Journal: Aging Cell

Article Title: Overactivation of Cdc42 GTPase Impairs the Cytotoxic Function of NK Cells From Old Individuals Towards Senescent Fibroblasts

doi: 10.1111/acel.70398

Figure Lengend Snippet: Natural killer cells from old adults reveal reduced cytotoxicity towards senescent fibroblasts. (A) Graphical illustration of the NK cell mediated target cell cytotoxicity assay. Target cells were first stained with calcein acetomethoxymethyl (AM), a vital fluorescent dye. Calcein AM is a non‐fluorescent compound that pass the intact cell membrane into the cytoplasm. Hydrolysis of calcein AM by intracellular esterases in live cells generates calcein, a hydrophilic, intensely fluorescent molecule which reliably stays in the cytoplasm. The stained target cells were next co‐cultured with NK cells isolated from young or old human or mice. NK cells exert their cytotoxicity towards target cells through the release of perforin and granzyme B. Upon lysis of target cells, the calcein dye is released and the loss of the dye is measured as a shift in fluorescence intensity by flow cytometry. Dead cells will appear to the left of the histogram, while alive cells on the right side. The percentage of dead cells can then simply be calculated and presented. (B) Graphical scheme depicts the experimental groups: Co‐cultures of NK cells from young adults with senescent human dermal fibroblasts in the top row and NK cells from old adults with senescent HDF in the bottom row. (C) Histogram (bi‐exponential scale) showing cytotoxicity of NK cells from young and old adults on different senescent HDF. RS, replicative senescent HDF, DIS, doxorubicin induced senescent HDF, IR, ionizing radiation induced senescence, CA, chronologically aged HDF (~75 years). The peak in the left part of the histogram showing the dead cell population and the percentage of dead cells. (D) The graph depicts the percentage of senescent HDF death ( y ‐axis) by NK cells isolated from young and old human adult. Data were represented as mean (Percentage of senescent fibroblast death) ± SEM. N = 5. Two tailed Student's t ‐test was used to assess the significance between young and old groups for each of senescence model. (E) Illustration of the experimental design showing cytotoxic activity of NK cells derived from bone marrow and spleen of young and old mice against aged murine dermal fibroblasts (MDF). (F) Histogram depicting cytotoxicity of NK cells from young and old mice on old MDF. The peak in the left part of the histogram showing the dead cell population and the percentage of dead cells. (G) The graph depicts the percentage of senescent MDF death ( y ‐axis) by NK cells isolated from young and old mice. Data were represented as mean (Percentage of senescent fibroblast death) ± SEM. N = 3. Each mouse NK cell sample used in the cytotoxicity assay was the pool of NK cells isolated from three different mice. One‐way ANOVA, followed by Bonferroni multiple comparison test was used to find the significance among the groups.

Article Snippet: Human dermal fibroblast , ATCC , Cat. #ATCC‐PCS‐201‐010.

Techniques: Cytotoxicity Assay, Staining, Membrane, Cell Culture, Isolation, Lysis, Fluorescence, Flow Cytometry, Two Tailed Test, Activity Assay, Derivative Assay, Comparison

Journal: Aging Cell

Article Title: Overactivation of Cdc42 GTPase Impairs the Cytotoxic Function of NK Cells From Old Individuals Towards Senescent Fibroblasts

doi: 10.1111/acel.70398

Figure Lengend Snippet: CASIN restores impairment of conjugation, degranulation and mitochondrial ATP generation in old Natural killer cells. (A) Synapse formation with conjugation of NK cells with the target senescent fibroblasts and the tubulin network pulling the perforin and granzyme B containing vesicles in the direction of the synapse. (B) Fusion of the NK cell derived secretory granules with the presynaptic membrane of NK cells and concomitant exposure of CD107a at the cell membrane and the release of perforin and granzyme B into the synaptic cleft towards the target cell. (C & D) Percentage of NK cell conjugation with senescent HDF when co‐cultured for (C) 60 and (D) 90 min at an effector to target (E:T) cell ratio of 1:1. Data were represented as mean (percentage of cell conjugation) ± SEM, N = 3. (E) Degranulation of NK cells when co‐cultured with senescent HDF for 7 h at an effector to target (E:T) cell ratio of 10:1. Data were represented as mean (mean fluorescence intensity) ± SEM, N = 6. One‐way ANOVA, followed by Bonferroni multiple comparison test was used to find the significance among the groups in C, D and E. (F) Seahorse flux analysis showed quantification of ATP generated by NK cells treated with vehicle from young donors and from NK cells treated with either vehicle or CASIN from old donors. The ATP generation either by glycolysis or by oxidative phosphorylation and total ATP was assessed. Data were represented as mean (ATP level) ± SEM, N = 7. Two‐way ANOVA, followed by Bonferroni multiple comparison test was used to find the significance among the groups. (G) Mitochondrial structure showing the chemical structure of the mitochondrial fluorescent probe JC‐1 that can form J‐aggregates (red) and J‐monomers (green) indicating high and low mitochondrial membrane potential, respectively. (H) Flow cytometry analysis of J‐aggregates (red) and J‐monomers (green) of young NK cells treated with vehicle, old NK cells treated with either vehicle or CASIN. (I) The graph depicts the percentage of J‐aggregates (Q2 population of figure H) of young NK treated with vehicle, and old NK cells treated with either vehicle or CASIN. Data were represented as mean (percentage of cell with J‐aggregate) ± SEM, N = 6. (J) Quantification of the ratio of J‐aggregates to J‐monomers from young and old NK treated with vehicle and old NK cells treated with CASIN. Data were represented as mean (ratio) ± SEM, N = 5. One‐way ANOVA, followed by Bonferroni multiple comparison test was used to find the significance among the groups in I and J.

