Enzyme mechanisms Abstract Aldehyde dehydrogenase ALDH assays measure the accumulated fluorescence of enzyme products. Without transporter inhibition, only small portions of both cell types were estimated to be ALDH-positive based on Aldefluor and AldeRed assays. Verapamil MDR inhibitor did not influence assay results. Limiting dilution assays demonstrated greater numbers of tumor-spheres formed by Aldefluor-positive compared to -negative CT26 cells selected in the presence of MK or novobiocin but not in their absence. These findings demonstrate that complete blockade of these transporters is important to ensure accurate ALDH assay results and to develop newer assay techniques. One mechanism for acquiring chemotherapeutic resistance is expression of ATP-binding cassette ABC transporters that mediate drug efflux 1 , 2.
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Enzyme mechanisms Abstract Aldehyde dehydrogenase ALDH assays measure the accumulated fluorescence of enzyme products. Without transporter inhibition, only small portions of both cell types were estimated to be ALDH-positive based on Aldefluor and AldeRed assays.
Verapamil MDR inhibitor did not influence assay results. Limiting dilution assays demonstrated greater numbers of tumor-spheres formed by Aldefluor-positive compared to -negative CT26 cells selected in the presence of MK or novobiocin but not in their absence.
These findings demonstrate that complete blockade of these transporters is important to ensure accurate ALDH assay results and to develop newer assay techniques. One mechanism for acquiring chemotherapeutic resistance is expression of ATP-binding cassette ABC transporters that mediate drug efflux 1 , 2. In stem cells, these transporters help maintain quiescence by stabilizing the internal milieu. ABC transporters are also highly expressed in cancer stem cells CSCs , a highly tumorigenic population 3 , where they contribute to treatment resistance of cancers of the brain 4 , lung 5 , ovary 6 , prostate 7 , and nasopharynx 8.
Given the crucial role of CSCs in tumor resistance and recurrence, targeting specific markers to recognize and isolate these cells has become an important tool for cancer research 9 , CSCs have several interesting characteristics, including the capacity for self-renewal and differentiation, high expression of organ-specific surface markers such as CD, CD44, and CD29, and increased Notch and Wnt pathways 3.
This underlines the importance of identifying factors that influence the results of Aldefluor assays. ALDH1 is an established marker of cancer stemness that strongly correlates with self-renewal capacity 12 and chemotherapy resistance in various cancer types 13 , Fluorescence-activated cell sorting FACS using fluorescent reagents is currently the standard method to identify cells that have increased ALDH activity.
In these assays, ALDH mediates conversion of reagent substrates into products that can no longer diffuse out of cells. This leads to increased intracellular accumulation of fluorescent signals for detection.
Aldefluor kits are the most widely employed ALDH assay method. This raises a potential problem for exploiting reagent accumulation to identify ALDH-positive cells. Specific inhibitors that reverse the ability of ABC transporters to pump out cognate substrates can offer valuable information.
We further assessed how activities of these transporters reduce reagent retention and lead to underestimation of ALDH positivity based on assays using Aldefluor and AldeRed Histograms of CT26 mouse colon cancer cells incubated with Efluxx-ID Green dye and analyzed by FACS showed a substantial right shift in the presence of verapamil and MK and a significant but mild right shift in the presence of novobiocin Fig.
Histograms of HT29 human colon cancer cells showed a substantial right shift due to MK and a mild shift due to novobiocin, but no response to verapamil Fig. Histograms of untreated control CT26 and HT29 cells are shown in purple, while green lines indicate histograms obtained after treatment with verapamil, MK, or novobiocin. Right-shifting of the latter compared to the former histogram represents inhibition of efflux through the corresponding ABC transporter representative of 3 samples per group.
Blots were cropped to combine into single figure. For full length blot pictures, see Supplementary Fig. Figure 2 ALDH expression at protein level.
Blots cropped for representative blots are shown. The resultant proportion of Aldefluor-positive cells at baseline was 6. This indicated that Aldefluor was not a substrate for MDR1-mediated reflux.
In Aldefluor assays performed with novobiocin, CT26 cells displayed a moderate increase in fluorescence Fig. DEAB was not used. ALDH-positive cells within black boxes are identified by low side scatter and high fluorescence. This raises the possibility that the concentration of novobiocin used was not sufficient to completely block BCRP activity in the presence of energy substrate-containing media.
