PMC

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).

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).

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.

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.

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.

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.