Efficacy of Lapatinib in Patients with HER2-Negative Metastatic Breast Cancer and HER2-Positive Circulating Tumor Cells-The DETECT III Clinical Trial.

BACKGROUND: The phenotypes of tumor cells change during disease progression, but invasive rebiopsies of metastatic lesions are not always feasible. Here we aimed to determine whether initially HER2-negative metastatic breast cancer (MBC) patients with HER2-positive circulating tumor cells (CTCs) benefit from a HER2-targeted therapy. METHODS: The open-label, interventional randomized phase III clinical trial (EudraCT Number 2010-024238-46, CliniclTrials.gov Identifier: NCT01619111) recruited from March 2012 until September 2019 with a follow-up duration of 19.5 months. It was a multicenter clinical trial with 94 participating German study centers. A total of 2137 patients with HER2-negative MBC were screened for HER2-positive CTCs with a final modified intention-to-treat population of 101 patients. Eligible patients were randomized to standard therapy with or without lapatinib. Primary study endpoints included CTC clearance (no CTCs at the end of treatment) and secondary endpoints were progression-free survival, overall survival (OS), and safety. RESULTS: In both treatment arms CTC clearance at first follow-up visit-although not being significantly different for both arms at any time point-was significantly associated with improved OS (42.4 vs 14.1 months; P = 0.002). Patients treated additionally with lapatinib had a significantly improved OS over patients receiving standard treatment (20.5 vs 9.1 months, P = 0.009). CONCLUSIONS: DETECT III is the first clinical study indicating that phenotyping of CTCs might have clinical utility for stratification of MBC cancer patients to HER2-targeting therapies. The OS benefit could be related to lapatinib, but further studies are required to prove this clinical observation. ClinicalTrials.gov Registration Number: NCT01619111.

A workflow for the enrichment, the identification, and the isolation of non-apoptotic single circulating tumor cells for RNA sequencing analysis.

Circulating tumor cells (CTCs) are constantly shed by tumor tissue and can serve as a valuable analyte for a gene expression analysis from a liquid biopsy. However, a high proportion of CTCs can be apoptotic leading to rapid mRNA decay and challenging the analysis of their transcriptome. We established a workflow to enrich, to identify, and to isolate single CTCs including the discrimination of apoptotic and non-apoptotic CTCs for further single CTC transcriptome analysis. Viable tumor cells-we first used cells from breast cancer cell lines followed by CTCs from metastatic breast cancer patients-were enriched with the CellSearch system from diagnostic leukapheresis products, identified by immunofluorescence analysis for neoplastic markers, and isolated by micromanipulation. Then, their cDNA was generated, amplified, and sequenced. In order to exclude early apoptotic tumor cells, staining with Annexin V coupled to a fluorescent dye was used. Annexin V staining intensity was associated with decreased RNA integrity as well as lower numbers of total reads, exon reads, and detected genes in cell line cells and CTCs. A comparative RNA analysis of single cells from MDA-MB-231 and MCF7 cell lines revealed the expected differential transcriptome profiles. Enrichment and staining procedures of cell line cells that were spiked into blood had only little effect on the obtained RNA sequencing data compared to processing of naïve cells. Further, the detection of transcripts of housekeeping genes such as GAPDH was associated with a significantly higher quality of expression data from CTCs. This workflow enables the enrichment, detection, and isolation of single CTCs for individual transcriptome analyses. The discrimination of apoptotic and non-apoptotic cells allows to focus on CTCs with a high RNA integrity to ensure a successful transcriptome analysis.

Progesterone-induced progesterone receptor membrane component 1 rise-to-decline changes are essential for decidualization.