Addition of verapamil did not influence fluorescence accumulation in either cell type, indicating that AldeRed was also a poor substrate for MDR1-mediated reflux. This raised the ALDH-positive rate from 6. Influence of ABC transporter inhibitors on tumor-sphere formation by ALDH-positive cells Limiting dilution assays were performed to assess whether Aldefluor-positive cells selected in buffers containing ABC transporter inhibitors had increased functional stemness.
The results revealed significantly greater numbers of tumor-spheres with Aldefluor-positive compared to Aldefluor-negative CT26 cells selected in the presence of MK or novobiocin. This was in contrast to cells selected in the absence of any ABC transporter inhibitor Fig. However, there no significant difference in the number of tumor-spheres formed between Aldefluor-positive and -negative cells selected in buffer containing verapamil or in Aldefluor assay buffer Fig.
Figure 5 Limiting dilution assays for assessment of functional stemness. Full size image Discussion Recent recognition that ALDH plays a central role in tumor progression and stemness has made it important to accurately measure the activity of this enzyme. To perform precise ALDH assays using presently available reagents and to develop newer assay reagents, a clear understanding of how individual ABC transporters influence their efflux from cancer cells is needed.
Efluxx-ID Green assays were used to yield measurements of transporter activity, which are more relevant to substrate efflux capacity than simple assessments of protein amount.
Western blots also confirmed that both cell types expressed more than one ABC transporter. Although it would have been useful to work with cells that express a single ABC transporter, it was difficult to select such cancer cells with certainty.
This is exemplified by a previous mRNA expression profile study demonstrating that virtually all cancer cell lines of the NCI panel express multiple ABC transporters Expression of multiple transporters by the cells in this study raises the issue of inhibitor specificity.
Inhibitor specificity is also supported by a recent study that showed efflux suppression in ovarian cancer cells by MK and novobiocin correlated with greater expression of MRP1 and BCRP, respectively Although inhibitor specificity against ABC transporters should be considered, the use of cells that express multiple ABC transporters is not likely to have significantly influenced the major findings of our study.
This kit also includes verapamil as a component of assay buffer. However, the low retention of these reagents in both CT26 and HT29 cells shown at baseline did not increase due to verapamil. This finding is not inconsistent with the patent results for BAAA, in which verapamil caused only a minor incremental increase in Aldefluor positive cells from 0. This shows that MRP is a major transporter through which Aldefluor and AldeRed products efflux out of cancer cells. The Aldefluor kit manual states that probenecid, which is considered an MRP inhibitor 33 , can be added for assays, although the circumstances when this is necessary are unspecified.
Our results indicated that probenecid or other MRP inhibitors should be added for assays of cells with MRP expression. The Aldefluor kit manual also states that 2-deoxy-D-glucose can be added for assays. Although this agent is generally thought to reduce drug resistance-related efflux, the precise mechanism of action remains unclear Increased background fluorescence of cells under different assay buffers that do not contain fluorescent substrates might influence FACS results.
MK, novobiocin, and Aldefluor assay buffer mildly increased fluorescence background, but the background fluorescent signal profiles were virtually identical whether or not DEAB was present Supplementary Fig. Since ALDH positive cells are detected by increased fluorescence in the absence of DEAB, the effects observed with ABC transporter inhibitors and Aldefluor assay buffer were not caused by increased background or auto-fluorescence.
Although the cause for this is unclear, MRP subtype differences may have contributed. In comparison, Aldefluor product efflux may be efficient for MRP1, 2, and 3. In contrast, little effect was observed for HT29 cells. However, it should be recognized that the band intensities in our Western blots fail to reflect ABC transport activity and also have limited usefulness for comparing ABC transporter levels.
Indeed, the amount of any two ABC transporters cannot be compared because they are blotted using different antibodies. The amount of the same ABC transporter cannot be compared in murine and human cells because the antibody likely binds human and murine protein with different affinities. This was illustrated when Western blots of protein from CT26 cells with an antibody specific for both murine and human BCRP resulted in faint protein bands, but repeating the Western blot with an antibody specific for murine BCRP detected some bands that matched the expected size of the murine protein Supplementary Fig.
The apparent inconsistencies mentioned above might have been due to inhibitor cross-reactivity with other ABC transporters. There are previous reports that verapamil exerts non-specific inhibition of BCRP 35 , Finally, since Aldefluor assay buffer likely contains one or more ABC inhibitors, we investigated its effect on reagent retention. The potency of these effects was similar to those achieved by MK, indicating that Aldefluor assay buffer likely contains an MRP inhibitor.
