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.

Esophageal adenocarcinoma (EAC), the predominant type of esophageal cancer in the United States, develops through Barrett’s esophagus (BE)-dysplasia-carcinoma cascade. Gastroesophageal reflux disease, where acidic bile salts refluxate into the esophagus, is the main risk factor for the development of BE and its progression to EAC. The NFE2-related factor 2 (NRF2) is the master cellular antioxidant regulator. We detected high NRF2 protein levels in the EAC cell lines and primary tissues. Knockdown of NRF2 significantly enhanced acidic bile salt-induced oxidative stress, DNA damage, and inhibited EAC cell growth. Brusatol, an NRF2 inhibitor, significantly inhibited NRF2 transcriptional activity and downregulated the NRF2 target genes. We discovered that in addition to inducing apoptosis, Brusatol alone or in combination with cisplatin (CDDP) induced significant lipid peroxidation and ferroptosis, as evidenced by reduced xCT and GPX4 expression, two known ferroptosis markers. The combination of Brusatol and CDDP significantly inhibited EAC tumor xenograft growth in vivo and confirmed the in vitro data showing ferroptosis as an important mechanism in the tumors treated with Brusatol or Brusatol and CDDP combination. Our data support the role of NRF2 in protecting against stress-induced apoptosis and ferroptosis in EACs. Targeting NRF2 in combination with platinum therapy can be an effective strategy for eliminating cancer cells in EAC.

Unfolded protein response (UPR) protects malignant cells from endoplasmic reticulum stress-induced apoptosis. We report that Aurora kinase A (AURKA) promotes cancer cell survival by activating UPR in esophageal adenocarcinoma (EAC). A strong positive correlation between AURKA and binding immunoglobulin protein (BIP) mRNA expression levels was found in EACs. The in vitro assays indicated that AURKA promoted IRE1α protein phosphorylation, activating prosurvival UPR in FLO-1 and OE33 cells. The use of acidic bile salts to mimic reflux conditions in patients induced high AURKA and IRE1α levels. This induction was abrogated by AURKA knockdown in EAC cells. AURKA and p-IRE1α protein colocalization was observed in neoplastic gastroesophageal lesions of the L2-IL1b mouse model of Barrett’s esophageal neoplasia. The combined treatment using AURKA inhibitor and tunicamycin synergistically induced cancer cell death. The use of alisertib for AURKA inhibition in the EAC xenograft model led to a decrease in IRE1α phosphorylation with a significant reduction in tumor growth. These results indicate that AURKA activates UPR, promoting cancer cell survival during ER stress in EAC. Targeting AURKA can significantly reverse prosurvival UPR signaling mechanisms and decrease cancer cell survival, providing a promising approach for the treatment of EAC patients.