Biology subjects

Kuhlers, P.

Publications and source records attributed to Kuhlers, P..

3 recordsLinked to original sources

bioRxiv2025

Recapitulating Patient-to-Patient Colorectal Cancer Tumor Heterogeneity Using Patient-Derived Xenograft Cells in an Engineered Tissue Model

Establishing in vitro cancer models that more closely recapitulate patient tumor microenvironmental heterogeneity, including variations in stromal cells and mechanical properties that influence colorectal cancer (CRC) progression, is crucial for advancing CRC research. This study evaluated the ability of 3D engineered CRC-PDX (3D-eCRC-PDX) tissues to recapitulate the heterogeneity found between patient-derived xenograft (PDX) tumors from three CRC patients (stage II, III-B, and IV). To form the 3D-eCRC-PDX tissues, CRC-PDX tumor cells were encapsulated in PEG-fibrinogen hydrogels and maintained for 29 days in vitro. 3D-eCRC-PDX tissues recapitulated key patient-specific tumor characteristics. During long-term culture, 3D-eCRC-PDX tissues mimicked the patient-specific growth rates of the originating CRC-PDX tumors. Importantly, tumor cellular subpopulations, including the ratio of human cancer cells to mouse stromal cells and the ratios of proliferative human cancer cells and CK20+ cells were maintained in 3D-eCRC-PDX tissues, unlike in 2D cell culture. Differences in mechanical stiffness between the originating CRC-PDX tumors were also recapitulated by the 3D-eCRC-PDX tissues. Principal component analysis of transcriptomic data clustered 3D-eCRC-PDX tissues and CRC-PDX tumors together by patient, indicating similar gene expression profiles. These findings highlight the potential of 3D-eCRC-PDX tissues as a promising tool for CRC research, capable of maintaining patient-specific tumor microenvironment heterogeneity. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/693817v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@16c19baorg.highwire.dtl.DTLVardef@d3a75org.highwire.dtl.DTLVardef@79026forg.highwire.dtl.DTLVardef@8ede45_HPS_FORMAT_FIGEXP M_FIG C_FIG Statement of SignificanceThis study establishes engineered colorectal cancer tissues formed using PEG-fibrinogen and patient-derived xenograft (PDX) tumor cells for modeling inter-patient tumor heterogeneity. In vitro models that preserve patient tumors biological and structural heterogeneity are central to the development of more predictive and reproducible preclinical cancer models. Here engineered tissues replicated patient-specific tumor growth dynamics, sustained key cancer and stromal cell subpopulations, recapitulated originating PDX tumor stiffness, and sustained patient-specific patterns of gene expression. This work demonstrates the long-term culture of cells from patient xenografts in engineered colorectal cancer tissues, enabling sustained preservation of critical patient tumor-specific microenvironmental characteristics in vitro.

bioengineering↗

bioRxiv2025

BAP1 deletion in hepatocytes primes an inflammatory transcriptional response

BackgroundBRCA1-associated protein 1 (BAP1) is a deubiquitinase, frequently altered in cancers including hepatocellular carcinoma and cholangiocarcinoma. While Bap1 has been shown to play key roles in metabolism, maintenance of tissue homeostasis, and immune cell development, little is known about its normal functions in the liver in vivo. This study aims to identify Bap1 specific effects on the livers immune microenvironment and biological functions. MethodsUsing AAV8-mediated CRISPR/CAS9 genome editing we generated a mouse hepatocyte-specific model of Bap1 knockout to define the changes that occur in liver biology in an in vivo system and characterize how loss of Bap1 alters the livers response to injury. Single-cell resolution spatial transcriptomics were performed in conjunction with immunohistochemistry to analyze cell-type composition and immune cell recruitment changes. Bulk RNA-sequencing was performed for further assessment of the impact of Bap1 loss on transcription. ResultsHepatocyte-specific depletion of Bap1 induced transcriptional changes shared with acute injury. We observed a strong dysregulation of inflammatory pathways associated with BAP1 loss. Moreover, the transcriptional response of Bap1 depletion in hepatocytes to damage was markedly different than in control liver, with Bap1-deleted livers showing a decreased hepatocyte identity based on gene expression. Spatial transcriptomics and quantitative texture analysis of immunohistochemistry revealed an altered immune environment prior to damage and an impaired recruitment of immune cells in Bap1 depleted livers after damage. ConclusionsUsing a hepatocyte-specific Bap1 deletion we identified Bap1 as a critical modulator in the livers immune cell response. We show that Bap1 loss leads to an inflammatory environment prior to damage and disrupts the recruitment immune cells. Our quantitative spatial analysis highlights the power of such approaches to characterize the spatial distribution of different cell types in a tissue.

genetics↗

bioRxiv2025

Menin-MLL1 complex cooperates with NF-Y to promote HCC survival

Identification of new therapeutic targets in hepatocellular carcinoma (HCC) remains critical. Chromatin regulating complexes are frequently mutated or aberrantly expressed in HCC, suggesting dysregulation of chromatin environments is a key feature driving liver cancer. To investigate whether the altered chromatin state in HCC cells could be targeted, we designed and utilized an epigenome-focused CRISPR library that targets genes involved in chromatin regulation. This focused approach allowed us to test multiple HCC cell lines in both 2D and 3D growth conditions, which revealed striking differences in the essentiality of genes involved in ubiquitination and multiple chromatin regulators vital for HCC cell survival in 2D but whose loss promoted growth in 3D. We found the core subunits of the menin-MLL1 complex among the strongest essential genes for HCC survival in all screens and thoroughly characterized the mechanism through which the menin-MLL1 complex promotes HCC cell growth. Inhibition of the menin-MLL1 interaction led to global changes in occupancy of the complex with concomitant decreases in H3K4me3 and expression of genes involved in PI3K/AKT/mTOR signaling pathway. Menin inhibition affected chromatin accessibility in HCC cells, revealing that increased chromatin accessibility at sites not bound by menin-MLL1 was associated with the recruitment of the pioneer transcription factor complex NF-Y. A CRISPR/Cas9 screen of chromatin regulators in the presence of menin inhibitor SNDX-5613 revealed a significantly increased cell death when combined with NFYB knockout. Together these data show that menin-MLL1 is necessary for HCC cell survival and cooperates with NF-Y to regulate oncogenic gene transcription.

cancer biology↗