Journal: Biomaterials

Article Title: Integrated genome and tissue engineering enables screening of cancer vulnerabilities in physiologically relevant perfusable ex vivo cultures

doi: 10.1016/j.biomaterials.2021.121276

Figure Lengend Snippet: Kinome-wide CRISPRko screens in MDA-MB-231 cells cultured in in vitro 2D conditions, in the ex vivo perfused 3D model, and in in vivo orthotopic locations in mice. (A) Schematic representation of the kinome-wide CRISPR knockout screen. MDA-MB-231 cells are lentivirally transduced with a 3152-element CRISPR knockout library. Cells are collected and distributed across three conditions: 2D-cell culture, perfused printed constructs, and tumors in mice injected orthotopically in the mammary fat pad. Conditions are sustained over 4 weeks, after which gDNA is extracted, amplified, sequenced, and processed. (B) Results of hierarchical clustering performed for top hit genes of all individual replicates for each treatment condition. (C) Venn diagram comparing top depletion and enrichment hits for cell culture, mammary tumor, and perfused print growth conditions, as determined by the MAGeCK algorithm. Criteria consisted of p-value < 0.0027, as well as a LFC value reflecting status as an enriched or depleted hit.

Article Snippet: Kinome-wide CRISPR knockout screen The Brunello Human Kinome CRISPR Knockout Library [ 86 ] (Addgene 75314) was transformed into Stbl4 chemically competent Escherichia coli (Invitrogen), which were subsequently incubated overnight at 37 °C in 100 mL of carbenicillin (50 mg/mL).

Techniques: Cell Culture, In Vitro, Ex Vivo, In Vivo, CRISPR, Knock-Out, Transduction, Construct, Injection, Amplification

Journal: Biomaterials

Article Title: Integrated genome and tissue engineering enables screening of cancer vulnerabilities in physiologically relevant perfusable ex vivo cultures

doi: 10.1016/j.biomaterials.2021.121276

Figure Lengend Snippet: Targeted CRISPRko screen in PDX medulloblastoma cells cultured in the ex vivo perfused 3D model, and in in vivo orthotopic locations in mice. (A) Overview of CRISPR knockout library design. Target genes of single-guide RNAs were assembled from a list of genes with FDA-approved drugs targeting their products. (B) Schematic representation of the targeted CRISPR knockout screen. PDX-medulloblastoma cells are isolated from tumors, and lentivirally transduced with a 74-element CRISPR knockout library. Cells are collected and distributed in perfused printed constructs and tumors in mice injected orthotopically in the cerebellum. Conditions are sustained over 6 weeks, after which gDNA is extracted, amplified, sequenced, and processed. (C) Results of a principal component analysis performed across all genes of all individual replicates for each treatment condition. (D) Comparison of effect sizes (Z-Score) for all 74 sgRNA targets in perfused prints and orthotopic tumors. AAVS1 and NTC respectively represent sgRNAs targeting the adeno-associated virus integration site 1 and non-targeting controls, both serving as controls. (E) Metabolic activity measurements of PDX medulloblastoma cells grown in perfused prints when treated with various drugs inhibiting top depletion targets of the CRISPRko screen (n = 3 with P values *P < 0.05 and **P < 0.01). (F) Timeline for a potential point-of-care functional oncology setting outlining the use of focused CRISPR screens in a perfused printed model for patient-specific cancer vulnerability screening.

Article Snippet: Kinome-wide CRISPR knockout screen The Brunello Human Kinome CRISPR Knockout Library [ 86 ] (Addgene 75314) was transformed into Stbl4 chemically competent Escherichia coli (Invitrogen), which were subsequently incubated overnight at 37 °C in 100 mL of carbenicillin (50 mg/mL).

Techniques: Cell Culture, Ex Vivo, In Vivo, CRISPR, Knock-Out, Isolation, Transduction, Construct, Injection, Amplification, Comparison, Virus, Activity Assay, Functional Assay