BACKGROUND: Decidualization of endometrial cells is the prerequisite for embryo implantation and subsequent placenta formation and is induced by rising progesterone levels following ovulation. One of the hormone receptors contributing to endometrial homeostasis is Progesterone Receptor Membrane Component 1 (PGRMC1), a non-classical membrane-bound progesterone receptor with yet unclear function. In this study, we aimed to investigate how PGRMC1 contributes to human decidualization. METHODS: We first analyzed PGRMC1 expression profile during a regular menstrual cycle in RNA-sequencing datasets. To further explore the function of PGRMC1 in human decidualization, we implemented an inducible decidualization system, which is achieved by culturing two human endometrial stromal cell lines in decidualization-inducing medium containing medroxyprogesterone acetate and 8-Br-cAMP. In our system, we measured PGRMC1 expression during hormone induction as well as decidualization status upon PGRMC1 knockdown at different time points. We further conferred proximity ligation assay to identify PGRMC1 interaction partners. RESULTS: In a regular menstrual cycle, PGRMC1 mRNA expression is gradually decreased from the proliferative phase to the secretory phase. In in vitro experiments, we observed that PGRMC1 expression follows a rise-to-decline pattern, in which its expression level initially increased during the first 6 days after induction (PGRMC1 increasing phase) and decreased in the following days (PGRMC1 decreasing phase). Knockdown of PGRMC1 expression before the induction led to a failed decidualization, while its knockdown after induction did not inhibit decidualization, suggesting that the progestin-induced 'PGRMC1 increasing phase' is essential for normal decidualization. Furthermore, we found that the interactions of prohibitin 1 and prohibitin 2 with PGRMC1 were induced upon progestin treatment. Knocking down each of the prohibitins slowed down the decidualization process compared to the control, suggesting that PGRMC1 cooperates with prohibitins to regulate decidualization. CONCLUSIONS: According to our findings, PGRMC1 expression followed a progestin-induced rise-to-decline expression pattern during human endometrial decidualization process; and the correct execution of this expression program was crucial for successful decidualization. Thereby, the results of our in vitro model explained how PGRMC1 dysregulation during decidualization may present a new perspective on infertility-related diseases.

Ultra-sensitive CTC-based liquid biopsy for pancreatic cancer enabled by large blood volume analysis.

The limited sensitivity of circulating tumor cell (CTC) detection in pancreatic adenocarcinoma (PDAC) stems from their extremely low concentration in the whole circulating blood, necessitating enhanced detection methodologies. This study sought to amplify assay-sensitivity by employing diagnostic leukapheresis (DLA) to screen large blood volumes. Sixty patients were subjected to DLA, with a median processed blood volume of ~ 2.8 L and approximately 5% of the resulting DLA-product analyzed using CellSearch (CS). Notably, DLA significantly increased CS-CTC detection to 44% in M0-patients and 74% in M1-patients, yielding a 60-fold increase in CS-CTC enumeration. DLA also provided sufficient CS-CTCs for genomic profiling, thereby delivering additional genomic information compared to tissue biopsy samples. DLA CS-CTCs exhibited a pronounced negative prognostic impact on overall survival (OS), evidenced by a reduction in OS from 28.6 to 8.5 months (univariate: p = 0.002; multivariable: p = 0.043). Additionally, a marked enhancement in sensitivity was achieved (by around 3-4-times) compared to peripheral blood (PB) samples, with positive predictive values for OS being preserved at around 90%. Prognostic relevance of CS-CTCs in PDAC was further validated in PB-samples from 228 PDAC patients, consolidating the established association between CTC-presence and reduced OS (8.5 vs. 19.0 months, p < 0.001). In conclusion, DLA-derived CS-CTCs may serve as a viable tool for identifying high-risk PDAC-patients and aiding the optimization of multimodal treatment strategies. Moreover, DLA enables comprehensive diagnostic profiling by providing ample CTC material, reinforcing its utility as a reliable liquid-biopsy approach. This high-volume liquid-biopsy strategy presents a potential pathway for enhancing clinical management in this malignancy.

Dynamics of CXCR4 positive circulating tumor cells in prostate cancer patients during radiotherapy.

