Journal: Clinical Cancer Research

Article Title: Genetically Targeted T Cells Eradicate Established Breast Cancer in Syngeneic Mice

doi: 10.1158/1078-0432.ccr-08-2381

Figure Lengend Snippet: Fig. 1. Expression of the scFv-CD28-~ chimeric antigen receptor in transduced mouseT lymphocytes. A, schematic representation of the scFv-CD28-~ chimeric antigen receptor.The scFv-CD28-~ chimeric antigen receptor consisted of the anti-erbB2 scFv, c-myc tag, part of the extracellular membrane, transmembrane, and cytoplasmic regions of the mouse CD28 signaling chain fused to the cytoplasmic region of CD3~ chain. B and C, the enriched mouseTcells (1 l06/mL) were stimulated in 24-well plates with 2 Ag/mL immobilized CD3 and 0.1 Ag/mL soluble CD28 mAbs for 48 h, followed by infection with high titer retrovirus expressing scFv-CD28-~ chimeric antigen receptor or control.The transduced cells were stained with APC-labeled anti-CD4, fluorescein isothiocyanate ^ labeled anti-CD8, and phycoerythrin-labeled anti ^ c-myc mAbs before flow cytometry analysis.The percentage of CD4+ or CD8+ Tcells in transduced cells or the surface expression of the scFv-CD28-~ chimeric antigen receptor in transduced cells is in the quadrants.

Article Snippet: In the assay for the blocking effect of soluble erbB2, RPMI-1640 with recombinant erbB2 protein (2 Ag/mL; R&D Systems) was used as the culture medium.

Techniques: Expressing, Membrane, Infection, Control, Staining, Labeling, Flow Cytometry

Journal: Journal of Advanced Research

Article Title: Rapid generation of a mouse model for evaluating on-target normal tissue toxicity of human CAR-T cells using replication-defective recombinant adenovirus

doi: 10.1016/j.jare.2022.08.008

Figure Lengend Snippet: The expression of human HER2 in murine liver by recombinant adenovirus delivery. ( A ) Design of the adenovirus vector carrying a truncated human HER2/ERBB2 ( tHER2 ) gene and a firefly luciferase gene, whose expression are driven by the CMV and SV40 promoter, respectively. ( B ) In vitro testing luciferase and HER2/ERBB2 expression in B16 cell (the murine melanoma cell line). A two-tailed unpaired t -test of luciferase and HER2/ERBB2 level was used for statistical analysis. ( C ) In vivo imaging of luciferase expression in mice injected with 0, 2.5E + 8, 5E + 8 and 1E + 9 PFU of Ad5-HER2 8 days post-infection (8dpi). ( D ) IHC staining of liver section from mice that received different dosage of Ad5-HER2 for human HER2 (brown). ( E ) Quantification of HER2 in the indicated liver tissue. Data presented as mean ± SEM, each group contained 4 mice. A one-way ANOVA with Dunnett's multiple comparisons test was used to test statistical significance of different groups. Statistical significance is denoted as * P ＜0.05, ** P ＜0.01, *** P ＜0.001, **** P ＜0.0001, ns not significant.

Article Snippet: Briefly, the slides were stained with anti-human HER2/ERBB2 (Cat #: 10004-T56, SinoBiological) or anti-human CD47 (ab218810, Abcam) for immunohistochemistry (IHC), and immunostained with anti-human CD3 (Cat #: ab11089, Abcam), anti-FLAG tag (Cat #: 637301, Biolegend) antibodies and DAPI for immunofluorescence, which performed by the immunoassay platform at the Shanghai Institute for Emerging and Re-emerging Infectious Diseases of SPHCC.

Techniques: Expressing, Recombinant, Plasmid Preparation, Luciferase, In Vitro, Two Tailed Test, In Vivo Imaging, Injection, Infection, Immunohistochemistry

Journal: Journal of Advanced Research

Article Title: Rapid generation of a mouse model for evaluating on-target normal tissue toxicity of human CAR-T cells using replication-defective recombinant adenovirus

doi: 10.1016/j.jare.2022.08.008

Figure Lengend Snippet: CAR-T cells are activated by human antigen and accumulated in murine liver. Human HER2 was expressed in murine liver following 0, 2.5E + 8, 5E + 8 and 1E + 9 PFU of Ad5-HER2 adenovirus delivery. Mice were then received either 5E + 6 HER2 CAR-T cells or UTD. ( A ) Systemic cytokine secretion by T cells was determined in murine serum (n = 3–4) by cytometric beads array 5 days after T cells transfer. ( B ) Frequency of HER2 CAR-T cells (up panel) and HER2 CAR-T cell counts (down panel) among live nucleated cells in the indicated tissues 7 days after T cells infusion. A one-way ANOVA with Dunnett's multiple comparisons test was used to test statistical significance of different groups. Statistical significance is denoted as * P ＜0.05, ** P ＜0.01, ns not significant.

Article Snippet: Briefly, the slides were stained with anti-human HER2/ERBB2 (Cat #: 10004-T56, SinoBiological) or anti-human CD47 (ab218810, Abcam) for immunohistochemistry (IHC), and immunostained with anti-human CD3 (Cat #: ab11089, Abcam), anti-FLAG tag (Cat #: 637301, Biolegend) antibodies and DAPI for immunofluorescence, which performed by the immunoassay platform at the Shanghai Institute for Emerging and Re-emerging Infectious Diseases of SPHCC.

