Journal: bioRxiv

Article Title: Cell-of-Origin, not Oncogenic Effect, Determines Desmoplastic Immune Exclusion in KRAS-Driven Liver Cancer

doi: 10.64898/2026.03.24.711280

Figure Lengend Snippet: Cholangiocyte-derived organoids harboring Trp53 deletion and Kras G12D mutation give rise to iCCA in a syngeneic orthotopic model. (A) Illustration of the syngeneic orthotopic CCA tumor model. Isolated wild-type chol-orgs were genetically engineered to harbor Trp53 deletion and Kras G12D mutation by CRISPR/Cas9 and implanted intrahepatically. (B) Bright-field microscopic images of wildtype (WT) chol-orgs and with Trp53 deletion (P) and Kras G12D mutation (PK). The CRISPR/Cas9-induced genetic modifications in Trp53 and Kras genes are indicated below. (C) Representative stains of WT and PK chol-orgs indicating positive biliary lineage marker expression (CK19) and upregulation of CD44 in chol-PK orgs. (D) Representative histopathology images of liver tumors obtained upon orthotopic implantation of chol-PK resembling CCA. Stains as indicated. Red dotted line demarcates the boundary between non-tumor liver (N) and tumor (T). All scale bars in (B) indicate 500 μm, and in (C) and (D) 100 μm.

Article Snippet: [ ] The pBabe-KrasG12D plasmid was reconstructed from pBabe-Kras G12D -puro (Addgene #58902) by removing the puromycin resistance gene through digestion with HindIII-HF and BspDI, blunt-ended by Klenow and re-ligation to restore plasmid circularity.

Techniques: Derivative Assay, Mutagenesis, Isolation, CRISPR, Marker, Expressing, Histopathology

Journal: bioRxiv

Article Title: Cell-of-Origin, not Oncogenic Effect, Determines Desmoplastic Immune Exclusion in KRAS-Driven Liver Cancer

doi: 10.64898/2026.03.24.711280

Figure Lengend Snippet: Hepatocyte-derived organoids harboring Trp53 deletion and Kras G12D overexpression give rise to HCC in a syngeneic orthotopic model. (A) Illustration of the syngeneic orthotopic HCC tumor model. Isolated wild-type hep-orgs were genetically engineered to harbor Trp53 deletion by CRISPR/Cas9 and Kras G12D via lentiviral transduction and implanted intrahepatically. (B) Bright-field microscopic images of wildtype (WT) hep-orgs and with Trp53 deletion (P) and Kras G12D overexpression (PK). The CRISPR/Cas9-induced genetic modifications in the Trp53 gene and the sequence of the overexpressing Kras G12D transgene are indicated below. (C) Representative stains of WT and PK hep-orgs indicating positive hepatocyte marker expression (HNF4α) and upregulation of CD44 in hep-PK orgs. (D) Bar plot of tumor penetrance of chol-PK and hep-PK organoids upon orthotopic tumor implantation. (E) Representative histopathology images of liver tumors obtained upon orthotopic implantation of hep-PK organoids resembling HCC. Stains as indicated. Red dotted line demarcates the boundary between non-tumor liver (N) and tumor (T). All scale bars in (B) indicate 500 μm, and in (C) and (E) 100 μm.

Article Snippet: [ ] The pBabe-KrasG12D plasmid was reconstructed from pBabe-Kras G12D -puro (Addgene #58902) by removing the puromycin resistance gene through digestion with HindIII-HF and BspDI, blunt-ended by Klenow and re-ligation to restore plasmid circularity.

Techniques: Derivative Assay, Over Expression, Isolation, CRISPR, Transduction, Sequencing, Marker, Expressing, Tumor Implantation, Histopathology