In contrast, since the effects were hugely divergent from those obtained with novobiocin, Aldefluor assay buffer is not likely to contain a BCRP inhibitor. Consistent with the FACS results, microscopic inspection confirmed positive fluorescence in the majority of both HT29 and CT26 cells in Aldefluor assay buffer, although the fluorescence intensity of individual cells was significantly stronger in HT29 cells data not shown. Aldefluor-positive cells selected in the presence of MK or novobiocin formed greater numbers of tumor-spheres compared to Aldefluor-negative cells.
This indicates that Aldefluor assays may need to be performed in buffer containing one or both of these ABC transporter inhibitors to better select cancer cells with increased functional stemness. There was no difference in the number of tumor-spheres formed between Aldefluor-positive and -negative cells selected in buffer containing verapamil or in AAB. This suggests that, depending on cell type, ALDH expression alone may not be sufficient to ensure greater functional stemness.
ABC transporters are also found in certain differentiated cancer cells. Further investigations are thus warranted to establish an optimal threshold for Aldefluor assays performed with appropriate ABC transporter inhibitors and to explore whether this might allow differentiation of CSCs from differentiated cancer cells with greater accuracy.
Whether cells isolated using different assay conditions differed in functional stemness is also an interesting question. However, this was beyond the scope of the present study, and further investigations are required to elucidate this issue. Fluorescent signals reduced by this effect resulted in substantial underestimation of the amount of ALDH-positive cancer cells. This finding underscores the importance of complete blockade of these transporters for accurate assessment of cancer cell ALDH activity and to develop new ALDH assay reagents.
The buffer contained one of three major ABC transporter inhibitors. The same concentrations of inhibitors were used for both CT26 and HT29 cells in all experiments. Efluxx-ID Green was excited at nm and fluorescence emission was detected at nm.
However, BCRP immunoblot for CT26 cells was performed with a murine-specific antibody because the antibody with reactivity against antigens of both human and murine proteins showed only faint BCRP bands in these cells. After washing 3 times for 10 min with tris-buffered saline with Tween 20, the membrane was incubated with secondary antibodies for 1 h at room temperature.
Immune reactive protein was finally detected with an enhanced chemiluminescence kit Thermo Fisher Scientific, MA. BC or AldeRed reagents. Since components of the Aldefluor assay buffer Stemcell Tech. The same amount of vehicle as in treatment groups was added to all null groups. After incubation, cells were washed and underwent FACS analysis as above. Aldefluor oxidation products were detected by a combination of nm laser as the excitation channel and a nm wavelength fluorescence emission detector channel.
ALDH positive cells in each inhibitor-treatment group were identified by fluorescence exceeding the region for control cells that were under identical conditions but with DEAB. Although the Aldefluor kit may have originally been designed for human cells, it is also widely used to detect ALDH activity as a stem cell marker in murine cells 37 , including CT26 cells 16 , Significant differences between groups were analyzed by Student t-tests for 2 groups and ANOVA with Tukey post hoc tests for 3 or more groups.
Data Availability All data generated or analyzed during this study are included in this published article. References 1.
ALDEFLUOR™ Assay Buffer
Optimal erythrocyte lysis can be achieved with buffers containing: Lo JF et al. Yae T et al. Colon Cancer Cells Lotti F et al. In the DEAB-treated control, fluorescence will reflect the size of the intracellular substrate pool.
To circumvent these issues, we derived MSCs from transgene-free human induced pluripotent stem cells iPSCs efficiently with a modified protocol that eliminated the need of flow cytometric sorting. Hanke M et al. Kut Q et al. Using various strategies to block neutrophil recruitment to the pre-metastatic site, we demonstrate that neutrophils specifically support metastatic initiation. Other ALDH inhibitors can aldeflior used as appropriate for the enzyme isoform expressed. For nonhematopoietic cells optimal incubation times may be different. Lo JF et al.
Will this cause a problem? No, the reagents in the kit are stable to freezing. Assay performance will not be affected. This is not recommended. The assay buffer incorporates an efflux pump inhibitor to produce optimal discrimination of the ALDHbr cells and to maximize fluorescent signal stability. Not using the assay buffer produces a proportionate loss in the assay signal, depending on the time and temperature at which the stained cells are held.