Ablative radiotherapy is a highly efficient treatment modality for patients with metastatic prostate cancer (PCa). However, a subset of patients does not respond. Currently, this subgroup with bad prognosis cannot be identified before disease progression. We hypothesize that markers indicative of radioresistance, stemness and/or bone tropism may have a prognostic potential to identify patients profiting from metastases-directed radiotherapy. Therefore, circulating tumor cells (CTCs) were analyzed in patients with metastatic PCa (n = 24) during radiotherapy with CellSearch, multicolor flow cytometry and imaging cytometry. Analysis of copy-number alteration indicates a polyclonal CTC population that changes after radiotherapy. CTCs were found in 8 out of 24 patients (33.3%) and were associated with a shorter time to biochemical progression after radiotherapy. Whereas the total CTC count dropped after radiotherapy, a chemokine receptor CXCR4-expressing subpopulation representing 28.6% of the total CTC population remained stable up to 3 months. At once, we observed higher chemokine CCL2 plasma concentrations and proinflammatory monocytes. Additional functional analyses demonstrated key roles of CXCR4 and CCL2 for cellular radiosensitivity, tumorigenicity and stem-like potential in vitro and in vivo. Moreover, a high CXCR4 and CCL2 expression was found in bone metastasis biopsies of PCa patients. In summary, panCK+ CXCR4+ CTCs may have a prognostic potential in patients with metastatic PCa treated with metastasis-directed radiotherapy.

Comparative analysis of EpCAM high-expressing and low-expressing circulating tumour cells with regard to their clonal relationship and clinical value.

BACKGROUND: Circulating tumour cells (CTCs) are mainly enriched based on the epithelial cell adhesion molecule (EpCAM). Although it was shown that an EpCAM low-expressing CTC fraction is not captured by such approaches, knowledge about its prognostic and predictive relevance and its relation to EpCAM-positive CTCs is lacking. METHODS: We developed an immunomagnetic assay to enrich CTCs from metastatic breast cancer patients EpCAM independently using antibodies against Trop-2 and CD-49f and characterised their EpCAM expression. DNA of single EpCAM high expressing and low expressing CTCs was analyzed regarding chromosomal aberrations and predictive mutations. Additionally, we compared CTC-enrichment on the CellSearch system using this antibody mix and the EpCAM based enrichment. RESULTS: Both antibodies acted synergistically in capturing CTCs. Patients with EpCAM high-expressing CTCs had a worse overall and progression-free survival. EpCAM high- and low-expressing CTCs presented similar chromosomal aberrations and mutations indicating a close evolutionary relationship. A sequential enrichment of CTCs from the EpCAM-depleted fraction yielded a population of CTCs not captured EpCAM dependently but harbouring predictive information. CONCLUSIONS: Our data indicate that EpCAM low-expressing CTCs could be used as a valuable tumour surrogate material-although they may be prognostically less relevant than EpCAM high-expressing CTCs-and have particular benefit if no CTCs are detected using EpCAM-dependent technologies.

Implementing microwell slides for detection and isolation of single circulating tumor cells from complex cell suspensions.

Cell loss during detection and isolation of circulating tumor cells (CTCs) is a challenge especially when label-free pre-enrichment technologies are used without the aid of magnetic particles. Although microfluidic systems can remove the majority of "contaminating" white blood cells (WBCs), their remaining numbers are still impeding single CTC isolation, thus making additional separation steps needed. This study aimed to develop a workflow from blood-to-single CTC for complex cell suspensions by testing two microwell formats. In the first step, different cell lines were used to compare the performances of Sievewell™ 370 K (TOK, Japan) and CellCelector™ Nanowell U25 (ALS Automated Lab Solutions, Germany) slides for cell labelling and single-cell micromanipulation. Confounding levels of auto-fluorescence inherent to different plastic materials used to cast the microwells, staining recovery rates, and cell isolation rates were determined. In the second step, three different blood preservation tubes were tested for RNA analysis. Lastly, the established workflow was applied to isolate CTCs from peripheral blood samples obtained from metastasized breast cancer (mBC) patients for single-cell DNA and RNA analysis. The detection of CTCs in Sievewell slides profit from better signal-to-noise ratios in the fluorescence channels mainly used for CTC detection. In addition, due to its design, Sievewell supports direct in situ CTC labelling, which minimizes cell loss and leads to single-cell recovery rates after staining of approx. 94%. Detection of PIK3CA mutations in single CTCs verified the applicability of the workflow for the analysis of genomic DNA of CTCs. Furthermore, combined with blood preservation up to 48 h at room temperature in LBguard tubes, panel RT-PCR transcript analysis was successful for single cell line cells and CTCs, respectively. The combined use of Sievewell microwell slides and CellCelector™ automated micromanipulation system improves single CTC detection, labelling and isolation from complex cell suspensions. This approach is especially valuable when samples of high cellular content are processed.