Article Snippet: Human dermal fibroblast , ATCC , Cat. #ATCC‐PCS‐201‐010.

Techniques: Conjugation Assay, Derivative Assay, Membrane, Cell Culture, Fluorescence, Comparison, Generated, Phospho-proteomics, Flow Cytometry

Journal: Aging Cell

Article Title: Overactivation of Cdc42 GTPase Impairs the Cytotoxic Function of NK Cells From Old Individuals Towards Senescent Fibroblasts

doi: 10.1111/acel.70398

Figure Lengend Snippet: CASIN treatment improves the cytotoxic ability of Natural killer cells from old humans and mice. (A) Graphical illustration of experimental plan, where young NK cells treated with vehicle and old NK cells treated with either vehicle or CASIN for 8 h and thereafter subjected to co‐culture with target senescent HDF exerting their differential killing ability. (B) Representative histograms depicting the killing ability of different experimental groups as measured by flow cytometry. Peak at the left side of histogram, showing the dead senescent HDF population with percentage of dead cells. (C) Quantification of the percentage of target senescent HDF death executed by young NK cells treated with vehicle, and old NK cells treated with either vehicle or CASIN. Data were represented as mean (Percentage of senescent fibroblast death) ± SEM. N = 4. (D) Representative histograms show the distribution of K562 killing by young NK cells treated with vehicle, and old NK cells treated with either vehicle or CASIN. Peak at the left side of histogram, showing the dead K562 population with percentage of dead cells. (E) Graph shows the percentage of target cell (K562) death mediated either by young NK cells treated with vehicle or by old NK cells treated with either vehicle or CASIN. Data were represented as mean (Percentage of K562 lysis) ± SEM. N = 4. One‐way ANOVA, followed by Bonferroni multiple comparison test was used to find the significance among the groups in C and E. (F) Illustration of the experimental design for treatment of young mice (average age 120 days) treated with vehicle and old mice (average age 650 days) treated with either vehicle or CASIN. Following treatment, NK cells were isolated from spleen and bone marrow and subjected to co‐cultures with murine dermal fibroblasts (MDF) derived from old mice (average age 650 days). (G) Flow cytometry with representative histograms depicting old/senescent MDF killing by NK cells isolated from bone marrow (left panel) and spleen (right panel) of vehicle and CASIN treated old mice. Peak at the left side of histogram, showing the dead old MDF population with percentage of dead cells. (H) Quantification of the percentage of old/senescent MDF killing by NK cells isolated from bone marrow and spleen of young and old mice treated with vehicle and old mice treated with CASIN. Data were represented as mean (percentage of old/senescent MDF lysis) ± SEM, N = 4, where each group contains pool of NK cells isolated from 4 different mice of same treatment group. Two‐way ANOVA, followed by Bonferroni multiple comparison test was used to find the significance among the groups. (I) Graphical summary. Unrestrained Cdc42 activity causes failure of old NK cells to kill senescent fibroblasts. Unrestrained Cdc42 activity disrupts the microtubular network and impaired mitochondrial ATP resulting in reduced conjugation, and impaired degranulation of lytic vesicles into the synaptic cleft with reduced cytotoxicity. CASIN can attenuate all these steps and in part attenuate the killing of senescent fibroblasts (senescent HDF).

Article Snippet: Human dermal fibroblast , ATCC , Cat. #ATCC‐PCS‐201‐010.

