Journal: bioRxiv

Article Title: In vivo metabolomics identifies CD38 as an emergent vulnerability in LKB1 -mutant lung cancer

doi: 10.1101/2023.04.18.537350

Figure Lengend Snippet: LKB1 deficient NSCLC show shifts in NAD + homeostasis and upregulation of the CD38 NADase. A , Metabolites that show significant changes in KL mouse lung tumors compared to KP tumors. Values are shown as z score of fold-change. B , C, Flow cytometry analysis showing quantification of CD38 expression in neoplastic (EPCAM+) cells in Kras / Lkb1 (KL) and Kras / Trp53 (KP) tumors. B , (left panel) percent positive tumor cells and (right panel) median fluorescent intensity (MFI). Student’s t test, two-tailed. ** P <0.01, **** P <0.0001. C , Representative FACS blots of CD38 expression. D , CD38 mRNA levels in human KP and KL lung cancer patient specimens, from TCGA dataset. Unpaired t test with Welch’s correction, two-tailed. ** P <0.01. E,F, Lung cancer patient specimens with LKB1 wt and mutant status were stained with anti-CD38 antibodies. E , Representative IHC images. Scale bar, 100 μm. F , Quantification of CD38 IHC score. Student’s unpaired t test, two tailed. *** P <0.001.

Article Snippet: Primary antibodies used are human CD38 (Cell Signaling Technology, Cat #51000S), mouse CD38 (Cell Signaling Technology, Cat #92457), LKB1 (Cell Signaling Technology, Cat #3047), β-actin (Sigma, Cat# A5441).

Techniques: Flow Cytometry, Expressing, Two Tailed Test, Mutagenesis, Staining

Journal: bioRxiv

Article Title: In vivo metabolomics identifies CD38 as an emergent vulnerability in LKB1 -mutant lung cancer

doi: 10.1101/2023.04.18.537350

Figure Lengend Snippet: LKB1 is a negative regulator of CD38 expression in lung cancer cells. A , B , A series of KRAS mutant NSCLC cell lines with or without LKB1 mutation were analyzed for CD38 expression via FACS. A , Representative FACS plots. B , Quantification. n=3 biological replicates for each cell line. C , Western blot analysis of CD38 protein levels in KRAS mutant lung cancer cell lines with or without LKB1 mutation. D , E, LKB1 mutant (Lx337) and LKB1 wildtype (Lx210) lung cancer PDXs were tested for CD38 expression by FACS analysis. D , Representative FACS plots. E , Quantification of percent CD38+ tumor cells in the PDXs. n=4 tumors per group. Student’s t test, two tailed. * P <0.05. F, G, A549 NSCLC cells were transduced with empty vector (pBABE) or ectopically expressing LKB1 wildtype (LKB1) or kinase-dead mutant (LKB1-KD). F , Cells were tested for CD38 expression by FACS analysis. n=3 each group. Each dot represents one independent experiment. One-way ANOVA Dunnett’s multiple comparisons test compared with pBABE group. *** P <0.001. G , Western blot analysis of CD38 protein levels.

Article Snippet: Primary antibodies used are human CD38 (Cell Signaling Technology, Cat #51000S), mouse CD38 (Cell Signaling Technology, Cat #92457), LKB1 (Cell Signaling Technology, Cat #3047), β-actin (Sigma, Cat# A5441).

Techniques: Expressing, Mutagenesis, Western Blot, Two Tailed Test, Transduction, Plasmid Preparation

Journal: bioRxiv

Article Title: In vivo metabolomics identifies CD38 as an emergent vulnerability in LKB1 -mutant lung cancer

