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: The primary antibodies used for the HCT116 H2B-GFP ABAT knock down validation were mouse anti-ABAT (1:500; Santa Cruz Technology, Inc., #sc-393769) and mouse anti-Actin (1:10000; Millipore Sigma, #A5441).

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: The primary antibodies used for the HCT116 H2B-GFP ABAT knock down validation were mouse anti-ABAT (1:500; Santa Cruz Technology, Inc., #sc-393769) and mouse anti-Actin (1:10000; Millipore Sigma, #A5441).

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

Journal: Nature Immunology

Article Title: Enhancing human NK cell antitumor function by knocking out SMAD4 to counteract TGFβ and activin A suppression

doi: 10.1038/s41590-025-02103-z

Figure Lengend Snippet: a , Mean ± s.e.m. percentage of activated caspase-3-positive HCT116 cells after coculture with control NK and SMAD4 KO NK cells treated with IL-2 ± TGFβ1 at the indicated effector:target (E:T) ratios analyzed by flow cytometry. Spontaneous active caspase-3 levels were subtracted. Data are from four independent experiments. b , c , Amount of CCL5 ( b ) and IFNγ ( c ) in cell-free culture supernatants of control NK and SMAD4 KO NK cells after coculture with the HCT116 cell line by ELISA. Each dot shows data from an independent experiment ( n = 4). d – f , HCT116-GFP + -Luc + spheroids were cocultured with PKH26-labeled control NK or SMAD4 KO NK cells previously exposed or not to TGFβ1. Images were taken at 6 h and 24 h. d , Representative image of one spheroid in each coculture at 24 h. e , Mean ± s.e.m. GFP intensity along time in each coculture. Data are from two independent experiments including five technical replicates each. Only the significance by two-way ANOVA followed by Tukey’s multiple-comparisons test between TGFβ1-treated control and SMAD4 KO NK cells at 30 h is indicated. f , Mean ± s.e.m. luciferase activity of remaining HCT116 spheroids after 24 h of coculture with NK cells. Each dot represents the average cytotoxic activity of NK cells in four independent experiments. g , h , Tumor growth kinetics of HCC1954 xenografts in NSG mice treated with: (i) trastuzumab (Tz)/pertuzumab (Pt) ( n = 4); (ii) control NK cells (2 × 10 5 , n = 5); (iii) SMAD4 KO NK cells (2 × 10 5 , n = 5); (iv) control NK cells (1 × 10 5 ) and trastuzumab/pertuzumab ( n = 5); or (v) SMAD4 KO NK cells (1 × 10 5 ) and trastuzumab/pertuzumab ( n = 5). g , Treatment schedule. h , Tumor volume fold change in each treatment group. Only differences between control and SMAD4 KO NK cells at last measurement are indicated. i , Tumor growth kinetics of HCT116 xenografts in NSG mice treated with either control or SMAD4 KO NK cells (2 × 10 5 ). Tumor volume fold change in each treatment group ( n = 5 in NT group, n = 6 in control and SMAD4 KO NK cell groups). Statistical significance by two-way ANOVA followed by Tukey’s multiple-comparisons test at last measurement for a , e , h and i ; and one-way ANOVA followed by Tukey’s multiple-comparisons test for b , c , f and g . RLUs, relative light units.

Article Snippet: Production of CCL5, TNF and IFNγ was measured after 2 h coculture with HCT116 cells and analyzed by ELISA using DY278, DY210 (R&D Systems) and 88-7316-88 (Invitrogen), following the manufacturer’s instructions.

