Journal: Neurobiology of aging

Article Title: Prostate stem cell antigen interacts with nicotinic acetylcholine receptors and is affected in Alzheimer's disease.

doi: 10.1016/j.neurobiolaging.2015.01.001

Figure Lengend Snippet: Fig. 1. Prostate stem cell antigen (PSCA) is soluble and co-purifies with the a4 nAChR subunit in human cortex. (A) The antiserum directed against PSCA recognizes the human recombinant GST-tagged PSCA protein (total molecular weight 34.2 kDa) from 1 to 16 ng/well at the expected band size. Endogenous PSCA from mouse and human cortical tissue (8 mg total protein/well) is detected at approximately 24 kDa, and the amount in the cortical samples was compared with the known concentrations of re- combinant PSCA protein. (B) Images of Western blot showing PSCA protein levels in cortical tissue from human (protein concentration 4, 8, and 12 mg/well) and mouse (8, 15, and 30 mg/well) in the absence (peptide) and the presence of PSCA peptide (þpeptide). (C) Representative images of Western blots showing PSCA and Lypd6 protein levels in soluble and membrane fractions of human temporal cortical tissue. The membrane receptor proteins b2 nAChR and GluR2 are used as control. (D) Mag- netic beads covalently coupled with PSCA recombinant protein were incubated with cortical homogenates from human temporal cortex followed by detection of nAChR subunits by Western blot. Homogenates before (input) and after affinity purification (output) as well as the negative control (CTRL) were loaded.

Article Snippet: Validation of PSCA antibody (1:1,000, Novus Biologicals) was done with co-incubation with the PSCA peptide, which had been used as immunogen (1:100, # NB100-91938PEP, Novus Biologicals).

Techniques: Recombinant, Molecular Weight, Western Blot, Protein Concentration, Membrane, Control, Incubation, Negative Control

Journal: Cancer Immunology Research

Article Title: Secreted Fas Decoys Enhance the Antitumor Activity of Engineered and Bystander T Cells in Fas Ligand–Expressing Solid Tumors

doi: 10.1158/2326-6066.cir-22-0115

Figure Lengend Snippet: Figure 1. CAR T cells are susceptible to suppression by tumor-expressed FasL. T cells from healthy donor PBMCs were transduced with SFG-based retrovirus engineered to express a second-generation CAR specific for human PSCA. A, Transduction efficiency was determined by flow cytometry on day 5 after transduction. Shown in the histogram are data representative of a single donor and in the bar graph are summary data (mean SEM, n ¼ 8). Statistical significance was calculated using an unpaired t test. B, Cytolytic activity of nontransduced control T cells (NT) and CAR PSCA T cells (CAR) was tested in a 51Cr-release assay using 5103 PSCA– 293T cells and PSCAþ CAPAN-1 and CFPAC-1 pancreatic cancer cells (mean SEM, n ¼ 3). Statistical difference was calculated using an unpaired t test. C and D, Expression of Fas (CD95) on T cells at baseline, upon activation, and after CAR transduction was determined by flow cytometry. Expression by T cells from a representative donor (C) and summary data (mean SEM, n ¼ 5; D). Significance was determined using one-way ANOVA. E, The viability of CAR PSCA T cells was determined by flow cytometry using Annexin V and 7-AAD staining after exposure to the indicated concentrations of recombinant FasL. The % viable cells (Annexin V–7-AAD–) under each condition is shown (mean SEM, n ¼ 3). Significance was determined using one-way ANOVA. F, FasL expression on CAPAN-1 and CFPAC-1 pancreatic cancer cell lines was assessed by flow cytometry during normal culture conditions and following 24-hour exposure to IFNg. Shown in the histograms are data representative of 3 independent analyses. G, CAPAN-1 tumor cells engineered to express PSCA-GFP were cultured with CAR PSCA T cells in the absence or presence of a Fas- blocking antibody(ZB4) before analysisby flow cytometry. Shown in the FACS plots are data representativeof a single sample and in the bar graph are summary data (mean SEM, n ¼ 4). Significance was determined using one-way ANOVA. H, Illustration demonstrating the susceptibility of CAR T cells to suppression by solid tumors via the Fas/FasL signaling axis. P > 0.05 ¼ nonsignificant (ns); , P ≤0.05; , P ≤0.01; , P ≤0.001, as indicated in the figure panels.

Article Snippet: Briefly, supernatant samples harvested from T cells activated in 24-well non-TC plates coated with recombinant PSCA antigen (CUSABIO; catalog no. CSB-YP018840HU) at indicated concentrations or irradiated K562-PSCA cells at indicated E:T. To measure the production of FDþ15 fusionmolecule, samples were used in soluble Fas and IL15 ELISAs (R&D Systems, catalog no. DY247) to measure the two components separately.