Techniques: Co-Culture Assay, Flow Cytometry, Lysis, Comparison, Isolation, Derivative Assay, Activity Assay, Conjugation Assay

Journal: Aging Cell

Article Title: Overactivation of Cdc42 GTPase Impairs the Cytotoxic Function of NK Cells From Old Individuals Towards Senescent Fibroblasts

doi: 10.1111/acel.70398

Figure Lengend Snippet: Natural killer cells from old adults reveal reduced cytotoxicity towards senescent fibroblasts. (A) Graphical illustration of the NK cell mediated target cell cytotoxicity assay. Target cells were first stained with calcein acetomethoxymethyl (AM), a vital fluorescent dye. Calcein AM is a non‐fluorescent compound that pass the intact cell membrane into the cytoplasm. Hydrolysis of calcein AM by intracellular esterases in live cells generates calcein, a hydrophilic, intensely fluorescent molecule which reliably stays in the cytoplasm. The stained target cells were next co‐cultured with NK cells isolated from young or old human or mice. NK cells exert their cytotoxicity towards target cells through the release of perforin and granzyme B. Upon lysis of target cells, the calcein dye is released and the loss of the dye is measured as a shift in fluorescence intensity by flow cytometry. Dead cells will appear to the left of the histogram, while alive cells on the right side. The percentage of dead cells can then simply be calculated and presented. (B) Graphical scheme depicts the experimental groups: Co‐cultures of NK cells from young adults with senescent human dermal fibroblasts in the top row and NK cells from old adults with senescent HDF in the bottom row. (C) Histogram (bi‐exponential scale) showing cytotoxicity of NK cells from young and old adults on different senescent HDF. RS, replicative senescent HDF, DIS, doxorubicin induced senescent HDF, IR, ionizing radiation induced senescence, CA, chronologically aged HDF (~75 years). The peak in the left part of the histogram showing the dead cell population and the percentage of dead cells. (D) The graph depicts the percentage of senescent HDF death ( y ‐axis) by NK cells isolated from young and old human adult. Data were represented as mean (Percentage of senescent fibroblast death) ± SEM. N = 5. Two tailed Student's t ‐test was used to assess the significance between young and old groups for each of senescence model. (E) Illustration of the experimental design showing cytotoxic activity of NK cells derived from bone marrow and spleen of young and old mice against aged murine dermal fibroblasts (MDF). (F) Histogram depicting cytotoxicity of NK cells from young and old mice on old MDF. The peak in the left part of the histogram showing the dead cell population and the percentage of dead cells. (G) The graph depicts the percentage of senescent MDF death ( y ‐axis) by NK cells isolated from young and old mice. Data were represented as mean (Percentage of senescent fibroblast death) ± SEM. N = 3. Each mouse NK cell sample used in the cytotoxicity assay was the pool of NK cells isolated from three different mice. One‐way ANOVA, followed by Bonferroni multiple comparison test was used to find the significance among the groups.

Article Snippet: Human dermal fibroblast , iXCells Biotechnologies , Cat. #10HU‐013.

Techniques: Cytotoxicity Assay, Staining, Membrane, Cell Culture, Isolation, Lysis, Fluorescence, Flow Cytometry, Two Tailed Test, Activity Assay, Derivative Assay, Comparison

Journal: Aging Cell

Article Title: Overactivation of Cdc42 GTPase Impairs the Cytotoxic Function of NK Cells From Old Individuals Towards Senescent Fibroblasts

doi: 10.1111/acel.70398

Figure Lengend Snippet: CASIN restores impairment of conjugation, degranulation and mitochondrial ATP generation in old Natural killer cells. (A) Synapse formation with conjugation of NK cells with the target senescent fibroblasts and the tubulin network pulling the perforin and granzyme B containing vesicles in the direction of the synapse. (B) Fusion of the NK cell derived secretory granules with the presynaptic membrane of NK cells and concomitant exposure of CD107a at the cell membrane and the release of perforin and granzyme B into the synaptic cleft towards the target cell. (C & D) Percentage of NK cell conjugation with senescent HDF when co‐cultured for (C) 60 and (D) 90 min at an effector to target (E:T) cell ratio of 1:1. Data were represented as mean (percentage of cell conjugation) ± SEM, N = 3. (E) Degranulation of NK cells when co‐cultured with senescent HDF for 7 h at an effector to target (E:T) cell ratio of 10:1. Data were represented as mean (mean fluorescence intensity) ± SEM, N = 6. One‐way ANOVA, followed by Bonferroni multiple comparison test was used to find the significance among the groups in C, D and E. (F) Seahorse flux analysis showed quantification of ATP generated by NK cells treated with vehicle from young donors and from NK cells treated with either vehicle or CASIN from old donors. The ATP generation either by glycolysis or by oxidative phosphorylation and total ATP was assessed. Data were represented as mean (ATP level) ± SEM, N = 7. Two‐way ANOVA, followed by Bonferroni multiple comparison test was used to find the significance among the groups. (G) Mitochondrial structure showing the chemical structure of the mitochondrial fluorescent probe JC‐1 that can form J‐aggregates (red) and J‐monomers (green) indicating high and low mitochondrial membrane potential, respectively. (H) Flow cytometry analysis of J‐aggregates (red) and J‐monomers (green) of young NK cells treated with vehicle, old NK cells treated with either vehicle or CASIN. (I) The graph depicts the percentage of J‐aggregates (Q2 population of figure H) of young NK treated with vehicle, and old NK cells treated with either vehicle or CASIN. Data were represented as mean (percentage of cell with J‐aggregate) ± SEM, N = 6. (J) Quantification of the ratio of J‐aggregates to J‐monomers from young and old NK treated with vehicle and old NK cells treated with CASIN. Data were represented as mean (ratio) ± SEM, N = 5. One‐way ANOVA, followed by Bonferroni multiple comparison test was used to find the significance among the groups in I and J.

