Maximilian Boesch

Drug Transporter-Mediated Protection of Cancer Stem Cells From Ionophore Antibiotics

Ionophore antibiotics were reported to selectively kill cancer stem cells and to overcome multidrug resistance, but mechanistic studies of the significance of drug transporters for treatment with these compounds are lacking. We applied chemosensitivity testing of well‐characterized human cancer cell lines to elaborate on whether drug transporters are involved in protection from the cytotoxic effects of the ionophore antibiotics salinomycin and nigericin. Our experiments demonstrated that ionophore antibiotics were ineffective against both stem‐like ovarian cancer side population cells (expressing either ABCB1 or ABCG2) and K562/Dox‐H1 cells, which constitute a genetically defined model system for ABCB1 expression. Considering that cancer stem cells often express high levels of drug transporters, we deduced from our results that ionophore antibiotics are less suited to cancer stem cell‐targeted treatment than previously thought.

DyeCycle violet used for side population detection is a substrate of P‐glycoprotein

Cancer stem cells (CSC) are increasingly recognized as key target cells for cancer therapy because they are both tumorigenic and chemoresistant and, therefore, can give rise to recurrent disease. Side population (SP) sorting using an ultraviolet (UV) laser is an established method to isolate CSC based on ABC drug transporter‐dependent (e.g., ABCG2) Hoechst 33342 efflux. We here show that Vybrant® DyeCycle™ Violet (DCV), a DNA‐binding fluorophor allowing SP sorting using a non‐UV laser (such as violet laser), is transported via P‐glycoprotein (PgP). Because PgP might be particularly abundant in multidrug‐resistant cancer cells rather than bona fide CSC, investigators using DCV should be aware that this strategy might also detect PgP‐expressing non‐CSC.

Protein signatures of oxidative stress response in a patient specific cell line model for autism

BackgroundKnown genetic variants can account for 10% to 20% of all cases with autism spectrum disorders (ASD). Overlapping cellular pathomechanisms common to neurons of the central nervous system (CNS) and in tissues of peripheral organs, such as immune dysregulation, oxidative stress and dysfunctions in mitochondrial and protein synthesis metabolism, were suggested to support the wide spectrum of ASD on unifying disease phenotype. Here, we studied in patient-derived lymphoblastoid cell lines (LCLs) how an ASD-specific mutation in ribosomal protein RPL10 (RPL10[H213Q]) generates a distinct protein signature. We compared the RPL10[H213Q] expression pattern to expression patterns derived from unrelated ASD patients without RPL10[H213Q] mutation. In addition, a yeast rpl10 deficiency model served in a proof-of-principle study to test for alterations in protein patterns in response to oxidative stress.MethodsProtein extracts of LCLs from patients, relatives and controls, as well as diploid yeast cells hemizygous for rpl10, were subjected to two-dimensional gel electrophoresis and differentially regulated spots were identified by mass spectrometry. Subsequently, Gene Ontology database (GO)-term enrichment and network analysis was performed to map the identified proteins into cellular pathways.ResultsThe protein signature generated by RPL10[H213Q] is a functionally related subset of the ASD-specific protein signature, sharing redox-sensitive elements in energy-, protein- and redox-metabolism. In yeast, rpl10 deficiency generates a specific protein signature, harboring components of pathways identified in both the RPL10[H213Q] subjects’ and the ASD patients’ set. Importantly, the rpl10 deficiency signature is a subset of the signature resulting from response of wild-type yeast to oxidative stress.ConclusionsRedox-sensitive protein signatures mapping into cellular pathways with pathophysiology in ASD have been identified in both LCLs carrying the ASD-specific mutation RPL10[H213Q] and LCLs from ASD patients without this mutation. At pathway levels, this redox-sensitive protein signature has also been identified in a yeast rpl10 deficiency and an oxidative stress model. These observations point to a common molecular pathomechanism in ASD, characterized in our study by dysregulation of redox balance. Importantly, this can be triggered by the known ASD-RPL10[H213Q] mutation or by yet unknown mutations of the ASD cohort that act upstream of RPL10 in differential expression of redox-sensitive proteins.