Techniques: Transduction, Cytometry, Activity Assay, Control, Release Assay, Expressing, Activation Assay, Staining, Recombinant, Cell Culture, Blocking Assay

Journal: Cancer Immunology Research

Article Title: Secreted Fas Decoys Enhance the Antitumor Activity of Engineered and Bystander T Cells in Fas Ligand–Expressing Solid Tumors

doi: 10.1158/2326-6066.cir-22-0115

Figure Lengend Snippet: Figure 2. Engineering a soluble Fas decoy receptor to sequester FasLCAR PSCA T cells were engineered to express Fas decoy (FD) by retroviral transduction and used for functional assessments. A, Diagram illustrating FD secreted by CAR T cells enhancing T-cell antitumor activity by blocking inhibitory FasL signaling. B, FD construct schematic is shown on top and CAR and FD coexpression in T cells from a representative donor assessed by flow cytometry on day 5 after transduction (density plots). C, Summary transduction data indicating expression of CAR PSCA and CAR.FD in T cells (mean SEM, n ¼ 8). D, Phenotype of CAR PSCA and CAR.FD T cells was compared 10 days after transduction by flow cytometry (mean SEM, n ¼ 3). No significant difference was observed between CAR PSCA and CAR.FD based on unpaired t test. E, Expansion of CAR PSCA and CAR.FD T cells stimulated with irradiated K562-PSCA tumor cells was monitored for six days using a flow cytometer (mean SEM, n ¼ 4). No significant difference was observed between CAR PSCA and CAR.FD based on an unpaired t test on days 3 and 6. F, Secretion of effector cytokines IFNg) and TNFa by CAR PSCA and CAR.FD T cells after 48-hour culture with K562 or K562-PSCA cells was measured using a multiplex cytokine assay (mean SEM, n ¼ 3). No significant difference was observed between CAR PSCA and CAR.FD based on an unpaired t test. G, Cytolytic function of CAR PSCA and CAR.FD T cells was compared in a chromium release assay using 293T, CAPAN-1, and CFPAC-1 cells as targets at indicated E:T (mean SEM, n ¼ 3). Unpaired t test indicated no significant difference between CAR PSCA and CAR.FD in tumor killing. H, FD secretion by CAR PSCA and CAR.FD T cells in the absence or presence of recombinant PSCA stimulation was measured in the supernatant by soluble Fas ELISA 48 hours after culture initiation (mean SEM, n ¼ 4). Statistical significance was calculated using an unpaired t test. P > 0.05 ¼ nonsignificant (ns); , P ≤0.05; , P ≤0.01; , P ≤0.001.

Article Snippet: Briefly, supernatant samples harvested from T cells activated in 24-well non-TC plates coated with recombinant PSCA antigen (CUSABIO; catalog no. CSB-YP018840HU) at indicated concentrations or irradiated K562-PSCA cells at indicated E:T. To measure the production of FDþ15 fusionmolecule, samples were used in soluble Fas and IL15 ELISAs (R&D Systems, catalog no. DY247) to measure the two components separately.

Techniques: Retroviral, Transduction, Functional Assay, Activity Assay, Blocking Assay, Construct, Cytometry, Expressing, Irradiation, Multiplex Assay, Cytokine Assay, Release Assay, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: Cancer Immunology Research

Article Title: Secreted Fas Decoys Enhance the Antitumor Activity of Engineered and Bystander T Cells in Fas Ligand–Expressing Solid Tumors