Techniques: Control, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Labeling, Luciferase, Activity Assay

Journal: Nature Immunology

Article Title: Enhancing human NK cell antitumor function by knocking out SMAD4 to counteract TGFβ and activin A suppression

doi: 10.1038/s41590-025-02103-z

Figure Lengend Snippet: a ) Mean ± SEM TNF in culture supernatants of control- and SMAD4 KO NK cells after 2 h coculture with HCT116 cells as analysed by ELISA. Statistical significance calculated by one-way ANOVA followed by Fisher’s test. b ) HCT116-Luc + -GFP+ spheroids were cocultured with control- or SMAD4 KO NK cells previously treated with IL-2 ± TGF-β. Mean ± SEM Luciferase activity of remaining HCT116 cells after 24 h coculture. Data from 3 independent experiments using NK cells from different donors. Each dot represents data from the 5 technical replicates included in each condition. Statistical significance calculated by one-way ANOVA followed by Turkey’s multiple comparisons test. c ) HCC1954 xenografts in NSG mice were treated with either: i) trastuzumab (Tz)/pertuzumab (Pt)(n = 4); ii) control NK cells (2 × 10 5 ,n = 5); iii) SMAD4 KO NK cells (2 × 10 5 ,n = 5); iv) control NK cells (1 × 10 5 ) and trastuzumab/pertuzumab (n = 5) or v) SMAD4 KO NK cells (1 × 10 5 ) and trastuzumab/pertuzumab (n = 5). Tumor volume in each treatment group. Statistical significance between control and SMAD4 KO NK cells treatments at the last measurment by two-way ANOVA followed by Turkey’s multiple comparisons test are indicated. d ) HCT116 xenografts in NSG mice were treated with either control or SMAD4 KO NK cells (2 × 10 5 ). Tumor volume in each treatment group (n = 5 in NT and n = 6 in NK cell treated groups). e ) HCT116-GFP + -Luc+ and HCC1954 GFP + -Luc+ spheroids were cultured with rIFNɣ, rIFN-β or rTNF. Mean ± SEM Luciferase activity of HCT116 nd HCC1954 spheroids after 24 h coculture. Statistical significance by one-way ANOVA followed by Turkey’s multiple comparisons test.

Article Snippet: Production of CCL5, TNF and IFNγ was measured after 2 h coculture with HCT116 cells and analyzed by ELISA using DY278, DY210 (R&D Systems) and 88-7316-88 (Invitrogen), following the manufacturer’s instructions.

Techniques: Control, Enzyme-linked Immunosorbent Assay, Luciferase, Activity Assay, Cell Culture

Journal: Nature Immunology

Article Title: Enhancing human NK cell antitumor function by knocking out SMAD4 to counteract TGFβ and activin A suppression

doi: 10.1038/s41590-025-02103-z

Figure Lengend Snippet: a , Mean transcript levels of adhesion molecules by RNA-seq analysis. b , Mean ± s.e.m. percentage or intensity of surface expression levels of CD11a ( n = 4), CD18 ( n = 3), CD49d ( n = 5), CD103 ( n = 6) and CD29 ( n = 5) by flow cytometry in control and SMAD4 KO NK cells treated or not with TGFβ1. Each dot indicates the results from an independent experiment with NK cells from different individuals. c – e , HCT116 spheroids were cocultured with control NK or SMAD4 KO NK cells previously exposed or not to TGFβ1. After 1 h coculture, spheroids and attached NK cells were fixed and processed for light-sheet imaging. HCT116 cells were labeled with an anti-EpCAM-FITC antibody and NK cells with an anti-CD45-Vio R667. c , Image of a representative HCT116 spheroid (green surface) cocultured with SMAD4 KO NK cells (red dots). d , Number of NK cells counted in spheroids in the indicated conditions. e , Quantification of the distance between the spheroid surface and each infiltrating NK cell. Each dot represents the measurement of one infiltrating NK cell in one experiment. f , Mean ± s.e.m. fluorescence intensity of surface CXCR3 ( n = 9), CCR5 ( n = 6) and CXCR4 ( n = 4) in control and SMAD4 KO NK cells treated or not with TGFβ1 by flow cytometry. Each dot shows data from independent experiments. g , Transcript expression levels of chemokine receptors in control NK and SMAD4 KO NK cells exposed or not to TGFβ1, according to RNA-seq data from three independent donors. The dashed lines in a and g separate genes downregulated from those upregulated by TGFβ in control NK cells. h , Mean ± s.e.m. percentage of transmigrating control or SMAD4 KO NK cells treated with TGFβ1 to CCL5, CXCL9, SDF-1/CXCL12 or the indicated chemokine combinations. Data are from four independent experiments with NK cells from different donors. In all assays, statistical significance was calculated by one-way ANOVA followed by Fisher’s test.

