a house keeping gene, was used as a control. The 3 experimental groups include: the parent H522 cell line (WT), and flow cytometry sorted H522 cells with either high (High) or low (Low) ALDH activity.

Figure 5

Comparison of primary xenograft formation in NOD/SCID mice by sorted H522 cells using Aldefluor flow cytometry assay. The curves reflect the increase in size of tumors growing under the skin from 10⁵ sorted H522 cells with either low (ALDH^lo, grey line) or high ALDH activity (ALDH^br, black line). Each time point represents the mean tumor size (mm³) calculated weekly from 3 similar animals in each experimental group.

Figure 6

Immunhistochemistry (IHC) staining for ALDH3A1 of a representative primary xenograft originating from ALDH^br H522 cells. The results demonstrate the clustering of cells containing high levels of ALDH3A1 around blood vessels (40X magnification). Similar results were obtained from IHC staining for ALDH1A1 of the same tumor sample (data not shown).