Label-Free Enrichment and Molecular Characterization of Viable Circulating Tumor Cells from Diagnostic Leukapheresis Products.

INTRODUCTION: Circulating tumor cells (CTCs) may be used to improve cancer diagnosis, prognosis, and treatment. However, because knowledge regarding CTC biology is limited and the numbers of CTCs and CTC-positive cancer patients are low, progress in this field is slow. We addressed this limitation by combining diagnostic leukapheresis (DLA) and microfluidic enrichment to obtain large numbers of viable CTCs from metastasized breast cancer patients. METHODS: DLA was applied to 9 patients, and 7.5 mL of peripheral blood was drawn. CTCs were enriched with the Parsortix™ system. The quality of CTCs from fresh and cryopreserved DLA products was tested, and CTCs were cultured in vitro. Single uncultured and cultured CTCs were isolated by micromanipulation to determine different parameters, such as genomic aberrations and mutation profiles of selected tumor-associated genes. Expression levels of estrogen receptor and HER2/neu were monitored during in vitro culture. RESULTS: Viable CTCs from peripheral blood and fresh or frozen DLA products could be enriched. DLA increased the likelihood of successful CTC culture. Cryopreserved DLA products could be stored with minimal CTC loss and no overt reduction in the tumor cell quality and viability during an observation period of up to 3 years. The analyzed parameters did not change during in vitro culture. DLA samples with high CTC numbers and lower ratios of apoptotic CTCs were more likely to grow in culture. CONCLUSIONS: The increased CTC numbers from fresh or cryopreserved DLA products facilitate multiple functional and molecular analyses and, thus, could improve our knowledge of their biology.

From in vitro to ex vivo: subcellular localization and uptake of graphene quantum dots into solid tumors.

Among various nanoparticles tested for pharmacological applications over the recent years, graphene quantum dots (GQDs) seem to be promising candidates for the construction of drug delivery systems due to their superior biophysical and biochemical properties. The subcellular fate of incorporated nanomaterial is decisive for transporting pharmaceuticals into target cells. Therefore a detailed characterization of the uptake of GQDs into different breast cancer models was performed. The demonstrated accumulation inside the endolysosomal system might be the reason for the particles' low toxicity, but has to be overcome for cytosolic or nuclear drug delivery. Furthermore, the penetration of GQDs into precision-cut mammary tumor slices was studied. These constitute a far closer to reality model system than monoclonal cell lines. The constant uptake into the depth of the tissue slices underlines the systems' potential for drug delivery into solid tumors.

Diagnostic leukapheresis for CTC analysis in breast cancer patients: CTC frequency, clinical experiences and recommendations for standardized reporting.

Diagnostic leukapheresis (DLA) is based on continuous centrifugation that collects mononuclear cells from peripheral blood with a density of 1.055-1.08 g/ml. As epithelial cells have a similar density, DLA cocollects circulating tumor cell (CTCs) along with the targeted mononuclear cells. Here, we report on our single center experience applying DLA in 40 nonmetastatic and metastatic breast cancer patients and its impact on CTC detection. We found that the use of just 5% of the DLA product (corresponding to a median peripheral blood volume of around 60 ml) in the CellSearch® assay already leads to a significant increase in CTC detection frequency and yield. The implementation of the method was unproblematic, and we did not observe any adverse events in our patient cohort. Extrapolating the CTC counts in the DLA samples to the whole DLA product indicated that enormous CTC numbers could be harvested by this approach (around 205x more CTCs than in the 7.5 ml blood sample in M1 patients). In conclusion, DLA is a clinically safe method to collect CTCs from liters of blood enabling a real liquid biopsy. Yet, further technical developments are required to process whole DLA products and exploit the full potential of this approach. As it is foreseeable that DLA will be used by several groups, and hopefully ultimately brought to the patients in a routine setting, we discuss recommendations on the minimum of required information for reporting on DLAs to allow comparison across different approaches. © 2018 International Society for Advancement of Cytometry.