doi: 10.1101/2023.04.18.537350

Figure Lengend Snippet: LKB1-SIK signaling suppresses CD38 transcriptionally. A , Relative phosphorylation levels of SIK3 T221 in human lungs cancer specimens with the indicated LKB1 genotypes (from the CPTAC dataset). Student’s unpaired t test, two tails. * P <0.05. B, FACS analysis showing CD38 expression on KP lung tumors with sgRNA mediated targeting of Lkb1 or Sik family members versus control. * P <0.05, *** P <0.001, **** P <0.0001. C , Representative FACS analysis of CD38 levels in KP lung cancer cells shown in ( B ). D , Western blot of CD38 levels in KP lines without Lkb1 or Sik family kinases expression, or KL lines without Lkb1 expression. E , Relative CD38 mRNA levels in the lines shown in panel B . One-way ANOVA, Dunnett’s multiple comparisons test compared with KP Cas9. ** P <0.01, **** P <0.0001. F , Representative MRI scan of lung tumor nodules (circled) with the indicated genotypes. H, heart. G , Quantification of tumor volume changes 6 weeks after tumor implantation. Unpaired student’s t test in comparison with KP sgCtrl group. * P <0.05. H , Metabolite changes with statistical significance comparing mouse KP lung tumor nodules with or without Sik1/2/3 loss. I , CREB reporter assay in the indicated cells. n=3 each cell line. Ordinary one-way ANOVA Dunnett’s multiple comparison test, each group compared with the KP Cas9 group. n=3 each group. * P <0.05, **** P <0.0001. J , EP300 ChIP-seq track of CD38 promoter using the A549 cell line from the ENCODE database.

Article Snippet: Primary antibodies used are human CD38 (Cell Signaling Technology, Cat #51000S), mouse CD38 (Cell Signaling Technology, Cat #92457), LKB1 (Cell Signaling Technology, Cat #3047), β-actin (Sigma, Cat# A5441).

Techniques: Phospho-proteomics, Expressing, Control, Western Blot, Tumor Implantation, Comparison, Reporter Assay, ChIP-sequencing

Journal: bioRxiv

Article Title: In vivo metabolomics identifies CD38 as an emergent vulnerability in LKB1 -mutant lung cancer

doi: 10.1101/2023.04.18.537350

Figure Lengend Snippet: Daratumumab induces killing of CD38 positive lung cancer cells through antibody-dependent cellular cytotoxicity (ADCC). A-B, Cytotoxicity assay in LKB1 mutant A549 ( A ), HCC44 ( B ) or LKB1 wild type H358 ( C ) target cells using calcein-acetoxymethyl (AM) release in the presence of NK cell line NK-92. Lytic activity was measured 4 hours after co-culturing (E:T=100:1) in the presence of Daratumumab (20 μg/ml). Student’s t test, two-tailed analysis was performed to compare with no NK cell control as indicated. D , Indicated cells were cultured with NK-92 cells (E:T=100:1) in the presence of increasing concentrations of daratumumab. The percentage of cell lysis for each cell line was measured 4 hrs after co-culturing. Paired student’s t test for LKB1 mutant cells (A549 or H1944) compared with LKB1 wild type control H358. E-F, Mouse A549 ( E ) or HCC44 ( F ) xenograft tumor growth in response to daratumumab treatment. For each mouse, 3x10 6 A549 cells ( E ) or 5x10 5 HCC44 ( F ) were implanted into nu/nu mice subcutaneously. Daratumumab or hIgG1 (12.5mg/kg, once per week) was dosed when tumor size reached100 mm 3 . Multiple unpaired student’s t test was performed for each of the time points indicated. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.

Article Snippet: Primary antibodies used are human CD38 (Cell Signaling Technology, Cat #51000S), mouse CD38 (Cell Signaling Technology, Cat #92457), LKB1 (Cell Signaling Technology, Cat #3047), β-actin (Sigma, Cat# A5441).

Techniques: Cytotoxicity Assay, Mutagenesis, Activity Assay, Two Tailed Test, Control, Cell Culture, Lysis

Journal: bioRxiv

Article Title: Non-invasive imaging of cell-based therapies using acoustic reporter genes