Article Snippet: Human dermal fibroblast , iXCells Biotechnologies , Cat. #10HU‐013.

Techniques: Conjugation Assay, Derivative Assay, Membrane, Cell Culture, Fluorescence, Comparison, Generated, Phospho-proteomics, Flow Cytometry

Journal: Aging Cell

Article Title: Overactivation of Cdc42 GTPase Impairs the Cytotoxic Function of NK Cells From Old Individuals Towards Senescent Fibroblasts

doi: 10.1111/acel.70398

Figure Lengend Snippet: CASIN treatment improves the cytotoxic ability of Natural killer cells from old humans and mice. (A) Graphical illustration of experimental plan, where young NK cells treated with vehicle and old NK cells treated with either vehicle or CASIN for 8 h and thereafter subjected to co‐culture with target senescent HDF exerting their differential killing ability. (B) Representative histograms depicting the killing ability of different experimental groups as measured by flow cytometry. Peak at the left side of histogram, showing the dead senescent HDF population with percentage of dead cells. (C) Quantification of the percentage of target senescent HDF death executed by young NK cells treated with vehicle, and old NK cells treated with either vehicle or CASIN. Data were represented as mean (Percentage of senescent fibroblast death) ± SEM. N = 4. (D) Representative histograms show the distribution of K562 killing by young NK cells treated with vehicle, and old NK cells treated with either vehicle or CASIN. Peak at the left side of histogram, showing the dead K562 population with percentage of dead cells. (E) Graph shows the percentage of target cell (K562) death mediated either by young NK cells treated with vehicle or by old NK cells treated with either vehicle or CASIN. Data were represented as mean (Percentage of K562 lysis) ± SEM. N = 4. One‐way ANOVA, followed by Bonferroni multiple comparison test was used to find the significance among the groups in C and E. (F) Illustration of the experimental design for treatment of young mice (average age 120 days) treated with vehicle and old mice (average age 650 days) treated with either vehicle or CASIN. Following treatment, NK cells were isolated from spleen and bone marrow and subjected to co‐cultures with murine dermal fibroblasts (MDF) derived from old mice (average age 650 days). (G) Flow cytometry with representative histograms depicting old/senescent MDF killing by NK cells isolated from bone marrow (left panel) and spleen (right panel) of vehicle and CASIN treated old mice. Peak at the left side of histogram, showing the dead old MDF population with percentage of dead cells. (H) Quantification of the percentage of old/senescent MDF killing by NK cells isolated from bone marrow and spleen of young and old mice treated with vehicle and old mice treated with CASIN. Data were represented as mean (percentage of old/senescent MDF lysis) ± SEM, N = 4, where each group contains pool of NK cells isolated from 4 different mice of same treatment group. Two‐way ANOVA, followed by Bonferroni multiple comparison test was used to find the significance among the groups. (I) Graphical summary. Unrestrained Cdc42 activity causes failure of old NK cells to kill senescent fibroblasts. Unrestrained Cdc42 activity disrupts the microtubular network and impaired mitochondrial ATP resulting in reduced conjugation, and impaired degranulation of lytic vesicles into the synaptic cleft with reduced cytotoxicity. CASIN can attenuate all these steps and in part attenuate the killing of senescent fibroblasts (senescent HDF).

Article Snippet: Human dermal fibroblast , iXCells Biotechnologies , Cat. #10HU‐013.

