Cancer Stem Cells (CSCs) are a sub-population of cells, identified in most tumors, responsible for the initiation, recurrence, metastatic potential, and resistance of different malignancies. In prostate cancer (PCa), CSCs were identified and thought to be responsible for the generation of the lethal subtype, commonly known as Castration-Resistant Prostate Cancer (CRPC). In vitro models to investigate the properties of CSCs in PCa are highly required. Sphere-formation assay is an in vitro method commonly used to identify CSCs and study their properties. Here, we report the detailed methodology on how to generate and propagate spheres from PCa cell lines and from murine prostate tissue. This model is based on the ability of stem cells to grow in non-adherent serum-free gel matrix. We also describe how to use these spheres in histological and immuno-fluorescent staining assays to assess the differentiation potential of the CSCs. Our results show the sphere-formation Assay (SFA) as a reliable in vitro assay to assess the presence and self-renewal ability of CSCs in different PCa models. This platform presents a useful tool to evaluate the effect of conventional or novel agents on the initiation and self-renewing properties of different tumors. The effects can be directly evaluated through assessment of the sphere-forming efficiency (SFE) over five generations or other downstream assays such as immuno-histochemical analysis of the generated spheres.

Cancer stem cells (CSCs) residing in colorectal cancer tissues have tumorigenic capacity and contribute to chemotherapeutic resistance and disease relapse. It is well known that the survival of colorectal CSCs after 5-fluorouracil (5-FU)-based therapy leads to cancer recurrence. Thus CSCs represent a promising drug target. Here, we designed and synthesized novel hybrid molecules linking 5-FU with the plant-derived compound thymoquinone (TQ) and tested the potential of individual compounds and their combination to eliminate colorectal CSCs. Both, Combi and SARB hybrid showed augmented cytotoxicity against colorectal cancer cells, but were non-toxic to organoids prepared from healthy murine small intestine. NanoString analysis revealed a unique signature of deregulated gene expression in response to the combination of TQ and 5-FU (Combi) and SARB treatment. Importantly, two principle stem cell regulatory pathways WNT/ß-Catenin and PI3K/AKT were found to be downregulated after Combi and hybrid treatment. Furthermore, both treatments strikingly eliminated CD133+ CSC population, accompanying the depleted self-renewal capacity by eradicating long-term propagated 3D tumor cell spheres at sub-toxic doses. In vivo xenografts on chicken eggs of SARB-treated HCT116 cells showed a prominent nuclear ß-Catenin and E-cadherin staining. This was in line with the reduced transcriptional activity of ß-Catenin and diminished cell adhesion under SARB exposure. In contrast to 5-FU, both, Combi and SARB treatment effectively reduced the angiogenic capacity of the remaining resistant tumor cells. Taken together, combination or hybridization of single compounds target simultaneously a broader spectrum of oncogenic pathways leading to an effective eradication of colorectal cancer cells.

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The high recurrence rates of colorectal cancer have been associated with a small population of cancer stem cells (CSCs) that are resistant to the standard chemotherapeutic drug, 5-fluorouracil (5FU). Thymoquinone (TQ) has shown promising antitumor properties on numerous cancer systems both in vitro and in vivo ; however, its effect on colorectal CSCs is poorly established. Here, we investigated TQ’s potential to target CSCs in a three-dimensional (3D) sphere-formation assay enriched for a population of colorectal cancer stem/progenitor cells. Our results showed a significant decrease in self-renewal potential of CSC populations enriched from 5FU-sensitive and resistant HCT116 cells at 10-fold lower concentrations when compared to 2D monolayers. TQ decreased the expression levels of colorectal stem cell markers CD44 and Epithelial Cell Adhesion Molecule EpCAM and proliferation marker Ki67 in colonospheres derived from both cell lines and reduced cellular migration and invasion. Further investigation revealed that TQ treatment led to increased TUNEL positivity and a dramatic increase in the amount of the DNA damage marker gamma H2AX particularly in 5FU-resistant colonospheres, suggesting that the diminished sphere forming ability in TQ-treated colonospheres is due to induction of DNA damage and apoptotic cell death. The intraperitoneal injection of TQ in mice inhibited tumor growth of spheres derived from 5FU-sensitive and 5FU-resistant HCT116 cells. Furthermore, TQ induced apoptosis and inhibited NF-κB and MEK signaling in mouse tumors. Altogether, our findings document TQ’s effect on colorectal cancer stem-like cells and provide insights into its underlying mechanism of action.

