Abstract
The failure of lung cancer treatments has been attributed mostly to the development of drug resistance, however the underlying cellular and molecular mechanisms are poorly understood. Cancer initiating stem cells (CSCs), present in tumors in a small percentage, play critical roles in the development of drug resistance, metastasis, and cancer relapse. Hence, novel treatments targeting both bulk cancer cells and CSCs are under intense investigation. Herein, we report that lung cancer cells grown on a 3D fibrous scaffold form tumoroids that resemble in vivo tumors, expand CSCs, and provide a platform to identify anti-CSC drugs. The screening of an NCI library of FDA-approved drugs using tumoroid cultures led to identification of Actinomycin D (AD) as a top CSC inhibitor. Since CSCs are mostly resident in the tumor’s inner core, AD was combined with an angiotensin receptor antagonist, Telmisartan (TS), which is known to increase drug permeability in tumors and was shown to have anti-CSC activity. Our results showed that AD + TS administered intra-tumorally was significantly more effective than either drug alone in both syngeneic and xenograft mouse models. The results of mechanistic studies revealed that CSC expansion in tumoroids was associated with activation of β catenin signaling and that AD + TS treatment reduced active β catenin levels in tumors. Together, these results establish the utility of the tumoroid culture system to expand CSCs ex vivo for targeted drug screening, to identify promising novel treatments with both anti-CSC and anti-cancer effects, and to individualize treatments for metastatic drug resistant lung cancer patients.
Highlights
The field of Cancer initiating stem cells (CSCs) research has been confronted with two major barriers
The most notable findings from our studies which provide evidence and new insights to the field include: (i) data showing a combination of Actinomycin D (AD) and TS synergistically inhibits tumor growth and CSC number and activity in vitro and in vivo, (ii) the results of transcriptomic analyses which identified the Wnt/β-catenin pathway as crucial for lung CSC development and re-confirmed the importance of known targets such as Sox[2], ALDH, and Nos[2]
The ex vivo tumoroid cultures allow the study of CSCs in an in vivo like TME and permits expansion and isolation of CSCs so that their unique properties can be studied in interaction with the non-CSC cells
Summary
The field of CSC research has been confronted with two major barriers. First, cell surface markers as well as functional assays used to identify CSCs vary for specific cancer types, a universal CSC marker is lacking[8]. Several approaches are under investigation to expand CSCs in an effort to overcome the limitations inherent to their rarity in culture These include (i) CSC expansion by successive rounds of growth in mouse, which is limited by strain variations, high cost, and time intensive protocols[15,16]; (ii) transformation of CSCs with reprogramming transcription factors such as Oct-4, c-myc and Sox-216; (iii) culturing CSCs on low attachment plates with growth factors such as EGF and FGF; and (iv) expansion of CSCs in organoid cultures by mimicking the 3D tumor microenvironment[17,18] which maximizes cell-matrix and cell-cell interactions and activates CSC signaling pathways[19]. Our data shows that a combination of AD and TS synergistically inhibited both tumor growth and expansion of CSCs in vitro and in vivo, and this inhibition is mediated by decreased signaling through Wnt/β-catenin pathway
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