Journal: bioRxiv

Article Title: Integration of Patient-Derived Organoids and Organ-on-Chip Systems: Investigating Colorectal Cancer Invasion within the Mechanical and GABAergic Tumor Microenvironment

doi: 10.1101/2023.09.14.557797

Figure Lengend Snippet: (A.) Schematic representing the initial events of the metastatic cascade that can be measured using the CRC-OOC. Tumor cells in the top channel (1) can be visualized and analyzed separately from tumor cells that have invaded and adhered in the endothelial compartment (2). Additionally, tumor cells that are found in the endothelial effluent (3; circulating tumor cells (“CTC-like” cells)) can also be collected and analyzed. (B.) Circulating tumor cells were collected from the endothelial effluent of stretched and not stretched HCT116 CRC-Chips and RNAseq was performed. GO Pathway analysis was performed on a subset of genes with either a 2-fold difference between stretched and not stretched CTCs or an FDR-adjusted p-value <0.1. N=2 biological replicates with 3 pooled chips in each replicate. (C.) Gene expression of neurotransmitter-related genes were measured by a neurotransmitter-specific PCR array. HCT116 tumor cells of stretched and not stretched chips were harvested on day 6 from the top epithelial channel and isolated via FACs. Data is displayed as the gene expression fold change of stretch versus not stretch conditions Expression was normalized to the average of 5 housekeeping genes. Genes that had a 1.5-fold increase or decrease in the stretched condition are displayed. N=3 biological replicates with 3 chips pooled per biological replicate. (D.) Schematic of the production of GABA from glutamate by the enzyme GAD1. In this figure, experiments related to GABA are indicated in purple and experiments related to GAD1 are indicated in light blue. (E.) Representative confocal immunofluorescent images of the epithelial (top; 1) or endothelial (bottom; 2) channel of the CRC-Chips stained for GABA (purple) on day 6. Invaded HCT116 H2B-GFP stain positive for GABA, while HCT116 H2B-GFP tumor cells that are in the top channel stain weakly for GABA. DAPI stains the nuclei of Caco2 C2BBe1 cells in the top channel and endothelial cells in the bottom channel. Scale bars represent 200 μm in the top channel image and 100 μm in the bottom channel images. Top channel images are maximum projections that span a 35 μm Z-height with a 5 μm step size. Bottom channel images are maximum projections that span a 10 μm Z-height with a 5 μm step size. (F.) RNAseq analysis was performed on CRC organoids and normalized GAD1 expression is shown. N=5 independent donors with 2-3 replicates each. Individual data points are shown and mean ± SEM is displayed. Analysis between US and UP data was performed using an unpaired t-test; ***p<0.001 ( G.) CRC organoids were isolated from stretched chips and qPCR analysis of GAD1 gene expression was performed. N=5 independent doners with 3 replicates each. Individual data points are shown and mean ± SEM is displayed. Analysis between US and UP data was performed using an unpaired t-test p<0.05. ( H.) GAD1 mRNA expression from TCGA in KRAS, NRAS, or BRAF mutant primary colon cancer tumors. N=196 patients with KRAS, NRAS, or BRAF mutant tumors; N=201 patients with KRAS, NRAS, or BRAF wildtype tumors. Individual data points are shown and median with interquartile range is represented. Data was analyzed with an unpaired t-test; ****p<0.0001. ( I.) Kaplan-Meier curve with univariate analysis of the survival of patients with KRAS, NRAS, or BRAF mutated CRC tumors based on high versus low expression of GAD1 (defined as above or below the median GAD1 mRNA expression z-score of 0.3). Data was extracted from the TCGA. N=254 patients. Data was analyzed using a log-rank (Mantel-Cox test). ( J.) Effluent from the epithelial channel of the patient-derived organoids was collected on day 0 (D0) and day 6 (D6). GABA intensity was analyzed from extracted metabolites N=6 chips per timepoint per patient; n=4 on D0 and 2 on D6 for UK. Data was analyzed using a two-way ANOVA; ***p<0.001; ****p<0.0001. ( K .) US-H2B-GFP (top) and UP-H2B-GFP (bottom) stretched tumor-chips were stained for GABA (purple). Scale bars represent 200 μm. L. Representative 10x immunofluorescence images of the 5 tumors stained for EpCAM (green), CK20 (red), and GABA (purple). Scale bars represent 500 μm and 200 μm for UK. All schematics were made in or are from BioRender.

Article Snippet: ABAT human shRNA lentiviral particles (Santa Cruz Biotechnology, Inc., #sc-93288-V) and control shRNA lentiviral particles (Santa Cruz Biotechnology, Inc., #sc-108080) were used to produce HCT116 H2B-GFP ABAT knock down cells at a multiplicity of infection (MOI) of 5 and 2 mg/mL of puromycin (Santa Cruz Biotechnology, #sc-108071) was used to select for knocked down cells.