Navigation through inter- and intratumoral heterogeneity of endocrine resistance mechanisms in breast cancer: A potential role for Liquid Biopsies?

The majority of breast cancers are hormone receptor positive due to the expression of the estrogen and/or progesterone receptors. Endocrine therapy is a major treatment option for all disease stages of hormone receptor-positive breast cancer and improves overall survival. However, endocrine therapy is limited by de novo and acquired resistance. Several factors have been proposed for endocrine therapy failures, which include molecular alterations in the estrogen receptor pathway, altered expression of cell-cycle regulators, autophagy, and epithelial-to-mesenchymal transition as a consequence of tumor progression and selection pressure. It is essential to reveal and monitor intra- and intertumoral alterations in breast cancer to allow optimal therapy outcome. Endocrine therapy navigation by molecular profiling of tissue biopsies is the current gold standard but limited in many reasons. "Liquid biopsies" such as circulating-tumor cells and circulating-tumor DNA offer hope to fill that gap in allowing non-invasive serial assessment of biomarkers predicting success of endocrine therapy regimen. In this context, this review will provide an overview on inter- and intratumoral heterogeneity of endocrine resistance mechanisms and discuss the potential role of "liquid biopsies" as navigators to personalize treatment methods and prevent endocrine treatment resistance in breast cancer.

A Novel Workflow to Enrich and Isolate Patient-Matched EpCAMhigh and EpCAMlow/negative CTCs Enables the Comparative Characterization of the PIK3CA Status in Metastatic Breast Cancer.

Circulating tumor cells (CTCs), potential precursors of most epithelial solid tumors, are mainly enriched by epithelial cell adhesion molecule (EpCAM)-dependent technologies. Hence, these approaches may overlook mesenchymal CTCs, considered highly malignant. Our aim was to establish a workflow to enrich and isolate patient-matched EpCAMhigh and EpCAMlow/negative CTCs within the same blood samples, and to investigate the phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) mutational status within single CTCs. We sequentially processed metastatic breast cancer (MBC) blood samples via CellSearch® (EpCAM-based) and via Parsortix™ (size-based) systems. After enrichment, cells captured in Parsortix™ cassettes were stained in situ for nuclei, cytokeratins, EpCAM and CD45. Afterwards, sorted cells were isolated via CellCelector™ micromanipulator and their genomes were amplified. Lastly, PIK3CA mutational status was analyzed by combining an amplicon-based approach with Sanger sequencing. In 54% of patients' blood samples both EpCAMhigh and EpCAMlow/negative cells were identified and successfully isolated. High genomic integrity was observed in 8% of amplified genomes of EpCAMlow/negative cells vs. 28% of EpCAMhigh cells suggesting an increased apoptosis in the first CTC-subpopulation. Furthermore, PIK3CA hotspot mutations were detected in both EpCAMhigh and EpCAMlow/negative CTCs. Our workflow is suitable for single CTC analysis, permitting-for the first time-assessment of the heterogeneity of PIK3CA mutational status within patient-matched EpCAMhigh and EpCAMlow/negative CTCs.

DanioCTC: Analysis of Circulating Tumor Cells from Metastatic Breast Cancer Patients in Zebrafish Xenografts.