Optimized Stem Cell Detection Using the DyeCycle-Triggered Side Population Phenotype

Tissue and cancer stem cells are highly attractive target populations for regenerative medicine and novel potentially curative anticancer therapeutics. In order to get a better understanding of stem cell biology and function, it is essential to reproducibly identify these stem cells from biological samples for subsequent characterization or isolation. ABC drug transporter expression is a hallmark of stem cells. This is utilized to identify (cancer) stem cells by exploiting their dye extrusion properties, which is referred to as the “side population assay.” Initially described for high-end flow cytometers equipped with ultraviolet lasers, this technique is now also amenable for a broader scientific community, owing to the increasing availability of violet laser-furnished cytometers and the advent of DyeCycle Violet (DCV). Here, we describe important technical aspects of the DCV-based side population assay and discuss potential pitfalls and caveats helping scientists to establish a valid and reproducible DCV-based side population assay. In addition, we investigate the suitability of blue laser-excitable DyeCycle dyes for side population detection. This knowledge will help to improve and standardize detection and isolation of stem cells based on their expression of ABC drug transporters.

Ionophore Antibiotics as Cancer Stem Cell-Selective Drugs: Open Questions

Ionophore Antibiotics as Cancer Stem Cell-Selective Drugs: Open Questions MAXIMILIAN BOESCH,* SIEGHART SOPPER,* DOMINIK WOLF Institute of Immunobiology, Kantonsspital St. Gallen, St. Gallen, Switzerland; Internal Medicine V, Medical University of Innsbruck, Innsbruck, Austria; Tyrolean Cancer Research Institute, Innsbruck, Austria; Medical Clinic III, University Clinic Bonn, Bonn, Germany *Contributed equally. Disclosures of potential conflicts of interest may be found at the end of this article.

Concise Review: Aggressive Colorectal Cancer: Role of Epithelial Cell Adhesion Molecule in Cancer Stem Cells and Epithelial‐to‐Mesenchymal Transition

Colorectal cancer (CRC) is one of the most common malignancies worldwide. In spite of various attempts to ameliorate outcome by escalating treatment, significant improvement is lacking particularly in the adjuvant setting. It has been proposed that cancer stem cells (CSCs) and the epithelial‐to‐mesenchymal transition (EMT) are at least partially responsible for therapy resistance in CRC. The epithelial cell adhesion molecule (EpCAM) was one of the first CSC antigens to be described. Furthermore, an EpCAM‐specific antibody (edrecolomab) has the merit of having launched the era of monoclonal antibody treatment in oncology in the 1990s. However, despite great initial enthusiasm, monoclonal antibody treatment has not proven successful in the adjuvant treatment of CRC patients. In the meantime, new insights into the function of EpCAM in CRC have emerged and new drugs targeting various epitopes have been developed. In this review article, we provide an update on the role of EpCAM in CSCs and EMT, and emphasize the potential predictive selection criteria for novel treatment strategies and refined clinical trial design. Stem Cells Translational Medicine 2018;7:495–501

Truncated isoform Vav3.1 is highly expressed in ovarian cancer stem cells and clinically relevant in predicting prognosis and platinum-response