doi: 10.1158/2326-6066.cir-22-0115

Figure Lengend Snippet: Figure 3. FD-engineered T cells exhibit superior function in the presence of FasL. FasL-neutralizing and T-cell function–enhancing properties of FD were assessed using in vitro immunoassays. A, FasL neutralization by FD was measured by incubating recombinant human FasL with fresh media, conditioned media obtained from 233T cells, or conditioned media obtained from 293T-FD cells and subsequently used in a human FasL ELISA (mean SEM, n ¼ 4). Statistical significance was calculated using one- way ANOVA. B and C, Suppression of FasL-mediated T-cell apoptosis by FD was assessed by measuring the viability of CAR PCSA T cells in different culture conditions—no FD or FasL [Control sup. (no FasL)]; no FD, in the presence of recombinant FasL [Control sup. (þ FasL)]; no FD in the presence of recombinant FasL and FasL-blocking antibody NOK-2 [Control sup. (þ FasL, þ NOK-2)]; recombinant FasL in the presence of FD [FD sup. (þ FasL)]. T-cell viability was measured by flow cytometry by Annexin V and 7-AAD staining after overnight culture. Flow cytometry plots demonstrating the viability of CAR PSCA T cells for a representative donor (B) and summary data (mean SEM, n ¼ 3) (C). Statistical significance was determined using one-way ANOVA. D and E, Antitumor activity of CAR PSCA and CAR.FD T cells was measured against CAPAN-1–PSCA tumor cells in a coculture assay (E:T of 1:10) and tumor as well as T-cell numbers were quantified by flow cytometry. Starting cell numbers (day 0) were used as reference to calculate fold change in cell numbers for all time points. Line graphs demonstrating fold change in tumor cell numbers (D) and fold change in T-cell numbers (mean SEM, n ¼ 4; E). Statistical significance was calculated using an unpaired t test. F, Pie chart depicting the percentage of polyfunctional (defined as cells producing 2 or more cytokines) CAR PSCA and CAR.FD T cells for a representative donor after 5-hour stimulation with CAPAN-1-PSCA cells (E:T of 1:4). Stimulated T cells were analyzed using IsoPlexis single-cell cytokine assay after stimulation. Numbers next to the pie charts indicate the number of cytokines produced by each fraction of T cells specified in the pie charts. Data representative of three independent donors. P > 0.05, nonsignificant (ns); , P ≤0.05; , P ≤0.01; , P ≤0.001.

Article Snippet: Briefly, supernatant samples harvested from T cells activated in 24-well non-TC plates coated with recombinant PSCA antigen (CUSABIO; catalog no. CSB-YP018840HU) at indicated concentrations or irradiated K562-PSCA cells at indicated E:T. To measure the production of FDþ15 fusionmolecule, samples were used in soluble Fas and IL15 ELISAs (R&D Systems, catalog no. DY247) to measure the two components separately.

Techniques: Cell Function Assay, In Vitro, Neutralization, Recombinant, Enzyme-linked Immunosorbent Assay, Control, Blocking Assay, Cytometry, Staining, Flow Cytometry, Activity Assay, Co-culture Assay, Cytokine Assay, Produced

Journal: Cancer Immunology Research

Article Title: Secreted Fas Decoys Enhance the Antitumor Activity of Engineered and Bystander T Cells in Fas Ligand–Expressing Solid Tumors

doi: 10.1158/2326-6066.cir-22-0115

Figure Lengend Snippet: Figure 4. FD and IL15 fusion protein (FDþ15) further enhance T-cell activity. T cells were retrovirally transduced to coexpress CAR PSCA and FDþ15 and then used in experiments to assess their function. A, Schematic diagram illustrating the FD and IL15 fusion (FDþ15) transgene. B and C, T-cell transduction to assess the expression of CAR PSCA, CAR PSCA, and FD, or CAR PSCA and FDþ15 was measured by flow cytometry 5 days after transduction. Flow cytometry density plots for a representative donor (B) and summary data indicating expression of the CAR PSCA, CAR.FD, and CAR.FDþ15 transgenes (mean SEM, n ¼ 8; C). D, Secretion of FDþ15 molecule by activated T cells was measured using Fas and IL15 ELISAs to separately measure the two components of the fusion molecule. Bar graphs demonstrate the concentration of Fas and IL15 in supernatant obtained from CAR.FDþ15 T cells cultured in the absence or presence of recombinant PSCA (mean SEM, n ¼ 4). Significance was calculated by unpaired t tests. E, Expansion of CAR PSCA, CAR.FD, and CAR.FDþ15 T cells stimulated with irradiated K562-PSCA tumor cells was monitored for 6 days using a flow cytometer (mean SEM, n ¼ 4). Statistical significance was calculated using one-way ANOVA. F and G, Antitumor activity of CAR PSCA, CAR.FD, and CAR.FDþ15 T cells was measured against CAPAN-1-PSCA tumor cells in a coculture assay (E:T of 1:10). Tumor as well as T-cell numbers were quantified by flow cytometry. Starting cell numbers (day 0) were used as a reference to calculate fold change in cell numbers for all time points. Graphs illustrate fold change in tumor cell numbers (F) and T-cell numbers (G) over time (mean SEM, n ¼ 4). Statistical significance was calculated using one-way ANOVA. P > 0.05 ¼ nonsignificant (ns); , P ≤0.05; , P ≤0.01; , P ≤0.001.