Circulating tumor cells (CTCs) serve as crucial metastatic precursor cells, but their study in animal models has been hindered by their low numbers. To address this challenge, we present DanioCTC, an innovative xenograft workflow that overcomes the scarcity of patient-derived CTCs in animal models. By combining diagnostic leukapheresis (DLA), the Parsortix microfluidic system, flow cytometry, and the CellCelector setup, DanioCTC effectively enriches and isolates CTCs from metastatic breast cancer (MBC) patients for injection into zebrafish embryos. Validation experiments confirmed that MDA-MB-231 cells, transplanted following the standard protocol, localized frequently in the head and blood-forming regions of the zebrafish host. Notably, when MDA-MB-231 cells spiked (i.e., supplemented) into DLA aliquots were processed using DanioCTC, the cell dissemination patterns remained consistent. Successful xenografting of CTCs from a MBC patient revealed their primary localization in the head and trunk regions of zebrafish embryos. DanioCTC represents a major step forward in the endeavors to study the dissemination of individual and rare patient-derived CTCs, thereby enhancing our understanding of metastatic breast cancer biology and facilitating the development of targeted interventions in MBC. Summary statement: DanioCTC is a novel workflow to inject patient-derived CTCs into zebrafish, enabling studies of the capacity of these rare tumor cells to induce metastases.

Clinical application of circulating tumor cells.

This narrative review aims to provide a comprehensive overview of the current state of circulating tumor cell (CTC) analysis and its clinical significance in patients with epithelial cancers. The review explores the advancements in CTC detection methods, their clinical applications, and the challenges that lie ahead. By examining the important research findings in this field, this review offers the reader a solid foundation to understand the evolving landscape of CTC analysis and its potential implications for clinical practice. The comprehensive analysis of CTCs provides valuable insights into tumor biology, treatment response, minimal residual disease detection, and prognostic evaluation. Furthermore, the review highlights the potential of CTCs as a non-invasive biomarker for personalized medicine and the monitoring of treatment efficacy. Despite the progress made in CTC research, several challenges such as standardization, validation, and integration into routine clinical practice remain. The review concludes by discussing future directions and the potential impact of CTC analysis on improving patient outcomes and guiding therapeutic decision-making in epithelial cancers.

Selective Adhesion and Switchable Release of Breast Cancer Cells via Hyaluronic Acid Functionalized Dual Stimuli-Responsive Microgel Films.

The detection of tumor cells from liquid biopsy samples is of critical importance for early cancer diagnosis, malignancy assessment, and treatment. In this work, coatings of hyaluronic acid (HA)-functionalized dual-stimuli responsive poly(N-isopropylacrylamide) (PNIPAM) microgels are used to study the specificity of breast cancer cell binding and to assess cell friendly release mechanisms for further diagnostic procedures. The microgels are established by straightforward precipitation polymerization with amine bearing comonomers and postfunctionalization with a UV-labile linker that covalently binds HA to the microgel network. Well-defined microgel coatings for cell binding are established via simple physisorption and annealing. The HA-presenting PNIPAM microgel films are shown to specifically adhere CD44 expressing breast cancer cell lines (MDA-MB-231 and MCF-7), where an increase in adhesion correlates with higher CD44 expression and HA functionalization. Upon cooling below the lower critical solution temperature of PNIPAM microgels, the cells could be released; however, 10-30% of the cells still remained on the surface even after prolonged cooling and mild mechanical agitation. A complete cell release is achieved after applying the light stimulus by short UV treatment cleaving HA units from the microgels. Owing to the comparatively straightforward preparation procedures, such dual-responsive microgel films could be considered for the effective capture, release, and diagnostics of tumor cells.

Murine leukemia virus resists producer cell APOBEC3A by its Glycosylated Gag but not target cell APOBEC3A.

The human APOBEC3A (A3A) polynucleotide cytidine deaminase has been shown to have antiviral activity against HTLV-1 but not HIV-1, when expressed in the virus producer cell. In viral target cells, high levels of endogenous A3A activity have been associated with the restriction of HIV-1 during infection. Here we demonstrate that A3A derived from both target cells and producer cells can block the infection of Moloney-MLV (MLV) and related AKV-derived strains of MLV in a deaminase-dependent mode. Furthermore, glycosylated Gag (glycoGag) of MLV inhibits the encapsidation of human A3A, but target cell A3A was not affected by glycoGag and exerted deamination of viral DNA. Importantly, our results clearly indicate that poor glycoGag expression in MLV gag-pol packaging constructs as compared to abundant levels in full-length amphotropic MLV makes these viral vectors sensitive to A3A-mediated restriction. This raises the possibility of acquiring A3A-induced mutations in retroviral gene therapy applications.

