Journal: Acta biomaterialia

Article Title: Bioconjugated liquid-like solid enhances characterization of solid tumor - chimeric antigen receptor T cell interactions.

doi: 10.1016/j.actbio.2023.09.042

Figure Lengend Snippet: Fig. 2. Expression of CD70 on solid tumors and confirmation of target expression and CAR T cell transduction efficiency. (A) Gene expression of CD70 in various solid tumors compared to normal matched tissues was analyzed using data from The University of Alabama at Birmingham Cancer data analysis (UALCAN) and The Cancer Genome Atlas program (TCGA). B-C) Top: confirmation of CD70 (target) expression on cancer cells post transduction – (B) glioblastoma (GBM) and (C) osteosarcoma (OS) models - and Bottom: transduction of CAR construct in (B) C57BL/6 mice - derived T cells (CAR T Kr158B ) and (C) in Balb/c mice - derived T cells (CAR T K7M2 ) as indicated by GFP reporter. On average, (D) the mean CD70 expression was found to be 73% for GBM and 99% for OS models. (E) The mean transduction efficiency was 66% for CAR T Kr158B and 60% for CAR T K7M2 . The names of the cell lines and tumor models, GBM for Kr158B and OS for K7M2, will be used interchangeably in the study. Number of biological replicates (N) is indicated on the graphs.

Article Snippet: Cell lines 293T cell line (ATCC, CRL-3216) was used for lentiviral plasid production encoding for mouse full length CD70 surface antien (GenBank: U78091.1).

Techniques: Expressing, Transduction, Gene Expression, Construct, Derivative Assay

Journal: Acta biomaterialia

Article Title: Bioconjugated liquid-like solid enhances characterization of solid tumor - chimeric antigen receptor T cell interactions.

doi: 10.1016/j.actbio.2023.09.042

Figure Lengend Snippet: Fig. 3. CAR T cell exhibit directed motion towards the target tumor. A) Representative confocal snapshots showed CD70-specific CAR T cells (green) navigating through supported COL1-LLS RhB microgels (red) and infiltrating the target GBM tumor (white), with blue arrows indicating the paths of CAR T cell migration. (B, D) Mean velocity of CAR T cells co-cultured with CD70-positive tumors and the wild-type (WT) control were quantified for GBM (B) and OS (D) tumor models. The number of tracks (n) and biological replicates (N) were indicated on the plots, and statistical significance was determined using an unpaired two-tailed Student’s t-test with p values indicated on the plots. (C, E) Tumor-infiltrating CAR T cells were quantified as a percentage of total CAR T cells on average from 0 to 72 h for GBM (C) and OS (E) tumors, displayed as box plots showing 25th and 75th percentiles, median, and mean, with whiskers representing the minimum to maximum observations. An unpaired two-tailed Student’s t-test was performed for statistical analysis, and p values were indicated on the plots. (F) Top panel: maximum intensity z projection showed snapshots of CAR T – GBM tumor interaction at 0, 24, and 72 h. Middle panel: the segmentation of CAR T with colors indicating individual CAR T cells at each frame. Bottom panel: maximum intensity projection of segmented CAR T cell velocity tracks over time with color-coded velocity gradient, revealing accumulation of CAR T cells inside the tumor. The segmentation employed a deep learning-based method, as discussed in the methods section, and the cells were tracked using a Linear Assignment Problem (LAP) tracker with maximum frame-to-frame linking and allowable track segment gap closing of 150 μm. (G) Evidence of chemotaxis and upregulation in migratory pathways for CAR T cells co-cultured with their target tumors was demonstrated for GBM (top panel) and OS (bottom panel) tumor models. Notably, evidence of immune-mediated cytotoxic function was shown through IFN γ detection. Error bars represent standard deviation.

Article Snippet: Cell lines 293T cell line (ATCC, CRL-3216) was used for lentiviral plasid production encoding for mouse full length CD70 surface antien (GenBank: U78091.1).

Techniques: Migration, Cell Culture, Control, Two Tailed Test, Chemotaxis Assay, Standard Deviation

Journal: Acta biomaterialia

Article Title: Bioconjugated liquid-like solid enhances characterization of solid tumor - chimeric antigen receptor T cell interactions.