Article Snippet: Briefly, supernatant samples harvested from T cells activated in 24-well non-TC plates coated with recombinant PSCA antigen (CUSABIO; catalog no. CSB-YP018840HU) at indicated concentrations or irradiated K562-PSCA cells at indicated E:T. To measure the production of FDþ15 fusionmolecule, samples were used in soluble Fas and IL15 ELISAs (R&D Systems, catalog no. DY247) to measure the two components separately.

Techniques: Activity Assay, Transduction, Expressing, Cytometry, Flow Cytometry, Concentration Assay, Cell Culture, Recombinant, Irradiation, Co-culture Assay

Journal: Cancer Immunology Research

Article Title: Secreted Fas Decoys Enhance the Antitumor Activity of Engineered and Bystander T Cells in Fas Ligand–Expressing Solid Tumors

doi: 10.1158/2326-6066.cir-22-0115

Figure Lengend Snippet: Figure 5. Decoy-engineered CAR T cells demonstrate enhanced in vivo antitumor activity. In vivo function of decoy-engineered T cells was performed using the CAPAN-1 xenograft model in NSG mice. A, Schematic representation of the in vivo model for the assessment of the decoy-engineered T cells. 5 106 CAPAN-1-PSCA tumor cells were engrafted subcutaneously in NSG mice, which were subsequently (approximately 4 weeks after tumor implantation) treated with i.v. injection of T cells. T cells used in the study were labeled with firefly luciferase to facilitate in vivo imaging. B, Change in body weight was used as an indicator of symptoms of toxicity associated with T-cell treatment and was monitored in all mice used in the study (mean SEM, n ¼ 7 per group). No statistical differences were observed between different treatment groups according to two-way ANOVA. C, Representative mice images (3 mice shown) demonstrating localization and expansion of the nontransduced (NT), CAR PSCA, CAR.FD, CAR.FDþ15, and CAR.FDþ15 cells by bioluminescence imaging. D and E, Tumor volumes were measured using calipers once a week for all mice in the study and reported as measurements for individual animals (D) and summarized data for each treatment group (mean SEM, n ¼ 7; E). F, T-cell expansion and persistence at the tumor were monitored by weekly bioluminescence imaging. Summary of T-cell luminescence at the tumor is demonstrated for each treatment group (mean SEM, n ¼ 7). Statistical significance between different treatment groups was calculated using two- way ANOVA. P > 0.05 ¼ nonsignificant (ns); , P ≤0.05; , P ≤0.01; , P ≤0.001.

Article Snippet: Briefly, supernatant samples harvested from T cells activated in 24-well non-TC plates coated with recombinant PSCA antigen (CUSABIO; catalog no. CSB-YP018840HU) at indicated concentrations or irradiated K562-PSCA cells at indicated E:T. To measure the production of FDþ15 fusionmolecule, samples were used in soluble Fas and IL15 ELISAs (R&D Systems, catalog no. DY247) to measure the two components separately.

Techniques: In Vivo, Activity Assay, Tumor Implantation, Injection, Labeling, Luciferase, In Vivo Imaging, Imaging

Journal: Cancer Immunology Research

Article Title: Secreted Fas Decoys Enhance the Antitumor Activity of Engineered and Bystander T Cells in Fas Ligand–Expressing Solid Tumors

doi: 10.1158/2326-6066.cir-22-0115

Figure Lengend Snippet: Figure 6. Decoy-secreting cells promote the effector activity of bystander T cells. Bystander effects of decoy-engineered T cells were assessed in a CFPAC-1 xenograft tumor model in NSG mice using CAR MUC1 T cells as bystander cells. A, Illustration of the in vivo model for the assessment of bystander effects of decoy-engineered T cells. NSG mice were engrafted subcutaneously with CFPAC-1 tumor cells and approximately 4 weeks after tumor inoculation, were treated with an i.v. injection of T cells. To facilitate in vivo tracking of both T-cell components administered to mice, CAR MUC1 cells were labeled with CBG99, and CAR PSCA, CAR.FD, and CAR.FDþ15 cells were labeled with Akaluc luciferases, respectively. B, Bioluminescence images of two representative mice per treatment group demonstrating localization and expansion of indicated T cells by bioluminescence imaging. Bystander CAR MUC1 T cells are depicted on the left and CAR PSCA, CAR.FD, and CAR.FDþ15 cells on the right. C, Tumor volumes were measured using calipers once a week for all mice in the study and illustrated in the line graphs for individual animals. D, Summary of CAR MUC1 T-cell luminescence indicating expansion and persistence of these cells in mice treated with CAR MUC1þCAR PSCA, CAR MUC1þCAR.FD, or CAR MUC1þCAR.FDþ15 T cells (mean SEM, n ¼ 7). E, Summary tumor volume for all mice in each treatment group illustrated in 6C (mean SEM, n ¼ 7). Statistical significance in tumor volumes between different treatment groups was calculated using two-way ANOVA. P > 0.05 ¼ nonsignificant (ns); , P ≤0.05; , P ≤0.01; , P ≤0.001.