PGRMC1 Promotes Progestin-Dependent Proliferation of Breast Cancer Cells by Binding Prohibitins Resulting in Activation of ERα Signaling.

In previous studies, we reported that progesterone receptor membrane component 1 (PGRMC1) is implicated in progestin signaling and possibly associated with increased breast cancer risk upon combined hormone replacement therapy. To gain mechanistic insight, we searched for potential PGRMC1 interaction partners upon progestin treatment by co-immunoprecipitation and mass spectrometry. The interactions with the identified partners were further characterized with respect to PGRMC1 phosphorylation status and with emphasis on the crosstalk between PGRMC1 and estrogen receptor α (ERα). We report that PGRMC1 overexpression resulted in increased proliferation of hormone receptor positive breast cancer cell lines upon treatment with a subgroup of progestins including norethisterone and dydrogesterone that promote PGRMC1-phosphorylation on S181. The ERα modulators prohibitin-1 (PHB1) and prohibitin-2 (PHB2) interact with PGRMC1 in dependency on S181-phosphorylation upon treatment with the same progestins. Moreover, increased interaction between PGRMC1 and PHBs correlated with decreased binding of PHBs to ERα and subsequent ERα activation. Inhibition of either PGRMC1 or ERα abolished this effect. In summary, we provide strong evidence that activated PGRMC1 associates with PHBs, competitively removing them from ERα, which then can develop its transcriptional activities on target genes. This study emphasizes the role of PGRMC1 in a key breast cancer signaling pathway which may provide a new avenue to target hormone-dependent breast cancer.

Multiparametric Circulating Tumor Cell Analysis to Select Targeted Therapies for Breast Cancer Patients.

BACKGROUND: The analysis of liquid biopsies, e.g., circulating tumor cells (CTCs) is an appealing diagnostic concept for targeted therapy selection. In this proof-of-concept study, we aimed to perform multiparametric analyses of CTCs to select targeted therapies for metastatic breast cancer patients. METHODS: First, CTCs of five metastatic breast cancer patients were analyzed by whole exome sequencing (WES). Based on the results, one patient was selected and monitored by longitudinal and multiparametric liquid biopsy analyses over more than three years, including WES, RNA profiling, and in vitro drug testing of CTCs. RESULTS: Mutations addressable by targeted therapies were detected in all patients, including mutations that were not detected in biopsies of the primary tumor. For the index patient, the clonal evolution of the tumor cells was retraced and resistance mechanisms were identified. The AKT1 E17K mutation was uncovered as the driver of the metastatic process. Drug testing on the patient's CTCs confirmed the efficacy of drugs targeting the AKT1 pathway. During a targeted therapy chosen based on the CTC characterization and including the mTOR inhibitor everolimus, CTC numbers dropped by 97.3% and the disease remained stable as determined by computer tomography/magnetic resonance imaging. CONCLUSION: These results illustrate the strength of a multiparametric CTC analysis to choose and validate targeted therapies to optimize cancer treatment in the future. Furthermore, from a scientific point of view, such studies promote the understanding of the biology of CTCs during different treatment regimens.

GLS-driven glutamine catabolism contributes to prostate cancer radiosensitivity by regulating the redox state, stemness and ATG5-mediated autophagy.