Techniques: Co-Culture Assay, Flow Cytometry, Lysis, Comparison, Isolation, Derivative Assay, Activity Assay, Conjugation Assay

Journal: Nature Communications

Article Title: Inhibition of iRhom1 by CD44-targeting nanocarrier for improved cancer immunochemotherapy

doi: 10.1038/s41467-023-44572-6

Figure Lengend Snippet: a Correlation of iRhom1 gene expression levels with survivals in BRCA-TNBC or COAD patients receiving chemotherapy. For BRCA-TNBC-Chemo: n(Low expression) = 52, n (High expression) = 33; For COAD-Chemo: n (Low expression)=89, n (High expression) = 56. b CTRP-based prediction of the correlation between iRhom1 gene expression levels and the responses to different types of anticancer drugs. HDACi: histone deacetylase inhibitor, MAi: microtube assembly inhibitor, Topoi: topoisomerase inhibitor, PLK1i: polo-like kinase 1 inhibitor, NAMPTi: nicotinamide phosphoribosyltransferase inhibitor. c The impact of siRNA-mediated iRhom1 knockdown on the cytotoxicity of DOX or CPT-SAHA in several cancer cell lines. n = 6 independent samples. d Changes in protein levels of iRhom1 following treatment with DOX or CPT-SAHA. n = 4 independent experiments and data were quantified by densitometry. e RNA-seq analysis of gene enrichment in various signaling pathways in iRhom1 KO CT26 cells compared to WT cells. f Changes in the protein levels of MAPK14-HSP27 axis in iRhom1 KO cells compared to WT cells. n = 5 independent experiments and data were quantified by densitometry. g Changes in the protein levels of MAPK14-HSP27 axis in iRhom1 OE cells compared to WT cells. n = 5 independent experiments and data were quantified by densitometry. h Changes in the protein levels of MAPK14-HSP27 axis after treatment with various concentrations of CPT-SAHA in iRhom1 KO cells and WT cells. n = 3 independent experiments and data were quantified by densitometry. i MTT cytotoxicity assay of treatments with various concentrations of CPT-SAHA in CT26 WT or CT26 iRhom1 OE cells, with or without knockdown of MAPK14 respectively. n = 6 independent samples. j Synergy between Ivermectin (IVM) and chemotherapy drug (CPT-SAHA or DOX). k Immunoprecipitation of HA-iRhom1 with anti-HA beads led to pulldown of p-38α (MAPK14) in HA-iRhom1 expressing 293 T cells and reciprocal immunoprecipitation of FLAG-MAPK14 with anti-FLAG beads led to pulldown of iRhom1 in FLAG-MAPK14 expressing 293 T cells. Data are presented as mean ± s.e.m. in ( c , i ) and mean ± s.d. in ( d , f , g ). Statistical analysis was performed by log rank test for comparison in ( a ), two-tailed Student’s t-test for comparison in ( c , f , g ), and one-way ANOVA with Tukey’s post hoc test for comparison in ( d and i ). Data are representative of two independent experiments in ( e ), i – k three independent experiments in ( c , h ), four independent experiments in ( d ), and five independent experiments in ( f , g ). Source data are provided as a file for ( c , d , f , g , h , i , j , k ).

Article Snippet: CT26 murine CRC cell lines (CRL-2638), HCT116 human CRC cell line (CCL-247), 4T1 murine BC cell line (CRL-2539) and MDA-MB-468 human BC cell line (HTB-132) were obtained from ATCC (Manassas, VA).

Techniques: Gene Expression, Expressing, Histone Deacetylase Assay, Knockdown, RNA Sequencing, Protein-Protein interactions, Cytotoxicity Assay, Immunoprecipitation, Comparison, Two Tailed Test

Journal: Nature Communications

Article Title: Inhibition of iRhom1 by CD44-targeting nanocarrier for improved cancer immunochemotherapy