doi: 10.1016/j.actbio.2023.09.042

Figure Lengend Snippet: Fig. 4. CAR T expansion, activation, and killing. (A) Representative confocal time-lapse images of the FITC channel (green) illustrating immune activation, expansion, and killing of the target tumors. CAR T cell clustering and rapid expansion were observed in almost all conditions with efficient anti-tumor activity. White arrows indicate CAR T cell clusters. (B, C) The number of CAR T cell clusters rapidly increased within the first 24 h and remained steady for over 72 h in (B) GBM and (C) OS models. (D, E) Cluster size in all co-cultures with target tumors steadily increased over time, but not in WT controls. (F, G) Expansion of CAR T cell clusters revealed an inverse correlation with tumor size. n = 3 for all CD70 pos samples and n = 2 for all WT samples. (H, I) Endpoint flow cytometry data measuring CAR T cell expansion after 96 h of co-culture with cancer cells in the 2D assay for both tumor models. (J, K) A comparison in IFN γ secretion (pg/mL) between 2D and 3D cultures for (J) GBM and (K) OS. Error bars indicate SD and the number of biological replicates is n = 2. (L) Top row: confocal 3D snapshots of GBM tumors after co-cultured with CAR T cells (E:T = 1:4) for 24 h, showing highly tortuous tumor margins in CD70 pos tumors compared to WT counterparts. Bottom row: cross-section view (at z depth: 70 μm) of the sample on the top row revealing infiltrating CAR T cells within the tumor mass. (M) Measurement of tumor tortuosity factors revealed more than 3-fold change for the CD70 pos tumors within the first 48 h of co-culture. The tumor tortuosity factor was calculated as the ratio between the perimeter of the tumor outline and the perimeter of a circle with the same pixel area. Data was obtained from GBM samples ( n = 3) at an initial E:T ratio of 1:2 and from osteosarcoma (K7M2) samples ( n = 3) at an initial E:T ratio of 1:1. Error bars represent standard deviation and biological samples ( n = 3) for each group were performed unless indicated otherwise.

Article Snippet: Cell lines 293T cell line (ATCC, CRL-3216) was used for lentiviral plasid production encoding for mouse full length CD70 surface antien (GenBank: U78091.1).

Techniques: Activation Assay, Activity Assay, Cytometry, Co-Culture Assay, Two-Dimensional Assay, Comparison, Cell Culture, Standard Deviation

Journal: Acta biomaterialia

Article Title: Bioconjugated liquid-like solid enhances characterization of solid tumor - chimeric antigen receptor T cell interactions.

doi: 10.1016/j.actbio.2023.09.042

Figure Lengend Snippet: Fig. 5. Quantification of CAR T–tumor interactions . Top rows: confocal timelapse images at 0, 24, 48, and 72 h showing dynamic immune-cancer interactions and antitumor activities of CAR T cells in (A) GBM and (B) OS tumor models. The bottom row of each group shows a digital reconstruction of the confocal data with distinct segmentation of tumor and immune cell populations. The initial CAR T to tumor cell ratio or effector-to-tar get (E:T) ratio is indicated on the graph. (C, D) Quantification of CAR T–tumor interactions revealing an inverse correlation between tumor size and infiltrating CAR T cells for (C) GBM and D) OS tumors. The percentage of tumor size (left axis) and CAR T cells infiltrating the tumor (right axis) were normalized to the original tumor size at time 0. (E, F) Killing rates over time were calculated as derivatives from C and D, respectively. (G, H) The measured CAR T to cancer cell area ratios (E:T) as a function of time. The E:T ratios dynamically changed over time, while for WT tumors, the ratios remained constant. (I) RNA sequencing data from single CAR T cells isolated from a 3D co-culture with target CD70 pos versus WT tumors. All genes shown in the results have a statistical significance of p ≤0.05. Error bars represent standard deviation and biological samples n = 3 for all CD70 pos samples and n = 2 for all WT samples.

Article Snippet: Cell lines 293T cell line (ATCC, CRL-3216) was used for lentiviral plasid production encoding for mouse full length CD70 surface antien (GenBank: U78091.1).

Techniques: RNA Sequencing, Isolation, Co-Culture Assay, Standard Deviation

Journal: Acta biomaterialia

Article Title: Bioconjugated liquid-like solid enhances characterization of solid tumor - chimeric antigen receptor T cell interactions.

