Biology subjects

Peeters, S.

Publications and source records attributed to Peeters, S..

5 recordsLinked to original sources

bioRxiv2025

Comparative characterization of OncoPro and Wnt-Based media reveals distinct phenotypic and pharmacologic states in patient-derived tumor organoids

BackgroundPatient-derived tumor organoids (PDTOs) are strongly influenced by culture medium. We compared OncoPro (OP) Tumoroid Culture medium with conventional Wnt/R-spondin/noggin (Wnt) medium. This recently developed OP medium offers a standardized, serum-free alternative to Wnt-based formulations. MethodsWe compared OP and Wnt media across 36 PDTO lines from various malignancies (colorectal, pancreatic, breast, lung, gastric, gastroesophageal junction, biliary head- and neck and uknown primary), assessing establishment success. Selected PDTO models were subjected to downstream characterization, including morphological assessment and bulk transcriptomic profiling with comparison to public single-cell RNA sequencing reference datasets (n=11), whole-exome sequencing (WES) (n=9), and pharmacological response profiling to a 33-drug panel (n=3). ResultsAdaptation from Wnt medium to OP succeeded in 83.3% (15/18), whereas de novo establishment favored Wnt (33.3% vs 11.1%). Key oncogenic driver alterations were retained across matched organoid cultures, supporting preservation of tumor-relevant genomic features. Transcriptomic profiling confirmed preserved tumor-identity across media, while revealing different epithelial state programs: Wnt upregulated proliferation/stemness-associated genes (e.g. LGR5) and OP enriched adhesion-associated genes and inflammatory/TGF-{beta} programs. In scRNA databases OP signatures preferentially mapped to malignant epithelial compartments in pancreatic cancer, whereas Wnt signatures were linked to non-malignant epithelium. Similarly, in colon cancer OP signature mapped predominantly to the malignant epithelial compartments. Drug (n=33) screening in pancreatic- and colorectal cancer PDTOs (n=3) demonstrated consistent medium-dependent shifts: Wnt-grown PDTOs were globally more sensitive in the screened subset, particularly to MAPK-axis inhibitors and apoptosis-sensitizers, while OP-grown PDTOs exhibited relative resistance. ConclusionsCulture medium composition is a key determinant of PDTO phenotype, transcriptome and drug sensitivity. Wnt medium was associated with drug-sensitive states, whereas OP medium was associated with adhesion- and inflammatory-related programs, relative resistance in the screened subset of 33 drugs and closer alignment with malignant epithelial programs in the analyzed pancreatic / colorectal cancer single-cell atlases.

cancer biology↗

bioRxiv2025

Nanobody-mediated modulation of long RSH enzymes Rel and RelA catalysis by restriction of their conformational landscape

Long RSH enzymes, Rel and RelA, are the master regulators of (p)ppGpp alarmone levels in bacteria. Their catalytic activity is governed by transitions between a compact, hydrolysis-competent (HDON) state and an elongated, synthesis-competent (SYNTHON) state. The equilibrium between these states is modulated by factors such as "starved" ribosomes and regulatory proteins DarB, EIIANTR, ACP and YtfK. Here, we identify and characterize camelid nanobodies that act as selective allosteric modulators by trapping Rel/RelA enzymes in distinct conformational states. Nanobodies that lock the TGS domain of RelA and prevent its activation by deacylated tRNA on starved ribosomes, strongly inhibit (p)ppGpp synthesis and suppress the virulence of E. coli in an animal model. Nb898 stabilizes Rel in the open SYNTHON state, enhancing synthesis activity while suppressing hydrolysis. Conversely, Nb585 traps Rel in a HDON conformation, strongly inhibiting alarmone synthesis while promoting (p)ppGpp hydrolysis. Structural and biochemical analyses reveal that nanobodies, like natural allosteric regulators, act by restricting the RSH enzymes conformational landscape. These findings establish nanobodies as powerful tools for dissecting RSH function and provide potential leads for developing protein-based RSH modulators.