doi: 10.1038/s41467-023-44572-6

Figure Lengend Snippet: a TCGA analysis of iRhom1 gene expression levels in various cancer subtypes. TNBC: triple negative breast cancer; non-TNBC: breast cancer patients other than TNBC patients; MSI-H: microsatellite instability-high; MSI-L: microsatellite instability-low; MSS: microsatellite stable. n (TNBC) = 115, n(non-TNBC) = 653, n(MSI-H) = 78, n(MSI-L) = 76, n (MSS) = 267. b Tumor growth curves of WT, iRhom1 KO, iRhom1 rescue and vector control CT26 cells in immunodeficient ( n = 4 mice) or immunocompetent mice ( n = 5 mice). c Activation of immune-related signaling pathways in iRhom1 −/− CT26 cells (RNAseq analysis). d Flow analysis of H2Kb/SIINFEKL presentation on B16-OVA and iRhom1 KO B16-OVA (B16-OVA KO) cells, with or without re-expression of iRhom1 (+piRhom). n = 3 independent samples. e Cytolysis of WT or iRhom1 KO B16-OVA cells co-cultured with CD8 + T cells isolated from spleens of OT-I mice, with or without blockade by H2K antibody (H2K Ab). n = 3 independent samples. f The correlation of protein expression levels between iRhom1 and various APP-related proteins in the tumor samples of TNBC patients. n = 25. g Changes in ERAP1 protein levels in various cancer cell lines following iRhom1 KO and OE. h Co-Immunoprecipitation of HA-iRhom1 with anti-HA antibody led to pulldown of ERAP1 in HA-iRhom1 expressing 293 T cells and reciprocal immunoprecipitation of GFP-ERAP1 with anti-GFP beads led to pulldown of iRhom1 in GFP-ERAP1 expressing 293 T cells. i Changes in ERAP1 protein levels in iRhom1 WT, KO and OE cells after treatment with Eeyarestatin I (4.5 μM). j The stability (t 1/2 ) of ERAP1 in iRhom1 WT and KO cells determined by cycloheximide chase experiment. The experiment was repeated three times independently and data were quantified by densitometry. k Representative WB of ERAP1 protein levels in cycloheximide chase experiments. Data are presented as mean ± s.e.m. in ( b , d , e , j ). Statistical analysis was performed by two-tailed Student’s t test for comparison in ( a (upper panel), b (immunodeficient)), and one-way ANOVA with Tukey’s post hoc test for comparison in ( a (lower panel), b (immunocompetent), d and e ). Data are representative of two independent experiments in ( b , c , d , e , g – i ) and three independent experiments in ( j , k ). Source data are provided as a file for ( b , d , e , g , h , i , j , k ).

Article Snippet: CT26 murine CRC cell lines (CRL-2638), HCT116 human CRC cell line (CCL-247), 4T1 murine BC cell line (CRL-2539) and MDA-MB-468 human BC cell line (HTB-132) were obtained from ATCC (Manassas, VA).

Techniques: Gene Expression, Plasmid Preparation, Control, Activation Assay, Protein-Protein interactions, Expressing, Cell Culture, Isolation, Immunoprecipitation, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: Inhibition of iRhom1 by CD44-targeting nanocarrier for improved cancer immunochemotherapy

doi: 10.1038/s41467-023-44572-6

Figure Lengend Snippet: a NIRF whole body imaging and ex vivo imaging of CT26 tumor-bearing mice at 24 h following i.v. administration of DiR-loaded, Cy5.5-labled PCL-CP NPs. n = 3 animals. b Plasma PK profile of free DOX or DOX loaded in DOX/pre-siRNA-coloaded PCL-CP NPs after i.v. administration. Non-compartment model was applied to generate the major pharmacokinetic profiles. AUCinf: area under the plasma concentration-time curve from time 0 to infinity; t 1/2 half-life; CL clearance; Vd volume of distribution. n = 3 biologically independent samples. c Biodistribution of free DOX or DOX loaded in DOX/pre-siRNA-coloaded PCL-CP NPs after i.v. administration (presented as percentage of injected dose). n = 3 biologically independent samples. d Biodistribution of free DOX or DOX loaded in DOX/pre-siRNA-coloaded PCL-CP NPs after i.v. administration (presented as DOX concentration normalized by tissue weight). n = 3 biologically independent samples. The biodistribution profile ( e ) and the plasma PK ( f ) of siRNA loaded in DOX/pre-siRNA-coloaded PCL-CP NPs after i.v. administration. n = 3 biologically independent samples. g Mice bearing CT26-luc tumors received i.v. administration of luciferase siRNA-loaded PCL-CP NPs at a dose of 2 mg siRNA/kg once every 3 days and the mice were subjected to whole body bioluminescence imaging the next day after each treatment. h The efficiency of knockdown after each treatment was also analyzed quantitatively. n = 3 animals. Data are presented as mean ± s.e.m. in ( a , b , c , d , e , h ). Statistical analysis was performed by one-way ANOVA with Tukey’s post hoc test for comparison in ( a ), and two-tailed Student’s t test for comparison in ( c , d , h ). Data are representative of two independent experiments in ( a – g ). Source data are provided as a file for ( c , d , e , f , h ).

Article Snippet: CT26 murine CRC cell lines (CRL-2638), HCT116 human CRC cell line (CCL-247), 4T1 murine BC cell line (CRL-2539) and MDA-MB-468 human BC cell line (HTB-132) were obtained from ATCC (Manassas, VA).