doi: 10.1016/j.actbio.2023.09.042

Figure Lengend Snippet: Fig. 7. Sensitivity of anti-tumor activity to various CAR T: cancer cell (E:T) ratios. (A,B) confocal time-lapse images of CAR T – tumor co-culture in iVITA at different E:T ratios. (C,D) show the digital image reconstruction of confocal data quantifying bulk tumor mass, migrating single cancer cells, immune cells, and immune cell clusters, and killing activity over time. (A,C) GBM CD70 pos tumors and (B,D) OS CD70 pos tumors were co-cultured with their respective CAR T cells at different concentrations corresponding to E:T of 1:4, 1:2, and 4:1 for the GBM model and E:T of 1:4, 1:1, and 4:1 for the OS model. (E–H) CAR T expansion as a function of initial E:T seeding ratios. (E,F) CAR T expansion on average in both models from 0 to 72 h. For each seeding E:T, CAR T expansion at each time point was normalized to the CAR T at time 0, and the average was calculated for all frames. G,H) the number of CAR T clusters counted every 1.5 h for 72 h for each group. The box plots display 25th and 75th percentiles, a line at the median a plus sign at the mean, from the minimum to the maximum observation. (I,J) Quantification of tumor size over time at the initial E:T = 1:4, 1:2, and 4:1 for I) GBM model and at E:T = 1:4, 1:1, and 4:1 for (J) OS model. (K,L) The E:T ratio dynamically changed over time. The CAR T expansion and tumor-killing were presented by the exponential increase of E:T ratios. (M,N) tumor killing rates were calculated as derivatives from (I) and (J), respectively. Error bars represent standard deviation. Statistical analysis was performed using Ordinary One-Way ANOVA. ( n = 3 unless indicated otherwise, ∗∗= p < 0.01, and ∗∗∗∗= p < 0.0 0 01).

Article Snippet: Cell lines 293T cell line (ATCC, CRL-3216) was used for lentiviral plasid production encoding for mouse full length CD70 surface antien (GenBank: U78091.1).

Techniques: Activity Assay, Co-Culture Assay, Cell Culture, Standard Deviation

Journal: Biology

Article Title: Iron Inhibits the Translation and Activity of the Renal Epithelial Sodium Channel

doi: 10.3390/biology11010123

Figure Lengend Snippet: Iron does not transcriptionally regulate ENaC alpha. The kidneys of 12-week-old WT or iron overloaded Hamp -/- mice were analyzed by real time PCR for gene expression of ENaC alpha. There was no significant difference in ENaC alpha gene expression of WT or spontaneously iron overloaded Hamp -/- mice under basal conditions ( A ). We confirmed that iron can regulate ENaC alpha gene transcription in mpkCCD cells, a cortical collecting duct cell line that is known to express ENaC alpha. 200 × 10 5 cells were serum-starved for 12 h and then cultured for 12 more hours in vehicle or FAC (100 uM). RNA was purified and then cDNA was synthesized. ENaC alpha gene expression was measured. Iron loading did not regulate ENaC alpha gene expression in mpkCCD cells ( B ), though it increased oxidative stress ( C ). One hundred uM FAC did not activate cell death program under the experimental conditions ( D ). Data from 2 independent experiments were pooled and analyzed. The statistical significance was determined by a 2-tailed Mann–Whitney test and is plotted as mean ± SEM. ** p < 0.001.

Article Snippet: The slides were then boiled in citrate buffer for 20 min (Vector Laboratories, Inc.; Burlingame, CA, USA) and washed for 3-min in type 1 water and then in 1X phosphate-buffered saline (1XPBS) (Corning; Manassas, VA, USA) for 5 min. After blocking with 2.5% normal horse serum (Vector Laboratories, Inc.) for 20 min the tissues were incubated for 60 min with a 1:500 dilution of primary rabbit polyclonal anti-MARCKS antibody (ab72459; Abcam, Boston, MA, USA) followed by a 60-min incubation with mouse anti-ENaC alpha antibody (StressMarq Biosciences Inc., Victoria BC, CA, Canada; SMC-242D), mouse anti-ENaC beta antibody (StressMarq Biosciences Inc., Victoria BC, CA, Canada; SMC-240D) or anti-ENaC gamma 2102 antibody conjugated with the Dylight NHS ester 594 kit (ThermoFisher, Waltham, MA, USA).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Cell Culture, Purification, Synthesized, MANN-WHITNEY

Journal: Biology

Article Title: Iron Inhibits the Translation and Activity of the Renal Epithelial Sodium Channel

doi: 10.3390/biology11010123

Figure Lengend Snippet: Immunohistochemistry analysis of ENaC alpha and MARCKS protein expression in Hamp -/- (KO) mice or wild-type (WT) mice. ( A ) ENaC alpha (red) protein expression and MARCKS (green) protein expression in WT control mice. ( B ) ENaC alpha (red) protein expression and MARCKS (green) protein expression in iron overloaded Hamp -/- mice. Data is representative of 3 mice in each group.