Article Snippet: Production of CCL5, TNF and IFNγ was measured after 2 h coculture with HCT116 cells and analyzed by ELISA using DY278, DY210 (R&D Systems) and 88-7316-88 (Invitrogen), following the manufacturer’s instructions.

Techniques: RNA Sequencing, Expressing, Flow Cytometry, Control, Imaging, Labeling, Fluorescence

Journal: Nature Immunology

Article Title: Enhancing human NK cell antitumor function by knocking out SMAD4 to counteract TGFβ and activin A suppression

doi: 10.1038/s41590-025-02103-z

Figure Lengend Snippet: a) Surface expression of LFA-1 (CD11a and CD18), VLA-4 (CD49d and CD29) and CD103 in control and SMAD4 KO NK cells treated with IL-2 ± TGF-β as analysed by flow cytometry. Representative histograms of each marker staining in NK cells from a representative individual. b ) HCT116 spheroids were cocultured with control- or SMAD4 KO NK cells previously treated with IL2 ± TGF-β. After 1 h coculture, spheroids and attached NK cells were fixed and processed for lightsheet imaging. HCT116 cells were labeled with an anti-Epcam-FITC antibody and NK cells with an anti-CD45-VioR667 antibody. Image of a representative HCT116 spheroid for the indicated conditions. Inset numbers correspond to the number of NK cells counted in each spheroid.

Article Snippet: Production of CCL5, TNF and IFNγ was measured after 2 h coculture with HCT116 cells and analyzed by ELISA using DY278, DY210 (R&D Systems) and 88-7316-88 (Invitrogen), following the manufacturer’s instructions.

Techniques: Expressing, Control, Flow Cytometry, Marker, Staining, Imaging, Labeling

Journal: Nature Immunology

Article Title: Enhancing human NK cell antitumor function by knocking out SMAD4 to counteract TGFβ and activin A suppression

doi: 10.1038/s41590-025-02103-z

Figure Lengend Snippet: a – d , SMAD4 KO and control NK cells were expanded in the presence of TGFβ1 for 7 days. Control NK cells were treated with the TGFBR-I inhibitor SB-431542 (SB inhib). a , b , SMAD4, pSMAD2 and β-actin levels in total cell extracts by western blot in SMAD4 KO and control NK cells. a , Representative western blots including NK cells from two different donors. b , Quantification of mean ± s.e.m. levels of SMAD4/β-actin and pSMAD2/β-actin ratios in NK cells from three different donors. Statistical significance was calculated by a two-tailed, unpaired Student’s t -test. c , Representative histograms showing the expression of GzmB, NKG2D and CD103 in the indicated NK cells by flow cytometry. d , e , SMAD4 KO and control NK cells treated with SB-431542 were cocultured with HCT116-GFP + -Luc + spheroids. Luciferase counts were analyzed at 24 h of coculture. d , Mean ± s.e.m. luciferase counts from one representative experiment. Dots show data from five technical replicates. e , Mean ± s.e.m. luciferase counts from three independent experiments using NK cells from different individuals. Each dot indicates the mean of five technical replicates for each condition/experiment. Statistical significance was calculated by one-way ANOVA followed by Tukey’s multiple-comparisons test for d and e .

Article Snippet: Production of CCL5, TNF and IFNγ was measured after 2 h coculture with HCT116 cells and analyzed by ELISA using DY278, DY210 (R&D Systems) and 88-7316-88 (Invitrogen), following the manufacturer’s instructions.

