Journal: Cancers

Article Title: Potent Antitumor Activity of Liposomal Irinotecan in an Organoid- and CRISPR-Cas9-Based Murine Model of Gallbladder Cancer

doi: 10.3390/cancers11121904

Figure Lengend Snippet: Gallbladder organoids express a biliary marker profile. ( A ) Technical outline: organoids were isolated from the gallbladders of adult mice, expanded in Matrigel, and genetically modified using CRISPR/Cas9 or by retroviral introduction of cDNAs. Genetically altered organoids were transplanted into recipient mice, either s.c. or orthotopically into the gallbladder. ( B ) Immunohistochemistry (IHC) confirms EpCAM expression within the epithelial layer of adult murine gallbladders. ( C ) Brightfield image of gallbladder organoids. ( D ) Flow cytometry analysis for EpCAM on single cell suspensions from adult mouse liver (left column), adult mouse gallbladder (middle column) and gallbladder organoids (right column). ( E ) Immunofluorescence on gallbladder organoids confirms expression of β-catenin (left), CK19 (middle), and SOX9 (right).

Article Snippet: For immunofluorescence, Santa Cruz Biotechnology: ß-Catenin (sc-7963), CK19 (sc-33111), and Sox9 (sc-20095); Cell Signaling Technology (Danvers, MA, USA): ERBB2, (2165s); Novus Biological (Centennial, CO, USA): p-ERBB2 (NB100-81960) were all diluted 1:50.

Techniques: Marker, Isolation, Genetically Modified, CRISPR, Retroviral, Immunohistochemistry, Expressing, Flow Cytometry, Immunofluorescence

Journal: Cancers

Article Title: Potent Antitumor Activity of Liposomal Irinotecan in an Organoid- and CRISPR-Cas9-Based Murine Model of Gallbladder Cancer

doi: 10.3390/cancers11121904

Figure Lengend Snippet: Genetically modified gallbladder organoids can give rise to gallbladder cancer (GBC) that resembles the human disease. ( A ) Schematic of plasmids used to transfect gallbladder organoids. Plasmids contain Cre recombinase, Cas9, and the respective sgRNA(s). ( B ) T7 endonuclease assay confirming cleavage after transfection and selection with blasticidin, first column: KCR8 organoids, second column: KP organoids, and third column: KPP organoids; arrows indicate cleaved bands. ( C ) Tumor volume 32 days after organoid implantation. No tumor development occurred in mice transplanted with KCR8 organoids during the four-month observation period. ( D ) Kaplan-Meyer curves of mice transplanted with KCR8, KP, and KPP organoids. Transplantation with KP and KPP organoids led to rapid tumor development (median survival: 69 days and 46 days for the KP and KPP cohorts, respectively). ( E ) Histological characteristics of GBC tumors derived from KP and KPP organoids. H&E staining of both genotypes shows GBCs classified as adenocarcinomas. IHC for CK19 confirms ductal differentiation and PTEN IHC detects loss of PTEN expression in sgPten-bearing epithelial tumor cells, but not in the surrounding stroma cells. IHC for αSMA confirms the presence of cancer-associated fibroblasts. Normal gallbladder tissue (H&E) for comparison. ( F ) Loss of p53 and PTEN confirmed on tumor-derived cell lines of the respective genotypes by immunoblotting. KCR8 organoids served as positive control. ( G ) Frequency of indels in the respective loci in preinjection organoids and in tumor derived cell lines shows enrichment of p53- and PTEN alterations during tumor development. ( H ) The relative stromal content of KP and KPP derived tumors (considering CK19 negative area as a surrogate for the relative stromal content) did not differ significantly (57.44% and 56.97%, respectively; p > 0.8669).

Article Snippet: For immunofluorescence, Santa Cruz Biotechnology: ß-Catenin (sc-7963), CK19 (sc-33111), and Sox9 (sc-20095); Cell Signaling Technology (Danvers, MA, USA): ERBB2, (2165s); Novus Biological (Centennial, CO, USA): p-ERBB2 (NB100-81960) were all diluted 1:50.