doi: 10.1101/2024.11.01.621111

Figure Lengend Snippet: a , Schematic of lentiviral vectors designed to report T cell activity state by expressing GVs downstream of the NFAT promoter. T cell activation is chemically induced by incubating cells with PMA and ionomycin, which trigger the NFAT promoter to express rtTA, and in the presence of doxycycline, activate transcription of GV genes. b , Representative BURST images (top) and signal quantification (bottom) of Jurkat cells transduced with the vectors in panel ( a ) with or without chemical activation, normalized to wild-type (WT) cells (not shown). c , Percentage of Jurkat cells in panel ( b ) expressing both fluorescent reporters (GFP and BFP), with and without activation. d , Imaging T cell activation in response to CD19-CD3 BTE-mediated engagement with CD19+ Raji cells, using cells engineered with the vectors shown in ( a ). e , Representative BURST images (top) and signal quantification (bottom) of Jurkat cells engineered to express GVs upon receptor-mediated activation in the presence of Dox. f , Percentage of cells from panel ( e ) expressing both fluorescent reporters (GFP and BFP), with and without receptor engagement. Statistical comparisons were performed using Fisher’s LSD test, with each condition compared to resting state T cells (without PMA/ionomycin or BTE stimulation). P-value for ( e ): 0.0173. All other p-values < 0.0001. Significance levels: *p<0 . 05, **p<0 . 01, ***p<0 . 001, ****p<0 . 0001 . Error bars represent mean ± s.e.m. of N = 3 biological replicates. Each data point represents the arithmetic mean of N = 2 technical replicates. Scale bars for all ultrasound images represent 1 mm.

Article Snippet: To induce receptor-mediated T cell cytotoxicity, CD19-CD3 BTE (1 ng/mL, BPS Bioscience) and IL-2 (100 U/mL) were added.

Techniques: Activity Assay, Expressing, Activation Assay, Transduction, Imaging

Journal: bioRxiv

Article Title: Non-invasive imaging of cell-based therapies using acoustic reporter genes

doi: 10.1101/2024.11.01.621111

Figure Lengend Snippet: a , Workflow for engineering T cells isolated from human PBMCs to express doxycycline-inducible GVs and imaging BTE-mediated homing of cytotoxic T cells into target tumors. Top: T cells are transduced with the 3-vector lentiviral system, and GFP expressing cells are sorted to select for transduction of the assembly factor vectors. The cells are then expanded in vitro for downstream characterization and in vivo injections. Bottom: T cells are systemically administered to immunocompromised mice bearing subcutaneous CD19+ Raji cell tumors, accompanied by intraperitoneal (IP) injections of BTE every 48 hours and IP injections of doxycycline for 72 hours prior to ultrasound imaging. b , BURST SBR (left) and representative images (right) of GV-expressing and WT T cells. Paired one-tailed t test, p = 0.0115. Error bars represent mean ± s.e.m. of N = 5 PBMC donors; each data point is the arithmetic mean of N = 3 technical replicates. c , Percentage of live Raji cells in a cytotoxicity assay, where T cells are co-cultured with CD19+ Raji cells at effector-to-target (E:T) ratios of 1:1 and 4:1. Welch’s two-tailed t-test, N = 6 biological replicates (2 PBMC donors and 3 replicates per donor). d , GFP and BFP fluorescence measurements from ( c ), normalized to the maximum mean fluorescence intensity (MFI) of each reporter. e , BURST images overlaid on anatomical B-mode grayscale images of representative Raji tumors in mice injected with GV-expressing T cells, either Dox-induced or uninduced, and mice injected with WT T cells. Red lines indicate tumor boundary used for signal quantification. f , Quantification of BURST signal inside tumors. Welch’s two-tailed t test, Dox+ vs. Dox-, p = 0.0067; Dox+ vs. WT, p = 0.0063. N = 10 mice for Dox+, N = 6 for Dox-, N = 6 for WT. g , Histology of an example tumor infiltrated by GV-expressing T cells and its corresponding BURST image. White: Raji-Antares cells; Magenta: CD8+ cytotoxic T cells; Green: GFP+ T cells; Blue: BFP+ T cells. h , Schematic of T cell distribution in the Dox-induced tumor from ( g ). i , Correlation of the BURST signal within tumors with the absolute numbers of GFP+ and BFP+ T cells in corresponding histological sections. Pearson correlation: r = 0.89, p = 0.0072, N = 7 mice. One mouse (gray data point) was excluded due to a significant number of T cells located outside the imaging plane whose GV expression was not captured by BURST (see ). All scale bars represent 1 mm.

Article Snippet: To induce receptor-mediated T cell cytotoxicity, CD19-CD3 BTE (1 ng/mL, BPS Bioscience) and IL-2 (100 U/mL) were added.

Techniques: Isolation, Imaging, Transduction, Plasmid Preparation, Expressing, In Vitro, In Vivo, One-tailed Test, Cytotoxicity Assay, Cell Culture, Two Tailed Test, Fluorescence, Injection