Article Snippet: Briefly, supernatant samples harvested from T cells activated in 24-well non-TC plates coated with recombinant PSCA antigen (CUSABIO; catalog no. CSB-YP018840HU) at indicated concentrations or irradiated K562-PSCA cells at indicated E:T. To measure the production of FDþ15 fusionmolecule, samples were used in soluble Fas and IL15 ELISAs (R&D Systems, catalog no. DY247) to measure the two components separately.

Techniques: Activity Assay, In Vivo, Injection, Labeling, Imaging

Journal: Oncology letters

Article Title: Correlation between prostate stem cell antigen gene expression and oral squamous cell carcinoma.

doi: 10.3892/ol.2018.8468

Figure Lengend Snippet: Figure 1. The rs2294008 polymorphism in PSCA. OSCC, oral squamous cell carcinoma; PSCA, prostate stem cell antigen.

Article Snippet: Human Prostate Stem Cell Antigen (PSCA) ELISA kit (cat no. CSB-EL018840HU) was purchased from CUSABIO BIOTECH (Stratech, Scientific, Ltd., UK).

Techniques:

Journal: Oncology letters

Article Title: Correlation between prostate stem cell antigen gene expression and oral squamous cell carcinoma.

doi: 10.3892/ol.2018.8468

Figure Lengend Snippet: Figure 2. PSCA expression pattern in eight cancer and two normal cell lines. OSCC, oral squamous cell carcinoma; PSCA, prostate stem cell antigen.

Article Snippet: Human Prostate Stem Cell Antigen (PSCA) ELISA kit (cat no. CSB-EL018840HU) was purchased from CUSABIO BIOTECH (Stratech, Scientific, Ltd., UK).

Techniques: Expressing

Journal: Oncology letters

Article Title: Correlation between prostate stem cell antigen gene expression and oral squamous cell carcinoma.

doi: 10.3892/ol.2018.8468

Figure Lengend Snippet: Figure 3. PSCA expression in OSCC tissues. (A and B) Representative images indicating PSCA expression in tissues from patients with OSCC and PSCA‑positive serum (IRS=6; A, 100x; B, 200x). (C and D) PSCA expression in tissues from patients with OSCC and PSCA‑negative serum (IRS=2; C, x100; D, x200). OSCC, oral squamous cell carcinoma; PSCA, prostate stem cell antigen.

Article Snippet: Human Prostate Stem Cell Antigen (PSCA) ELISA kit (cat no. CSB-EL018840HU) was purchased from CUSABIO BIOTECH (Stratech, Scientific, Ltd., UK).

Techniques: Expressing

Journal: Oncology letters

Article Title: Correlation between prostate stem cell antigen gene expression and oral squamous cell carcinoma.

doi: 10.3892/ol.2018.8468

Figure Lengend Snippet: Figure 4. PSCA expression in normal epithelium. Representative images from normal tissue with an IRS=0 (A, x100; B, x200). OSCC, oral squamous cell carcinoma; PSCA, prostate stem cell antigen.

Article Snippet: Human Prostate Stem Cell Antigen (PSCA) ELISA kit (cat no. CSB-EL018840HU) was purchased from CUSABIO BIOTECH (Stratech, Scientific, Ltd., UK).

Techniques: Expressing

Journal: Oncology letters

Article Title: Correlation between prostate stem cell antigen gene expression and oral squamous cell carcinoma.

doi: 10.3892/ol.2018.8468

Figure Lengend Snippet: Figure 5. PSCA expression in OSCC epithelium with different genotypes. (A and B) Representative images indicating PSCA expression in tissues from OSCC patients presenting the CC genotype (A, x100; B, x200). (C and D) Representative images indicating PSCA expression in OSCC patients presenting the CT genotype (C, x100; D, x200). Representative images indicating PSCA expression in tissues from OSCC patients presenting the TT genotype (E, x100; F, x200). OSCC, oral squamous cell carcinoma; PSCA, prostate stem cell antigen.

Article Snippet: Human Prostate Stem Cell Antigen (PSCA) ELISA kit (cat no. CSB-EL018840HU) was purchased from CUSABIO BIOTECH (Stratech, Scientific, Ltd., UK).

Techniques: Expressing