Techniques: Genetically Modified, Transfection, Selection, Transplantation Assay, Derivative Assay, Staining, Expressing, Comparison, Western Blot, Positive Control

Journal: Cancers

Article Title: Potent Antitumor Activity of Liposomal Irinotecan in an Organoid- and CRISPR-Cas9-Based Murine Model of Gallbladder Cancer

doi: 10.3390/cancers11121904

Figure Lengend Snippet: Genetically altered gallbladder organoids lead to metastasis upon orthotopic implantation. ( A ) Both s.c. and orthotopically implanted KPP organoids lead to GBCs classified as adenocarcinomas. ( B ) Orthotopic GBCs presented with a larger stromal compartment as assessed by quantification of the CK19 negative area (54.15% and 62.69%, respectively (n = 9 and n = 7, respectively, p = 0.0421)). ( C ) Lung metastases were present in 5/10 mice after orthotopic transplantation and in 0/10 mice after s.c. transplantation with KPP organoids. ( D ) H&E and CK19 staining of lung metastasis. ( E ) Compared to the parental orthotopic tumors, CK19 negative area as a surrogate for relative stromal content is significantly reduced in lung metastases (62.69% and 38.48% respectively, n = 7 and n = 3, respectively, p = 0.0026).

Article Snippet: For immunofluorescence, Santa Cruz Biotechnology: ß-Catenin (sc-7963), CK19 (sc-33111), and Sox9 (sc-20095); Cell Signaling Technology (Danvers, MA, USA): ERBB2, (2165s); Novus Biological (Centennial, CO, USA): p-ERBB2 (NB100-81960) were all diluted 1:50.

Techniques: Transplantation Assay, Staining

Journal: Cancers

Article Title: Potent Antitumor Activity of Liposomal Irinotecan in an Organoid- and CRISPR-Cas9-Based Murine Model of Gallbladder Cancer

doi: 10.3390/cancers11121904

Figure Lengend Snippet: Mutant ERBB2 cooperates with loss of p53 and leads to papillary GBC in recipient mice. ( A ) Top: Schematic of human ERBB2, indicating the location of two point mutants (S310F and V777L). Bottom: retroviral vector used to transduce organoids, that had been treated with an sgp53-containing plasmid (px459) to induce loss of p53. ( B ) Immunofluorescence for ERBB2 (top) and phospho-ERBB2 (bottom) on organoids harboring the indicated genetic alterations. ( C ) Tumor volumes 36 days after s.c. implantation of the respective organoids into recipient mice. All mice transplanted with sgp53;ERBB2 S310F - and sgp53;ERBB2 V777L organoids exhibited tumor development, whereas sgp53;empty vector- and sgp53;ERBB2 wildtype organoids did not give rise to tumors over a four-month observation period. There was no significant difference in the tumor burden of mice transplanted with sgp53;ERBB2 S310F - and sgp53;ERBB2 V777L organoids ( p = 0.999). ( D ) Mice transplanted with sgp53;ERBB2 S310F - and sgp53;ERBB2 V777L organoids reached endpoint criteria with a median survival of 79.5 days and 58.5 days, respectively. ( E ) H&E and IHC for CK19 and EGFP on tumors generated with sgp53;ERBB2 S310 - and sgp53;ERBB2 V777L organoids.

Article Snippet: For immunofluorescence, Santa Cruz Biotechnology: ß-Catenin (sc-7963), CK19 (sc-33111), and Sox9 (sc-20095); Cell Signaling Technology (Danvers, MA, USA): ERBB2, (2165s); Novus Biological (Centennial, CO, USA): p-ERBB2 (NB100-81960) were all diluted 1:50.

Techniques: Mutagenesis, Retroviral, Plasmid Preparation, Transduction, Immunofluorescence, Generated