Journal: bioRxiv

Article Title: Cell-of-Origin, not Oncogenic Effect, Determines Desmoplastic Immune Exclusion in KRAS-Driven Liver Cancer

doi: 10.64898/2026.03.24.711280

Figure Lengend Snippet: Cell-of-origin is the dominant determinant of transcriptional identity in Trp53-deleted , Kras G12D -mutant liver cancer organoids. (A) Messenger RNA expression of fibroblast activation markers in mHSCs treated for 24 hours with CCM from chol-or hep-derived organoids (WT or PK). (B) Schematic of multi-factorial transcriptome analysis in chol and hep organoids. Gene expression was modelled with lineage (chol vs hep), oncogenic effect (PK vs WT), and their interaction to identify lineage-specific PK effects. (C) Principal component analysis (PCA) plot of transcriptomes from indicated lines. Shown are PC1 (79.2% variance) versus PC3 (3.9%), which separate samples primarily by cell lineage and, to a lesser extent, oncogenic activation. (D) Distribution of variance in normalized expression of DEGs explained by lineage and oncogenic effect shown as violin plot with integrated box plot. For each gene, we fit a linear model on normalized counts with lineage, oncogenic effect, their interaction (lineage × oncogenic effect), and mouse strain as predictors. Residuals capture remaining variation. DEGs are the union across the four contrasts: chol-PK vs chol-WT, chol-PK vs hep-PK, chol-WT vs hep-WT, and hep-PK vs hep-WT. (E) Scatter plot showing upregulated DEGs in chol-PK organoids from multi-factorial analysis. Dot color indicates the significance of the interaction effect (adjusted p-value), and dot size reflects the significance of PK versus WT comparison in chol-orgs (adjusted p-value). (F) Gene Set Enrichment Analysis (GSEA) of Hallmark pathways specifically enriched in the lineage-dependent PK effect. The pathways selected for cancer-related cell cycle and oncogenic signaling, tumor-stroma crosstalk, and tumor-immune and inflammation with positive enrichment (i.e. PK effects stronger in chol-orgs) are shown.

Article Snippet: [ ] The pBabe-KrasG12D plasmid was reconstructed from pBabe-Kras G12D -puro (Addgene #58902) by removing the puromycin resistance gene through digestion with HindIII-HF and BspDI, blunt-ended by Klenow and re-ligation to restore plasmid circularity.

Techniques: Mutagenesis, RNA Expression, Activation Assay, Derivative Assay, Gene Expression, Expressing, Comparison

Journal: bioRxiv

Article Title: Cell-of-Origin, not Oncogenic Effect, Determines Desmoplastic Immune Exclusion in KRAS-Driven Liver Cancer

doi: 10.64898/2026.03.24.711280

Figure Lengend Snippet: Cholangiocyte-derived organoids harboring Trp53 deletion and Kras G12D mutation give rise to iCCA in a syngeneic orthotopic model. (A) Illustration of the syngeneic orthotopic CCA tumor model. Isolated wild-type chol-orgs were genetically engineered to harbor Trp53 deletion and Kras G12D mutation by CRISPR/Cas9 and implanted intrahepatically. (B) Bright-field microscopic images of wildtype (WT) chol-orgs and with Trp53 deletion (P) and Kras G12D mutation (PK). The CRISPR/Cas9-induced genetic modifications in Trp53 and Kras genes are indicated below. (C) Representative stains of WT and PK chol-orgs indicating positive biliary lineage marker expression (CK19) and upregulation of CD44 in chol-PK orgs. (D) Representative histopathology images of liver tumors obtained upon orthotopic implantation of chol-PK resembling CCA. Stains as indicated. Red dotted line demarcates the boundary between non-tumor liver (N) and tumor (T). All scale bars in (B) indicate 500 μm, and in (C) and (D) 100 μm.

Article Snippet: The Lenti-CRISPR-sgRNA( Trp53 ), pBabe- Kras G12D , or Lenti-luciferase-P2A-Neo plasmid (Addgene #105621) was co-transfected with packaging plasmid psPAX2 and pMD2.G (both from PlasmidFactory GmbH & Co. KG, Bielefeld, Germany) into HEK293T cells by TransIT LT1 transfection reagent according to the manufacturer’s instructions (Mirus Bio, Madison, WI, USA).