microbiology↗

bioRxiv2024

Generation of human appetite-regulating neurons and tanycytes from stem cells

The balance between energy intake and expenditure is controlled by the hypothalamus, a small brain region characterised by high neuronal diversity. Specifically, the arcuate nucleus (ARC) and ventromedial hypothalamus (VMH) are key hypothalamic nuclei controlling appetite through behavioural response to circulating humoral signals. Yet, despite their physiological importance, the cellular and functional characteristics of this highly specialised neural region has been studied mainly in animals due to a lack of human models. Here, we fine-tuned the differentiation of human pluripotent stem cells toward the ARC and VMH hypothalamic nuclei and identified key subtype-specific progenitor markers of these subregions. We demonstrate that the timing for initiation and termination of bone morphogenetic protein (BMP) signalling is essential for controlling subregional specification of tuberal hypothalamic progenitors along the anterior-posterior axis, balancing VMH versus ARC fates. A particular population of SHH-/NKX2.1+/FGF10high/RAXhigh/TBX3high posterior tuberal progenitors was identified as the source for generation of ARC-associated agouti-related peptide (AGRP) neurons and tanycytes whilst anterior tuberal SHH+/NKX2.1+/FGF10low/RAXlow/TBX3low progenitors generated VMH phenotypes including NR5A1 neurons. Upon maturation in vitro and in xenografts, ARC-patterned progenitors gave rise to key appetite-regulating cell types including those producing AGRP, prepronociceptin (PNOC), growth hormone-releasing hormone (GHRH), thyrotropin-releasing hormone (TRH) and pro-opiomelanocortin (POMC), as well as tanycyte glial cells. Differentiated ARC cultures showed high transcriptomic similarity to the human ARC and displayed evidence of functionality by AGRP secretion and responsiveness to leptin and fibroblast growth factor 1 (FGF1). In summary, our work provides insights into the developmental lineages underlying hypothalamic subregional specification and enables access to highly characterised human ARC and VMH cultures, which will provide novel opportunities for investigating the cellular and molecular pathways triggered by obesity-associated genetic variants and weight-regulating stimuli.

neuroscience↗

bioRxiv2024

Fine-tuning of a CRISPRi screen in the seventh pandemic Vibrio cholerae

Vibrio cholerae O1 El Tor, the etiological agent responsible for the last cholera pandemic, has become a well-established model organism for which some genetic tools exist. While CRISPRi has been applied in V. cholerae, improvements were necessary to upscale it and enable pooled screening by high-throughput sequencing in this bacterium. In this study, we introduce a pooled genome wide CRISPRi library construction specifically optimized for this V. cholerae strain, characterized by minimal cytotoxicity and streamlined experimental setup. This library allows the depletion of 3, 674 (98.9%) annotated genes from the V. cholerae genome. To confirm its effectiveness, we screened for essential genes during exponential growth in rich medium and identified 368 genes for which guides were significantly depleted from the library (log2FC < - 2). Remarkably, 82% of these genes had previously been described as hypothetical essential genes in V. cholerae or in a closely related bacterium, V. natriegens. We thus validated the robustness and accuracy of our CRISPRi-based approach for assessing gene fitness in a given condition. Our findings highlight the efficacy of the developed CRISPRi platform as a powerful tool for high-throughput functional genomics studies of V. cholerae. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=72 SRC="FIGDIR/small/601881v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@bab54aorg.highwire.dtl.DTLVardef@1d412c6org.highwire.dtl.DTLVardef@1cba6borg.highwire.dtl.DTLVardef@12d11d_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2023

Uncovering the hidden threat: single-organoid analysis reveals clinically relevant treatment-resistant and invasive subclones in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal diseases, characterized by a treatment-resistant and invasive nature. In-line with these inherent aggressive characteristics, only a subset of patients show a clinical response to the standard of care therapies, thereby highlighting the need for a more personalized treatment approach. In this study, we comprehensively unraveled the intra-patient response heterogeneity and intrinsic aggressive nature of PDAC on bulk and single-organoid resolution. We leveraged a fully characterized PDAC organoid panel (N=8) and matched our artificial intelligence-driven, live-cell organoid image analysis with retrospective clinical patient response. In-line with the clinical outcomes, we identified patient-specific sensitivities to the standard of care therapies (gemcitabine-paclitaxel and FOLFIRINOX) using a growth rate-based and normalized drug response metric. Moreover, the single-organoid analysis was able to detect resistant as well as invasive PDAC organoid clones, which was orchestrates on a patient, therapy, drug, concentration and time-specific level. Furthermore, our in vitro organoid analysis indicated a strong correlation with the matched patient progression-free survival (PFS) compared to the current, conventional drug response readouts. This work not only provides valuable insights on the response complexity in PDAC, but it also highlights the potential applications (extendable to other tumor types) and clinical translatability of our approach in drug discovery and the emerging era of personalized medicine.

cancer biology↗