Techniques: Control, Inhibition, Western Blot, Two Tailed Test, Expressing, Flow Cytometry, Luciferase

Journal: Nature Immunology

Article Title: Enhancing human NK cell antitumor function by knocking out SMAD4 to counteract TGFβ and activin A suppression

doi: 10.1038/s41590-025-02103-z

Figure Lengend Snippet: a , Relative transcript expression of activin receptor genes in the indicated NK cells. Average expression in NK cells from three independent individuals as analyzed by RNA-seq. b – d , SMAD4 KO and control NK cells were incubated with TGFβ1 or activin A for 7 days. b , Expression of GzmB at day 7 by flow cytometry. c , Mean ± s.e.m. granzyme B levels in SMAD4 KO and control NK cells. Data are from experiments with NK cells from eight different individuals. d , SMAD4 KO and control NK cells previously incubated with TGFβ1 or activin A (Activ) were cocultured with HCT116 cells for 2 h. Mean ± s.e.m. percentage of active caspase-3 + (aCasp3) HCT116 cells in the indicated conditions. Data are from five independent experiments with NK cells from different individuals. Statistical significance was calculated by one-way ANOVA followed by Fisher’s test.

Article Snippet: Production of CCL5, TNF and IFNγ was measured after 2 h coculture with HCT116 cells and analyzed by ELISA using DY278, DY210 (R&D Systems) and 88-7316-88 (Invitrogen), following the manufacturer’s instructions.

Techniques: Expressing, RNA Sequencing, Control, Incubation, Flow Cytometry

Journal: Nature Immunology

Article Title: Enhancing human NK cell antitumor function by knocking out SMAD4 to counteract TGFβ and activin A suppression

doi: 10.1038/s41590-025-02103-z

Figure Lengend Snippet: a – c , Control and SMAD4 KO NK cells transduced with anti-CD19-CAR lentivirus were cultured with IL-2 ± TGFβ1 for 6 days. a , Mean ± s.e.m. percentage of control or SMAD4 KO NK cells transduced with the anti-CD19-CAR by flow cytometry. Data are from three independent experiments. b , c , CAR19-transuced control or SMAD4 KO NK cells treated with IL-2 ± TGFβ1 were cocultured with Nalm6-GFP + -Luc + for 3 h at a 4:1 E:T ratio. b , Mean ± s.e.m. luciferase counts at the end of the coculture in the indicated conditions. Dots show data from technical replicates ( n = 4) in a representative experiment. c , Mean ± s.e.m. percentage of Nalm6 cell killing in the indicated conditions. Dots show data of independent experiments with CD19-CAR NK cells from different donors ( n = 4). d – f , Control and SMAD4 KO GTA002 cells were cultured in IL-2 ± TGFβ1 for 5 days. Data are from four independent experiments. GTA002 cells from different cord-blood units are presented as independent dots. d , SMAD4 MFI in control and SMAD4 KO GTA002 cells at day 5. e , Mean ± s.e.m. percentage of NKG2D + control or SMAD4 KO GTA002 cells in the indicated conditions. f , Mean luciferase activity remaining after 24 h coculture of HCT116-GFP + -Luc + spheroids with control or SMAD4 KO GTA002 cells in the indicated conditions. Sph, spheroid. g – i , Control or SMAD4 KO ADAPT-NK cells were cultured for 5 days with IL-2 ± TGFβ1. Degranulation and IFNγ production were analyzed by coculturing control or SMAD4 KO ADAPT-NK cells with K562 cells at a 1:1 E:T ratio for 4 h. Dots represent data from independent experiments using ADAPT-NK cells from different donors. g , SMAD4 MFI in control and SMAD4 KO ADAPT-NK cells at day 5. h , i , Mean ± s.e.m. percentage of CD107 + ( h ) and IFNγ + ( i ) control and SMAD4 KO ADAPT-NK cells in the indicated conditions. Basal degranulation and IFNγ production in the absence of target were subtracted. Statistical significance was calculated by a two-tailed, paired Student’s t -test in d and g , and by one-way ANOVA followed by Fisher’s test in b , c , e , f , h and i .

Article Snippet: Production of CCL5, TNF and IFNγ was measured after 2 h coculture with HCT116 cells and analyzed by ELISA using DY278, DY210 (R&D Systems) and 88-7316-88 (Invitrogen), following the manufacturer’s instructions.

Techniques: Control, Transduction, Cell Culture, Flow Cytometry, Luciferase, Activity Assay, Two Tailed Test