Article Snippet: The slides were then boiled in citrate buffer for 20 min (Vector Laboratories, Inc.; Burlingame, CA, USA) and washed for 3-min in type 1 water and then in 1X phosphate-buffered saline (1XPBS) (Corning; Manassas, VA, USA) for 5 min. After blocking with 2.5% normal horse serum (Vector Laboratories, Inc.) for 20 min the tissues were incubated for 60 min with a 1:500 dilution of primary rabbit polyclonal anti-MARCKS antibody (ab72459; Abcam, Boston, MA, USA) followed by a 60-min incubation with mouse anti-ENaC alpha antibody (StressMarq Biosciences Inc., Victoria BC, CA, Canada; SMC-242D), mouse anti-ENaC beta antibody (StressMarq Biosciences Inc., Victoria BC, CA, Canada; SMC-240D) or anti-ENaC gamma 2102 antibody conjugated with the Dylight NHS ester 594 kit (ThermoFisher, Waltham, MA, USA).

Techniques: Immunohistochemistry, Expressing

Journal: EBioMedicine

Article Title: Salt-deficient diet exacerbates cystogenesis in ARPKD via epithelial sodium channel (ENaC)

doi: 10.1016/j.ebiom.2019.01.006

Figure Lengend Snippet: ENaC protein and mRNA expression in PCK rats fed diets with varying sodium content. (a) Western blotting showing expression levels of α-ENaC in the renal cortex of the PCK rats fed a SD, NS and HS diets, and a summary graph with densitometry values (normalized to β-actin expression for the same samples). Each lane on the blot is one animal. (b) mRNA expression for α-, β-, and γ-ENaC in the renal cortex of the PCK rats fed a SD, NS and HS diets. (c) Western blotting showing expression levels of AQP2 in the renal cortex of the PCK rats fed a SD, NS and HS diets, and a summary graph with densitometry values (normalized to β-actin expression for the same samples). (d) miRNAs found to be differentially expressed ( p < .05) in the cortical tissues of the PCK rats fed a SD, NS and HS diets. *, p < .05, ** p < .01. (e) Western blots demonstrating total ENaC subunit expression in cultured mCCD cells transfected with a control scrambled miR sequence or miR9a-5p, with summary densitometry graphs (normalized to β-Actin and relative to control mCCD expression). Both β- and γ-ENaC are significantly decreased in mCCD cells over expressing miR9a-5p. * p < .05. Values in (b) and (c) were compared using a one-way ANOVA with a Tukey post-hoc test. For (d), differential expression was determined by DESeq2 method . False discovery rate within statistical analysis was controlled for using the Benjamini-Hochberg method, p value cutoff was 0.05. Graphs with whiskers show mean value in each group and SEM. In (e), following a Shapiro-Wilk normality test, control and experimental values were compared using an unpaired student t -test.

Article Snippet: Antibodies: α-ENaC (ext) – Alomone Cat #: ASC-030; AQP-2 – SCBT Cat # sc-9882; β-Actin – SCBT Cat # sc-1616 HRP, α-, β-, γ-ENaC –Stressmarq Cat#: SPC-403, SPC-404, and SPC-405 respectively.

Techniques: Expressing, Western Blot, Cell Culture, Transfection, Control, Sequencing, Quantitative Proteomics

Journal: EBioMedicine

Article Title: Salt-deficient diet exacerbates cystogenesis in ARPKD via epithelial sodium channel (ENaC)

doi: 10.1016/j.ebiom.2019.01.006

Figure Lengend Snippet: ENaC activity is altered in the miR-9a-5 overexpressed experimental group. (a) Representative gap-free current traces from cell-attached patches made on mCCD cells of naïve, untransfected cells (upper), control (cont) miR with a non-specific targeting sequence (middle) and miR-9a-5 (lower) group. The closed (c) and open (o) states are denoted with dashed lines. Currents were recorded at the membrane voltage of −60 mV. # denotes p < .05 between control group and miR9a-5 group. (b) Current-voltage relationship and representative examples of ENaC single-channel current at different membrane potentials made on mCCD cells of miR group. (c,d,e) Summary graphs of NP o (c), P o (d), and N (e) for ENaC activity in naïve (white), control miR (dark grey) and miR-9a-5 (black) groups. * P < .05, ** P < .01, ns – not statistically significant, miR9a-5 versus control-miR group by ANOVA . Values were compared using a one-way ANOVA with a Tukey post-hoc test. In box plots, the box is SEM, whiskers represent SD, and line within the box shows median value.

Article Snippet: Antibodies: α-ENaC (ext) – Alomone Cat #: ASC-030; AQP-2 – SCBT Cat # sc-9882; β-Actin – SCBT Cat # sc-1616 HRP, α-, β-, γ-ENaC –Stressmarq Cat#: SPC-403, SPC-404, and SPC-405 respectively.

Techniques: Activity Assay, Control, Sequencing, Membrane