Techniques: Imaging, Ex Vivo, Clinical Proteomics, Concentration Assay, Injection, Luciferase, Knockdown, Comparison, Two Tailed Test

Journal: Nature Communications

Article Title: Inhibition of iRhom1 by CD44-targeting nanocarrier for improved cancer immunochemotherapy

doi: 10.1038/s41467-023-44572-6

Figure Lengend Snippet: a NIRF whole body imaging, ex vivo imaging of CD44 WT or KO tumor-bearing CD44 WT or KO mice at 24 h following i.v. administration of DiR-loaded, Cy5.5-labled PCL-CP NPs. b Quantification of ex vivo imaging data from ( a ). n = 3 animals. c Fluorescence images of frozen tumor core sections from CD44 WT or KO tumor grown on CD44 WT or KO mice at 24 h after treatment with Cy5.5-labled PCL-CP NPs. CD31 was stained with FITC-labeled antibody to show the endothelial cells. Bar = 50 μm. d Biodistribution of DOX loaded in DOX/pre-siRNA-coloaded PCL-CP NPs in CD44 WT or KO tumor-bearing CD44 WT or KO mice at 24 h following i.v. administration. n = 3 independent samples. e Quantitative analysis of biodistribution of DOX in ( d ). n = 3 independent samples. f Illustration of transwell study. The figure is created with BioRender.com. Transwell assay of transmigration of Cy5.5 labeled PCL NPs, PCL-C NPs or PCL-CP NPs (with or without pretreatment with a transcytosis inhibitor) across HUVEC cells ( g ), CT26 cells ( h ), CD44 KO HUVEC cells ( i ), or CD44 KO CT26 cells ( j ). Fluorescence intensity of the medium in the lower chamber was measured to calculate the percentage of transmigration at indicated time points. n = 3 independent experiments. k Confocal z-stack images of CT26 tumor cell spheroids after 18 h incubation with Cy5.5-labeled PCL NPs, PCL-C NPs or PCL-CP NPs (with or without pretreatment with a transcytosis inhibitor). White circles highlight the efficient penetration of CS-coated NPs. Bar = 150 μm. l Confocal z-stack images of the middle sections of CD44 WT or KO CT26-GFP spheroids after 18 h incubation with Cy5.5-labeled PCL-CP NPs. Bar=200 μm. m Proposed role of the CD44 in tumor ECs and tumor cells in mediating tumor targeting and penetration of CS-coated NPs. The figure is created with BioRender.com. Data are presented as mean ± s.e.m. in ( b , d , g , h , i , j ). Statistical analysis was performed by one-way ANOVA with Tukey’s post hoc test for comparison in ( b , d , i , j ). Data are representative of two independent experiments in ( a – e , k , l ) and three independent experiments in ( g – j ). Source data are provided as a file for ( b , d , e , g , h , i , j ).

Article Snippet: CT26 murine CRC cell lines (CRL-2638), HCT116 human CRC cell line (CCL-247), 4T1 murine BC cell line (CRL-2539) and MDA-MB-468 human BC cell line (HTB-132) were obtained from ATCC (Manassas, VA).

Techniques: Imaging, Ex Vivo, Fluorescence, Staining, Labeling, Transwell Assay, Transmigration Assay, Incubation, Comparison

Journal: Nature Communications

Article Title: Inhibition of iRhom1 by CD44-targeting nanocarrier for improved cancer immunochemotherapy

doi: 10.1038/s41467-023-44572-6

Figure Lengend Snippet: a Cellular uptake of Cy5.5-PCL NPs with or without CS coating in various WT or iRhom1 KO cell lines. n = 3 independent samples. b Transwell assay of transmigration of Cy5.5-PCL NPs with or without CS coating across WT or iRhom1 KO CT26 cells. n = 3 independent samples. c NIRF whole body imaging of WT or iRhom1 KO tumor-bearing mice following i.v. administration of DOX-loaded, Cy5.5-PCL-CP NPs and d the corresponding quantification. n = 7 animals. e Quantitative analysis of biodistribution of DOX in WT or iRhom1 KO tumor-bearing mice (WT). n = 3 independent samples. f CD44 expression levels measured by flow cytometry by staining with an antibody recognizing the CD44 external region. n = 3 independent samples. g Fluorescence images of CD44 external region expression in WT or iRhom1 KO CT26 cells. Bar = 20 μm. h Changes in protein levels of full-length CD44 (CD44-Full) and the cleaved, CD44 membrane-bound fragment (CD44-EXT) in WT or iRhom1 knockout/knockdown cells, with or without ADAM17 inhibitor pretratment. i Changes in protein levels of CD44-Full and the CD44-EXT in WT, iRhom1 knockout and iRhom1 rescue cells, with or without ADAM17 inhibitor treatment. j Cellular uptake of Cy5.5 labeled PCL-CS NPs in WT or iRhom1 knockout/knockdown cells with or without ADAM17 inhibitor pretreatment. n = 3 independent samples. k Cellular uptake of Cy5.5 labeled PCL-CS NPs in WT, iRhom1 knockout/knockdown or iRhom1 rescue cells with or without ADAM17 inhibitor pretreatment. n = 3 independent samples. Data are presented as mean ± s.e.m. in ( a , b , d , e , f , j , k ). Statistical analysis was performed by one-way ANOVA with Tukey’s post hoc test for comparison in ( a , j , k ). and two-tailed Student’s t-test for comparison in ( b , d , e , f ). Data are representative of two independent experiments in ( c – e , g – i ) and three independent experiments in ( a , b , f , j , k ). Source data are provided as a file for ( a , b , d , e , f , h , i , j , k ).

