Biology subjects

Niederacher, D.

Publications and source records attributed to Niederacher, D..

4 recordsLinked to original sources

bioRxiv2024

Development of a Bipyrimidineamide based α-Helix Mimetic Lead Compound for efficient Targeting of MDM2 in Triple-Negative Breast Cancer

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=155 SRC="FIGDIR/small/582899v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@89ffd0org.highwire.dtl.DTLVardef@1f9cba2org.highwire.dtl.DTLVardef@a821b3org.highwire.dtl.DTLVardef@3c7b6a_HPS_FORMAT_FIGEXP M_FIG C_FIG Triple-negative breast cancer (TNBC) represents the most aggressive form among breast carcinoma subtypes. Due to limited therapy options, identification of novel active pharmacological compounds is an urgent medical need. A promising approach in cancer treatment is the pharmacological inhibition of murine double minutes 2 (MDM2)-p53/p73 interactions inducing apoptosis in tumors. We here describe a novel bipyrimidineamide based -helix mimetic 9 (VWK603) which was designed as a lead candidate to target MDM2. 9 (VWK603) potently induced cell death in the TNBC cell lines MDA-MB-231, MDA-MB-436 and MDA-MB-468 with IC50 values ranging between 3.7 {micro}M and 6.6 {micro}M. The anti-tumor activity was about four more potent higher than determined for the MDM2-specific inhibitor Nutlin-3a. Mechanistic analysis revealed induction of cellular apoptosis as the underlying mode of action of 9 (VWK603) anti-tumor activity. Since toxicity was observed to be reduced in non-cancerous breast cells, these studies make 9 (VWK603) a promising candidate for further preclinical MDM2 inhibitor development.

cancer biology↗

bioRxiv2023

Lysosomal acid lipase-activity as a novel target to efficiently address triple-negative breast cancer high malignancy

Increased metabolism of neutral lipids, e.g. triglycerides and cholesterol esters, is a hallmark of malignant cancers such as triple-negative breast cancer (TNBC). Predominantly, cancer cells with a high epigenetic stem cell-associated signature increasingly utilize neutral lipids to maintain their high degree of tumor stemness, linking metabolic aberrations to epigenetically dysregulated differentiation processes. Lysosomal acid lipase (LIPA) is a central enzyme in the cellular utilization of exogenous and endogenous neutral lipids; however, the role of LIPA-activity in TNBC remains unexplored. We here show for the first time that pharmacological inhibition of LIPA, highly expressed in TNBC, reduces the expression markers of breast cancer stemness in cell culture models of TNBC. A role of LIPA in maintaining TNBC high cellular stemness was stressed by specific siRNA knock-down. Furthermore, inhibition of LIPA sensitized TNBC cells to therapy with Paclitaxel and Doxorubicin, two important chemotherapeutics in current TNBC treatment. When LIPA-activity was inhibited in a three-dinensional (3D) patient derived organoid model, we observed a significant reduction in TNBC cellular viability. Importantly, LIPA inhibition prevented tumor metastasis in a TNBC-zebrafish xenograft model in vivo. These findings introduce LIPA-activity as a novel pharmacological target in TNBC therapy to specifically address its high cancer malignancy with a potential for implementation of LIPA inhibitors into personalized treatment in the future.

cancer biology↗

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↗