Techniques: Gene Expression, Isolation, Expressing, Staining, Mutagenesis, Derivative Assay, Immunofluorescence

Journal: bioRxiv

Article Title: Integration of Patient-Derived Organoids and Organ-on-Chip Systems: Investigating Colorectal Cancer Invasion within the Mechanical and GABAergic Tumor Microenvironment

doi: 10.1101/2023.09.14.557797

Figure Lengend Snippet: (A.) Invasion of HCT116 tumor-chips in the presence or absence of exogenous GABA (flowed through the epithelial channel) was measured on day 6 (D6) of the experiment and normalized to day 0 (D0) invasion. N=6 chips. Individual data are shown, with mean ± SEM represented and analyzed using a one-way ANOVA; ****p<0.0001. (B.) Intracellular [ 13 C 4 ]GABA or unlabeled GABA was measured via mass spectrometry-based metabolomics in the HCT116 tumor-chips after the addition of exogenous GABA for six days. N=3 chips. (C.) Schematic of GABA catabolism by ABAT, subsequent entry into the TCA cycle, and inhibition of ABAT activity by vigabatrin. In this figure, experiments related to GABA are indicated as purple, and experiments related to ABAT are indicated as teal. (D.) Western blot analysis of ABAT in shRNA control or ABAT shRNA HCT116 tumor cells. Cropped western blot (left) and quantification (right) confirm knockdown of ABAT. (E.) Growth rate of ABAT-knockdown or control HCT116 tumor cells when grown in traditional cell culture methods. N=3. Individual data are shown and mean ± SEM are represented. Data was analyzed using a t-test; **p<0.01. (F.) Numbers of ABAT-knockdown or control HCT116 tumor cells in the top channel on-chip as measured via fluorescence microscopy and quantified on day 0 (D0) and day 6 (D6). N=5-6 chips. Individual data are shown and mean ± SEM are represented. Data was analyzed using a two-way ANOVA; *p<0.05;***p<0.001. (G.) Invasion of ABAT knockdown (KD) or control shRNA HCT116 tumor-chips in the presence or absence of stretching was measured on day 6 (D6) of the experiment and normalized to day 0 (D0) invasion. N=5-6 chips. Individual data points are shown and mean ± SEM are represented. Data was analyzed using a one-way ANOVA; *p<0.05. (H.) Numbers of HCT116 tumor cells in the top channel on-chip in the presence or absence of stretching, with or without vigabatrin was measured via fluorescence microscopy and quantified on day 0 (D0) and day 6 (Day 6). Individual data are shown and mean ± SEM are represented. N=4 chips. Data was analyzed using a two-way ANOVA; **p<0.01. (I.) Invasion of HCT116 tumor-chips in the presence or absence of stretching, with or without vigabatrin was measured on day 6 (D6) of the experiment and normalized to day 0 (D0) invasion. N=4 chips. Individual data points are shown and mean ± SEM are represented. Data was analyzed using a one-way ANOVA; ***p<0.001. (J.) Numbers of US-H2B-GFP (red) or UP-H2B-GFP (blue) tumor cells in the top channel on-chip in the presence or absence of stretching, with or without vigabatrin was measured via fluorescence microscopy and quantified on day 0 (D0) and day 6 (Day 6). N=4-5 chips. Individual data are shown and mean ± SEM are represented. Data was analyzed using a two-way ANOVA; ns=p>0.05. (K.) Invasion of US-H2B-GFP or UP-H2B-GFP organoid-tumor chips in the presence or absence of stretching, with or without vigabatrin was measured on day 6 (D6) of the experiment and normalized to day 0 (D0) invasion. N=4-5 chips. Individual data are shown and mean ± SEM are represented. Data was analyzed using a one-way ANOVA; **p<0.01. All schematics were made in or are from BioRender.

Article Snippet: ABAT human shRNA lentiviral particles (Santa Cruz Biotechnology, Inc., #sc-93288-V) and control shRNA lentiviral particles (Santa Cruz Biotechnology, Inc., #sc-108080) were used to produce HCT116 H2B-GFP ABAT knock down cells at a multiplicity of infection (MOI) of 5 and 2 mg/mL of puromycin (Santa Cruz Biotechnology, #sc-108071) was used to select for knocked down cells.

Techniques: Mass Spectrometry, Inhibition, Activity Assay, Western Blot, shRNA, Control, Knockdown, Cell Culture, Fluorescence, Microscopy