Article Snippet: CT26 murine CRC cell lines (CRL-2638), HCT116 human CRC cell line (CCL-247), 4T1 murine BC cell line (CRL-2539) and MDA-MB-468 human BC cell line (HTB-132) were obtained from ATCC (Manassas, VA).

Techniques: Transwell Assay, Transmigration Assay, Imaging, Expressing, Flow Cytometry, Staining, Fluorescence, Membrane, Knock-Out, Knockdown, Labeling, Comparison, Two Tailed Test

Journal: Nature Communications

Article Title: Inhibition of iRhom1 by CD44-targeting nanocarrier for improved cancer immunochemotherapy

doi: 10.1038/s41467-023-44572-6

Figure Lengend Snippet: Changes in tumor volumes ( a ) and body weights ( b ) in mice bearing 4T1 orthotopic tumors receiving various treatments. n = 5 animals. c Representative flow cytometric analysis and the quantification of the relative abundance of CD8 + T-cells, CD4 + T-cells, CD8 + IFNγ + T-cells, CD8 + Granzyme B + T-cells and CD8 + PD1 + T-cells in 4T1 tumor tissues after various treatments. n = 8 biologically independent samples. Changes in tumor volumes ( d ) and body weights ( e ) in mice bearing CT26 tumors receiving various treatments. n = 5 animals. f Flow cytometric analysis and the quantification of the relative abundance of CD8 + T-cells, CD4 + T-cells, CD8 + IFNγ + T-cells, CD8 + Granzyme B + T-cells and CD8 + PD1 + T-cells in CT26 tumor tissues after various treatments. n = 8 biologically independent samples. g Histogram and the quantitative analysis of MHCI expression in CT26 tumor tissues after various treatments. n = 3 biologically independent samples. h Tumor growth inhibition and survival of CT26 tumor-bearing mice receiving various treatments. n = 8 animals. Data are presented as mean ± s.e.m. in all panels. Statistical analysis was performed by one-way ANOVA with Tukey’s post hoc test for comparison in ( a , b , c , d , e , f , g , h (left)) and log rank test for comparison in ( h ) (right). Data are representative of two independent experiments in ( a – h ). Source data are provided as a file for ( a – h ).

Article Snippet: CT26 murine CRC cell lines (CRL-2638), HCT116 human CRC cell line (CCL-247), 4T1 murine BC cell line (CRL-2539) and MDA-MB-468 human BC cell line (HTB-132) were obtained from ATCC (Manassas, VA).

Techniques: Expressing, Inhibition, Comparison

Journal: Drug Delivery and Translational Research

Article Title: Current status and future of delivery systems for prevention and treatment of infections in the oral cavity

doi: 10.1007/s13346-021-00961-2

Figure Lengend Snippet: Drug delivery systems for treatment of fungal infection-related conditions in the oral cavity

Article Snippet: Propolis , Periodontal pocket , Magnetic nanoparticle in liquid crystalline , Nanoparticle: Iron oxide Liquid crystal: Isopropyl myristate, polyoxyethylene oleyl ether , - Drug release Periodontal pocket simulator apparatus with a flow system - Antifungal activity broth macrodilution test - Cytotoxicity fibroblasts cell line (ATCC CCL-1.3) , - , - Prolonged drug release - Fungicide activity against Candida spp. - Very low cytotoxicity , [ ] .

Techniques: Infection, In Vitro, In Vivo, Diffusion-based Assay, Activity Assay, Modification, Ex Vivo, Clinical Proteomics, In Situ, Stripping Membranes, MTT Assay, Membrane, Concentration Assay, Calcium Carbonate, Histopathology, Maltodextrin, Dissolution, Inhibition, Formulation