In this study, we developed an image-based assay to specifically analyze the HDECC population in cancer cells. The assay can be easily adopted for high-throughput studies using a regular automated fluorescence microscope. This capability was demonstrated by a screening on the LOPAC library which successfully identified 12 compounds that can potently inhibit HDECCs in lung cancer cells. Our study focused on lung cancer cells but the assay can also be performed on adherent cancer cells of other origins ().
The nature of HDECCs is still unclear. Many recent studies showed that they are more tumorigenic when injected into immunodeficient mice, suggesting that they are selectively enriched with stem-like cancer cells (6
). Whether there is correlation between the two cell populations remains controversial, our results that four compounds inhibiting HDECCs reduced the tumorigenicity of cancer cells provided new evidence for this correlation. Moreover, several proteins targeted by the inhibitors identified in this work have been shown by independent studies to play key roles in stem-like cancer cells. One of the targets, PRL-3, has been associated with tumorigenesis and metastasis by many studies (39
). Compounds modulating the other target, dopamine receptor, can potently inhibit the growth of cancerous neural progenitor cells (35
). These evidences support an effect of the HDECC inhibitors on stem-like cancer cells and a relationship between HDECCs and stem-like cancer cells. However, it should be noted that none of the inhibition of tumorigenicity was complete, possibly because the two cell populations are not identical although highly overlapping. Drug screening on cancer stem cells is challenging due to the rarity of these cells as well as their instability in culture such that few advances have been made so far (41
), the current reported system may provide a new angle for caner stem cell research, as well as drug discovery.
In addition to their correlation with stem-like cancer cells, HDECCs represent a major obstacle for chemotherapy as they cause MDR. Traditionally, development of drugs to overcome resistance has been done on induced resistant cell lines generated by continuous or pulsed exposure to drugs (42
). However, such induced cells often are associated with changes which are partly characteristic of resistance to certain drugs instead of MDR (44
). We adopted a different approach by selectively studying an intrinsic MDR cell population in the context of the whole cancer cell population. Nine compounds were identified by the reported HCS system that can nonspecifically enhance the efficacy of chemotherapeutic drugs in vitro
and two of them were further shown to be also effective in vivo
(), highlighting its potential for MDR drug discovery. However, MDR can be caused by a huge variety of transporters and many of them may not be revealed by the Hoechst efflux assay, as well as mechanisms other than drug-efflux (5
), so that ineffective compounds may be identified in our screening. Indeed, three inhibitors failed to improve chemotherapy efficacy either in vitro
or in vivo
although they were able to reduce Hoechst efflux. Nevertheless, most inhibitors identified significantly improved the efficacy for all the chemotherapy drugs we tested at least in vitro
, suggesting that Hoechst efflux may be used as an effective indicator for MDR. None of the effective inhibitors kill HDECCs directly, our system was able to identified these non-toxic effectors because the image-based approach allowed more comprehensive measurement of the responses to perturbance than simple viability assay. Our study focused on drug-efflux and cell viability but actually a complete morphology phenotype profile can be generated from the images (22
) which may greatly facilitate related mechanism study in the future.
High-throughput compound library screening usually identifies effectors that result in desired phenotype without revealing their mechanism. In the absence of the knowledge, it is hard to predict the therapeutic potential of the compounds as they may be associated with severe side effects. For this concern we used LOPAC library of which the compound has been extensively characterized with known protein-substrate interactions. Such approach may not only allow quick translation of the screening result to clinical application, but also reveal the “chemical genetics” (45
) to provide insight into the related molecular mechanisms. The latter potential is demonstrated by the consistency of our screening results with previous reports on individual protein targets, like the aforementioned roles of dopamine receptor and PRL-3 for stem-like cancer cells. Reduction of HDECC by IGF-1R inhibitor PQ401 is also consistent with previous reports that IGF-1R inhibition may sensitize lung cancer cells to chemotherapy (46
), suggesting a key role of IGF-1R for MDR. The finding that retinoic acid can significantly increase HDECC is surprising, but retinoic acid has long been noticed to be able to induce the expression of MDR genes and cause drug resistance (47
), although it has been used clinically for leukemia treatment to induce stem cell differentiation. Other mechanisms such like the reduction of tumorigenicity by DMCM is unclear and need to be further investigated. With a more complete library with most major signaling pathways targeted by multiple structurally divergent chemicals, such chemical genetics approach would help to reveal the whole repertoire of signaling pathways underlying HDECCs.