Biology subjects

Franken, A.

Publications and source records attributed to Franken, A..

4 recordsLinked to original sources

bioRxiv2023

ZeptoCTC - Sensitive Protein Analysis of True Single Cell Lysates using Reverse Phase Protein Arrays (RPPA)

Circulating Tumor Cells (CTCs) are commonly analyzed through genomic profiling, which does not capture posttranslational and functional alterations of encoded proteins. To address this limitation, we developed ZeptoCTC, a single-cell protein analysis workflow that combines established technologies for single-cell isolation and sensitive Reverse Phase Protein Array (RPPA) analysis to assess multiple protein expression and activation in individual CTCs. The workflow involves single cell labeling, isolation, lysis, and printing of the true single cell lysates onto a ZeptoChip using a modified micromanipulator CellCelectorTM. Subsequently, the printed lysates undergo fluorescence immunoassay RPPA protein detection using a ZeptoReader followed by signal quantification with Image J software. ZeptoCTC was successfully optimized, beginning with the measurement of EpCAM protein expression--a standard marker for CTC detection. As expected, mean fluorescence signals for EpCAM levels were significantly higher in single MCF-7 cells compared to MDA-MB-231 cells. Next, Capivasertib-treated MCF-7 cells exhibited an approximately 2-fold increase in the pAkt/Akt ratio compared to non-treated control cells. This finding was consistent with a co-performed western blot analysis of pooled MCF-7 cells. Application of ZeptoCTC to the analysis of single CTCs derived from a metastasized breast cancer (MBC) patient indicated a significantly higher level of pAkt, accompanied by a corresponding increase in pErk level when compared to patient-matched WBC. Finally, the current workflow successfully indicated the detectable pAkt and Akt signal difference in CTCs from two MBC patients: one with an Akt1 wild-type genotype, and the other harboring approximately 80% Akt1(E17K) mutated CTCs. The mutated CTCs revealed clearly elevated pAkt levels (1.8-fold), along with an even more strongly elevated total Akt (3.4-fold) when compared to the respective signals measured in wild-type CTCs. In conclusion, ZeptoCTC is a highly sensitive method for measuring the expression and phosphorylation of treatment-relevant proteins in key cancer-driving signaling pathways from true single cell samples.

molecular biology↗

bioRxiv2023

DanioCTC: Injection of circulating tumor cells from metastatic breast cancer patients in zebrafish xenografts for analysis of metastasis

Circulating tumor cells (CTCs) are considered as metastatic precursor cells, and zebrafish xenografts provide an in vivo model to study cancer cell spread. Currently, the low number of patient-derived CTCs limits their analysis in animal models. We present DanioCTC, a xenograft workflow for injecting CTCs from metastatic breast cancer (MBC) patients into zebrafish embryos to study cell dissemination in vivo. The study successfully adapts existing workflows and combines diagnostic leukapheresis (DLA), the Parsortix microfluidic system, flow cytometry, and the automated cell micromanipulator CellCelector setup to enrich and isolate MBC-derived CTCs and to finally inject them into Zebrafish embryos, where their dissemination was tracked up to 3 days post-injection. MDA-MB-231 cells were used as a standard xenotransplantation control, and these cells were frequently found in the head and blood-forming regions of the tail. Using DLA aliquots spiked with MBA-MB-231 cells, the newly established DanioCTC workflow confirmed the dissemination of MDA-MB-231 cells into these regions. CTCs from an MBC patient were then enriched by DLA, Parsortix, and flow cytometry, isolated with the CellCelectorTM and xenografted into zebrafish embryos. CTCs were mainly detected in the head and trunk, unlike MDA-MB-231 cells, which were present in the head and tail. DanioCTC presents a significant breakthrough in the use of zebrafish embryos as a model to study CTC dissemination in vivo, which can be used for patient-derived CTCs instead of cell culture-derived cancer cells as a crucial step towards understanding the biology of metastatic breast cancer. Statement of significanceDanioCTC is a novel workflow to inject patient-derived CTCs into zebrafish, enabling studies on CTC dissemination and personalized treatment in vivo, therefore advancing our toolkit to fight metastatic cancer.

cancer biology↗

bioRxiv2021

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. Here, we found that radioresistant PCa cells and prostate cancer stem cells (CSCs) have a high glutamine demand. Glutaminase (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 glutaminase (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 (-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. Our findings demonstrate that GLS-driven glutaminolysis is a prognostic biomarker and therapeutic target for PCa radiosensitization.

cancer biology↗

bioRxiv2020

A Pan-Cancer Blueprint of the Heterogeneous Tumour Microenvironment Revealed by Single-Cell Profiling

AO_SCPLOWBSTRACTC_SCPLOWThe stromal compartment of the tumour microenvironment consists of a heterogeneous set of tissue-resident and tumour-infiltrating cells, which are profoundly moulded by cancer cells. An outstanding question is to what extent this heterogeneity is similar between cancers affecting different organs. Here, we profile 233,591 single cells from patients with lung, colorectal, ovary and breast cancer (n=36) and construct a pan-cancer blueprint of stromal cell heterogeneity using different single-cell RNA and protein-based technologies. We identify 68 stromal cell populations, of which 46 are shared between cancer types and 22 are unique. We also characterise each population phenotypically by highlighting its marker genes, transcription factors, metabolic activities and tissue-specific expression differences. Resident cell types are characterised by substantial tissue specificity, while tumour-infiltrating cell types are largely shared across cancer types. Finally, by applying the blueprint to melanoma tumours treated with checkpoint immunotherapy and identifying a naive CD4+ T-cell phenotype predictive of response to checkpoint immunotherapy, we illustrate how it can serve as a guide to interpret scRNA-seq data. In conclusion, by providing a comprehensive blueprint through an interactive web server, we generate a first panoramic view on the shared complexity of stromal cells in different cancers.

cancer biology↗