Radiotherapy is one of the curative treatment options for localized prostate cancer (PCa). The curative potential of radiotherapy is mediated by irradiation-induced oxidative stress and DNA damage in tumor cells. However, PCa radiocurability can be impeded by tumor resistance mechanisms and normal tissue toxicity. Metabolic reprogramming is one of the major hallmarks of tumor progression and therapy resistance. Specific metabolic features of PCa might serve as therapeutic targets for tumor radiosensitization and as biomarkers for identifying the patients most likely to respond to radiotherapy. The study aimed to characterize a potential role of glutaminase (GLS)-driven glutamine catabolism as a prognostic biomarker and a therapeutic target for PCa radiosensitization. Methods: We analyzed primary cell cultures and radioresistant (RR) derivatives of the conventional PCa cell lines by gene expression and metabolic assays to identify the molecular traits associated with radiation resistance. Relative radiosensitivity of the cell lines and primary cell cultures were analyzed by 2-D and 3-D clonogenic analyses. Targeting of glutamine (Gln) metabolism was achieved by Gln starvation, gene knockdown, and chemical inhibition. Activation of the DNA damage response (DDR) and autophagy was assessed by gene expression, western blotting, and fluorescence microscopy. Reactive oxygen species (ROS) and the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) were analyzed by fluorescence and luminescence probes, respectively. Cancer stem cell (CSC) properties were investigated by sphere-forming assay, CSC marker analysis, and in vivo limiting dilution assays. Single circulating tumor cells (CTCs) isolated from the blood of PCa patients were analyzed by array comparative genome hybridization. Expression levels of the GLS1 and MYC gene in tumor tissues and amino acid concentrations in blood plasma were correlated to a progression-free survival in PCa patients. Results: Here, we found that radioresistant PCa cells and prostate CSCs have a high glutamine demand. GLS-driven catabolism of glutamine serves not only for energy production but also for the maintenance of the redox state. Consequently, glutamine depletion or inhibition of critical regulators of glutamine utilization, such as GLS and the transcription factor MYC results in PCa radiosensitization. On the contrary, we found that a combination of glutamine metabolism inhibitors with irradiation does not cause toxic effects on nonmalignant prostate cells. Glutamine catabolism contributes to the maintenance of CSCs through regulation of the alpha-ketoglutarate (α-KG)-dependent chromatin-modifying dioxygenase. The lack of glutamine results in the inhibition of CSCs with a high aldehyde dehydrogenase (ALDH) activity, decreases the frequency of the CSC populations in vivo and reduces tumor formation in xenograft mouse models. Moreover, this study shows that activation of the ATG5-mediated autophagy in response to a lack of glutamine is a tumor survival strategy to withstand radiation-mediated cell damage. In combination with autophagy inhibition, the blockade of glutamine metabolism might be a promising strategy for PCa radiosensitization. High blood levels of glutamine in PCa patients significantly correlate with a shorter prostate-specific antigen (PSA) doubling time. Furthermore, high expression of critical regulators of glutamine metabolism, GLS1 and MYC, is significantly associated with a decreased progression-free survival in PCa patients treated with radiotherapy. Conclusions: Our findings demonstrate that GLS-driven glutaminolysis is a prognostic biomarker and therapeutic target for PCa radiosensitization.

Bioinformatic Identification of a Breast-Specific Transcript Profile.

PURPOSE: To identify a breast-specific transcript profile for the first time, and present an updated bioinformatics strategy for searching tissue-specific transcripts and predicting their significance in cancer. EXPERIMENTAL DESIGN: The RNA-seq data of 49 311 transcripts in 88 human tissues from the GTEx, the Illumina Body Map, and the RIKEN FANTOM5 project are integrated to screen breast-specific transcripts. Gene Expression Profiling Interactive Analysis, TGCA, and Kaplan-Meier Plotter are used to examine their expression in cancer tissues and values for prognosis prediction. RESULTS: Only 96 transcripts in human genome are breast-specific for women. Among them, ankyrin repeat domain 30A (ANKRD30A) and long intergenic non-protein coding RNA 993 (LINC00993) are further analyzed. The two transcripts are also breast-specific in 33 types of common female cancer and are often dysregulated in breast cancer tissues. Their expression is higher in the luminal breast cancer while significantly downregulated in triple-negative breast cancer. Moreover, the high expression levels of ANKRD30A and LINC0993 in breast cancer tissues indicate a better prognosis of patients with breast cancer. CONCLUSIONS AND CLINICAL RELEVANCE: Breast-specific transcripts in human genome are rare and poorly understood currently. The data indicate that these breast-specific biomarkers are promising candidates for screening early cancer, assessing treatment response, monitoring recurrence, identifying metastatic tumor origin, and serving as potential targets for immunotherapy.