Techniques: Derivative Assay, Mutagenesis, Isolation, CRISPR, Marker, Expressing, Histopathology

Journal: bioRxiv

Article Title: Cell-of-Origin, not Oncogenic Effect, Determines Desmoplastic Immune Exclusion in KRAS-Driven Liver Cancer

doi: 10.64898/2026.03.24.711280

Figure Lengend Snippet: Hepatocyte-derived organoids harboring Trp53 deletion and Kras G12D overexpression give rise to HCC in a syngeneic orthotopic model. (A) Illustration of the syngeneic orthotopic HCC tumor model. Isolated wild-type hep-orgs were genetically engineered to harbor Trp53 deletion by CRISPR/Cas9 and Kras G12D via lentiviral transduction and implanted intrahepatically. (B) Bright-field microscopic images of wildtype (WT) hep-orgs and with Trp53 deletion (P) and Kras G12D overexpression (PK). The CRISPR/Cas9-induced genetic modifications in the Trp53 gene and the sequence of the overexpressing Kras G12D transgene are indicated below. (C) Representative stains of WT and PK hep-orgs indicating positive hepatocyte marker expression (HNF4α) and upregulation of CD44 in hep-PK orgs. (D) Bar plot of tumor penetrance of chol-PK and hep-PK organoids upon orthotopic tumor implantation. (E) Representative histopathology images of liver tumors obtained upon orthotopic implantation of hep-PK organoids resembling HCC. Stains as indicated. Red dotted line demarcates the boundary between non-tumor liver (N) and tumor (T). All scale bars in (B) indicate 500 μm, and in (C) and (E) 100 μm.

Article Snippet: The Lenti-CRISPR-sgRNA( Trp53 ), pBabe- Kras G12D , or Lenti-luciferase-P2A-Neo plasmid (Addgene #105621) was co-transfected with packaging plasmid psPAX2 and pMD2.G (both from PlasmidFactory GmbH & Co. KG, Bielefeld, Germany) into HEK293T cells by TransIT LT1 transfection reagent according to the manufacturer’s instructions (Mirus Bio, Madison, WI, USA).

Techniques: Derivative Assay, Over Expression, Isolation, CRISPR, Transduction, Sequencing, Marker, Expressing, Tumor Implantation, Histopathology

Journal: bioRxiv

Article Title: Cell-of-Origin, not Oncogenic Effect, Determines Desmoplastic Immune Exclusion in KRAS-Driven Liver Cancer

doi: 10.64898/2026.03.24.711280

Figure Lengend Snippet: Cell-of-origin is the dominant determinant of transcriptional identity in Trp53-deleted , Kras G12D -mutant liver cancer organoids. (A) Messenger RNA expression of fibroblast activation markers in mHSCs treated for 24 hours with CCM from chol-or hep-derived organoids (WT or PK). (B) Schematic of multi-factorial transcriptome analysis in chol and hep organoids. Gene expression was modelled with lineage (chol vs hep), oncogenic effect (PK vs WT), and their interaction to identify lineage-specific PK effects. (C) Principal component analysis (PCA) plot of transcriptomes from indicated lines. Shown are PC1 (79.2% variance) versus PC3 (3.9%), which separate samples primarily by cell lineage and, to a lesser extent, oncogenic activation. (D) Distribution of variance in normalized expression of DEGs explained by lineage and oncogenic effect shown as violin plot with integrated box plot. For each gene, we fit a linear model on normalized counts with lineage, oncogenic effect, their interaction (lineage × oncogenic effect), and mouse strain as predictors. Residuals capture remaining variation. DEGs are the union across the four contrasts: chol-PK vs chol-WT, chol-PK vs hep-PK, chol-WT vs hep-WT, and hep-PK vs hep-WT. (E) Scatter plot showing upregulated DEGs in chol-PK organoids from multi-factorial analysis. Dot color indicates the significance of the interaction effect (adjusted p-value), and dot size reflects the significance of PK versus WT comparison in chol-orgs (adjusted p-value). (F) Gene Set Enrichment Analysis (GSEA) of Hallmark pathways specifically enriched in the lineage-dependent PK effect. The pathways selected for cancer-related cell cycle and oncogenic signaling, tumor-stroma crosstalk, and tumor-immune and inflammation with positive enrichment (i.e. PK effects stronger in chol-orgs) are shown.

Article Snippet: The Lenti-CRISPR-sgRNA( Trp53 ), pBabe- Kras G12D , or Lenti-luciferase-P2A-Neo plasmid (Addgene #105621) was co-transfected with packaging plasmid psPAX2 and pMD2.G (both from PlasmidFactory GmbH & Co. KG, Bielefeld, Germany) into HEK293T cells by TransIT LT1 transfection reagent according to the manufacturer’s instructions (Mirus Bio, Madison, WI, USA).

Techniques: Mutagenesis, RNA Expression, Activation Assay, Derivative Assay, Gene Expression, Expressing, Comparison