Vav3 is a key modulator of GTP‐hydrolases of the Rho/Rac family, which are crucially involved in cell proliferation. Vav3 is alternatively spliced in full‐length Vav3‐alpha and N‐terminal truncated Vav3.1 lacking its self‐regulatory domains. The aim of our study was to estimate the clinical impact of Vav3 and all other Vav family members in ovarian cancer. Purification of a stem‐cell like side‐population (SP) from ovarian cancer cell lines was performed by flow cytometry/FACS. Differences in gene expression between SP and NSP were assessed by Gene Array analysis and confirmed by RT‐PCR and immunoblot. In addition, Vav mRNA expression was determined in 150 epithelial ovarian cancers. Clinicopathological parameters, platinum‐sensitivity and survival were analyzed and associated with Vav expression. SP fractions of ovarian cancer cell lines exhibited marked overexpression of Vav3.1 (p < 0.001). Vav1 and Vav2 did not prove to be of clinicopathologic relevance in ovarian cancer. High Vav3.1 expression correlated with higher FIGO stage and residual disease. Furthermore, Vav3.1 overexpression was associated with poor progression‐free (HR = 2.820, p = 0.0001) and overall survival (HR = 2.842, p = 0.0001). Subgroup analyses revealed an impact of Vav3.1 on survival in Type‐II but not in Type‐I cancers. Notably, platinum‐refractory cancers showed marked overexpression of Vav3.1 compared to other subsets of platinum‐sensitivity (15.848 vs. 6.653, p = 0.0001). In conclusion, Vav3.1 is over‐expressed in stem‐cell like SP fractions and is clinically relevant in the pathophysiology of ovarian cancer. The N‐terminal truncated Vav3.1 may be decisively involved in mechanisms causing genuine multi‐drug resistance.

High prevalence of side population in human cancer cell lines.

Cancer cell lines are essential platforms for performing cancer research on human cells. We here demonstrate that, across tumor entities, human cancer cell lines harbor minority populations of putative stem-like cells, molecularly defined by dye extrusion resulting in the side population phenotype. These findings establish a heterogeneous nature of human cancer cell lines and argue for their stem cell origin. This should be considered when interpreting research involving these model systems.

CCL19-producing fibroblastic stromal cells restrain lung carcinoma growth by promoting local antitumor T-cell responses

Background A particular characteristic of non–small cell lung cancer is the composition of the tumor microenvironment with a very high proportion of fibroblastic stromal cells (FSCs). Objective Lapses in our basic knowledge of fibroblast phenotype and function in the tumor microenvironment make it difficult to define whether FSC subsets exist that exhibit either tumor‐promoting or tumor‐suppressive properties. Methods We used gene expression profiling of lung versus tumor FSCs from patients with non–small cell lung cancer. Moreover, CCL19‐expressing FSCs were studied in transgenic mouse models by using a lung cancer metastasis model. Results CCL19 mRNA expression in human tumor FSCs correlates with immune cell infiltration and intratumoral accumulation of CD8+ T cells. Mechanistic dissection in murine lung carcinoma models revealed that CCL19‐expressing FSCs form perivascular niches to promote accumulation of CD8+ T cells in the tumor. Targeted ablation of CCL19‐expressing tumor FSCs reduced immune cell recruitment and resulted in unleashed tumor growth. Conclusion These data suggest that a distinct population of CCL19‐producing FSCs fosters the development of an immune‐stimulating intratumoral niche for immune cells to control cancer growth. Graphical abstract Figure. No Caption available.

Interleukin 7-expressing fibroblasts promote breast cancer growth through sustenance of tumor cell stemness

ABSTRACT The tumor microenvironment harbors cancer-associated fibroblasts that function as major modulators of cancer progression. Here, we assessed to which extent distinct cancer-associated fibroblast subsets impact mammary carcinoma growth and cancer cell stemness in an orthotopic murine model. We found that fibroblasts expressing the Cre recombinase under the control of the interleukin 7 promoter occupied mainly the tumor margin where they physically interacted with tumor cells. Intratumoral ablation of interleukin 7-expressing fibroblasts impaired breast tumor growth and reduced the clonogenic potential of cancer cells. Moreover, cDNA expression profiling revealed a distinct oncogenic signature of interleukin 7-producing fibroblasts. In particular, Cxcl12 expression was strongly enhanced in interleukin 7-producing fibroblasts and cell type-specific genetic ablation and systemic pharmacological inhibition revealed that the CXCL12/CXCR4 axis impacts breast tumor cell stemness. Elevated expression of CXCL12 and other stem cell factors in primary human breast cancer-associated fibroblasts indicates that certain fibroblast populations support tumor cell stemness and thereby promote breast cancer growth.