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Background: Prostate cancer (PC) is the most frequently diagnosed cancer among men worldwide. The poor prognosis of PC is largely due to late diagnosis of the disease when it has progressed to advanced stages marked by androgen-independence. We interrogated proteomic signatures that embody the transition of PC from an androgen-dependent (AD) to an androgen-independent (AI) state. Methods: We have previously established AD and AI murine PC cell lines, PLum-AD and PLum-AI, respectively, which recapitulate primary and progressive PC at phenotypic and subcellular levels. We statistically surveyed global protein expression profiles in these cell lines. Differential profiles were functionally interrogated by pathways and protein–protein interaction network analyses. Results: Protein expression pattern analysis revealed a total of 683 proteins, among which 99 were significantly differentially altered in PLum-AI cells as compared to PLum-AD cells (45 increased and 54 decreased). Principal component analysis (PCA) revealed that the two different cell lines clearly separated apart, indicating a significant proteome expression difference between them. Four of the proteins (vimentin, catalase, EpCAM, and caspase 3) that were differentially expressed in PLum-AI cells compared to PLum-AD cells were subjected to biochemical validation by Western blotting. Biological process gene ontology (GO) analysis of the differentially expressed proteins demonstrated enrichment of biological functions and pathways in PLum-AI cells that are central to PI3 kinase and androgen receptor pathways. Besides, other relevant biological processes that are enriched in PLum-AI cells included cell adhesion and cell migration processes, cell and DNA damage, apoptosis, and cell cycle regulation. Conclusions: Our protein expression analysis of a murine in vitro model of PC progression identified differential protein spots that denote this progression and that comprise high-potential targets for early treatment of PC with a personalized patient-specific approach. Efforts are underway to functionally assess the potential roles of these proteins as therapeutic targets for PC progression.

Resistance of cancer cells and normal tissue toxicity of ionizing radiation (IR) are known to limit the success of radiotherapy. There is growing interest in using IR with natural compounds to sensitize cancer cells and spare healthy tissues. Thymoquinone (TQ) was shown to radiosensitize several cancers, yet no studies have investigated its radiosensitizing effects on colorectal cancer (CRC). Here, we combined TQ with IR and determined its effects in two-dimensional (2D) and three-dimensional (3D) culture models derived from HCT116 and HT29 CRC cells, and in patient-derived organoids (PDOs). TQ sensitized CRC cells to IR and reduced cell viability and clonogenic survival and was non-toxic to non-tumorigenic intestinal cells. TQ sensitizing effects were associated with G2/M arrest and DNA damage as well as changes in key signaling molecules involved in this process. Combining a low dose of TQ (3 µM) with IR (2 Gy) inhibited sphere formation by 100% at generation 5 and this was associated with inhibition of stemness and DNA repair. These doses also led to ~1.4- to ~3.4-fold decrease in organoid forming ability of PDOs. Our findings show that combining TQ and IR could be a promising therapeutic strategy for eradicating CRC cells.

Ichthyosis Follicularis, Atrichia, and Photophobia (IFAP) is a severe rare genetic disorder caused by mutations in the gene encoding the Membrane-Bound Transcription Factor Peptidase, Site 2 (MBTPS2). Olmsted syndrome is another rare genetic disease with overlapping clinical features caused by mutations in the gene encoding the Transient Receptor Potential Cation Channel, subfamily V (TRPV3). Mutations in MBTPS2 have been recently reported in Olmsted syndrome, underscoring the overlap and the confusion in separating Olmsted from IFAP syndrome. We studied a Lebanese family with IFAP syndrome both, clinically and molecularly, and investigated whether there is a cross relation between TRPV3 and MBTPS2. We identified a recurrent mutation designated p.F475S in MBTPS2 in the affected individuals. This mutation was not found in 100 control individuals from the same population. We determined that TRPV3 regulatory region is a target for MBTPS2. In addition, there was an increased cell death in the cells transfected with the mutant versus the wild-type MBTPS2. In conclusion, we identified a direct regulatory effect of MBTPS2 on TRPV3 which can partially contribute to the overlapping clinical features of IFAP and Olmsted syndromes under a common signaling pathway.