Techniques:

Journal: Journal of Advanced Research

Article Title: Rapid generation of a mouse model for evaluating on-target normal tissue toxicity of human CAR-T cells using replication-defective recombinant adenovirus

doi: 10.1016/j.jare.2022.08.008

Figure Lengend Snippet: HER2 CAR-T cells cause acute on-target liver toxicity in mice. ( A ) Flow chart of the experimental design for evaluating on-target, liver injury of CAR-T cells. NSG mice received 0, 2.5E + 8, 5E + 8 and 1E + 9 PFU of Ad5-HER2 and then infused with either 5E + 6 HER2 CAR-T cells or untransduced T cells (UTD) 8dpi. An n = 3–4 mice per group is shown in each panel unless stated otherwise. ( B ) Weight change shown by percent change from initial weight ± SEM in mice that received different dosage of Ad5-HER2 and then either HER2 CAR-T cells or UTD. ( C ) Survival curves of NSG mice that received different dosage of Ad5-HER2 and then T cells transfer. Statistical analysis was performed using a log-rank Mantel-Cox test. ( D ) Liver function profile as determined by serum ALT and AST levels collected days as indicated after T cells infusion. Mean ALT/AST ± SEM in mice received different dosage of Ad5-HER2 and then T cells injection. A one-way ANOVA with Dunnett's multiple comparisons test was used to test statistical significance of different groups. Statistical significance is denoted as * P ＜0.05, ** P ＜0.01, *** P ＜0.001, **** P ＜0.0001, ns not significant.

Article Snippet: Briefly, the slides were stained with anti-human HER2/ERBB2 (Cat #: 10004-T56, SinoBiological) or anti-human CD47 (ab218810, Abcam) for immunohistochemistry (IHC), and immunostained with anti-human CD3 (Cat #: ab11089, Abcam), anti-FLAG tag (Cat #: 637301, Biolegend) antibodies and DAPI for immunofluorescence, which performed by the immunoassay platform at the Shanghai Institute for Emerging and Re-emerging Infectious Diseases of SPHCC.

Techniques: Injection

Journal: Journal of Advanced Research

Article Title: Rapid generation of a mouse model for evaluating on-target normal tissue toxicity of human CAR-T cells using replication-defective recombinant adenovirus

doi: 10.1016/j.jare.2022.08.008

Figure Lengend Snippet: Hypoxia-response CAR-T cells don’t cause acute on-target liver toxicity in mice expressing high levels of human antigen. ( A ) Flow chart of the experimental design for evaluating on-target, liver injury of CAR-T cells. NSG mice received 1E + 9 PFU of Ad5-HER2 and then infused with either 5E + 6 HER2 CAR-T cells or hypoxia-response CAR-T cells. A human antigen negative control group of NSG mice received 5E + 6 HER2 CAR-T cells but no Ad5-HER2 (i.e. Ad-). An n = 3–4 mice per group is shown in each panel unless stated otherwise. ( B ) Weight change shown by percent change from initial weight ± SEM in mice that received 1E + 9 PFU of Ad5-HER2 and then either CAR-T cells or hypoxia-response CAR-T cells. ( C ) Survival curves of NSG mice that received 1E + 9 PFU of Ad5-HER2 and then T cells transfer. Statistical analysis was performed using a log-rank Mantel-Cox test. ( D ) Liver function profile as determined by serum ALT levels collected days as indicated after T cells infusion. Mean ALT ± SEM in mice (n = 3–4) received 1E + 9 PFU of Ad5-HER2 and then T cells injection. A one-way ANOVA with Dunnett's multiple comparisons test was used to test statistical significance between UTD group and other groups. Statistical significance is denoted as * P ＜0.05, ** P ＜0.01, *** P ＜0.001, **** P ＜0.0001, ns not significant.

Article Snippet: Briefly, the slides were stained with anti-human HER2/ERBB2 (Cat #: 10004-T56, SinoBiological) or anti-human CD47 (ab218810, Abcam) for immunohistochemistry (IHC), and immunostained with anti-human CD3 (Cat #: ab11089, Abcam), anti-FLAG tag (Cat #: 637301, Biolegend) antibodies and DAPI for immunofluorescence, which performed by the immunoassay platform at the Shanghai Institute for Emerging and Re-emerging Infectious Diseases of SPHCC.

Techniques: Expressing, Negative Control, Injection

Journal: Journal of Advanced Research

Article Title: Rapid generation of a mouse model for evaluating on-target normal tissue toxicity of human CAR-T cells using replication-defective recombinant adenovirus

doi: 10.1016/j.jare.2022.08.008

Figure Lengend Snippet: Hypoxia-response CAR-T cells are not activated and accumulated by human antigen in murine liver. Human HER2 was expressed in murine liver following 1E + 9 PFU of Ad5-HER2 delivery. Mice were then received 5E + 6 HER2 CAR-T cells or hypoxia-response HER2 CAR-T cells. Control groups included mice that had hepatic HER2 expression but untransduced T cells (UTD) and mice that received HER2 CAR-T cells but lacked hepatic HER2 expression (i.e. Ad-). ( A ) Systemic cytokine secretion by T cells was determined in murine serum (n = 4–13) by cytometric beads array 7 days after T cells transfer. ( B ) Quantitation of inflammatory infiltration lesions in the liver 7 days after T cells transfer. ( C ) Frequency of CAR-T cells (up panel) and CAR-T cell counts (down panel) among live nucleated cells in the indicated tissues 7 days after T cells infusion. A one-way ANOVA with Dunnett's multiple comparisons test was used to test statistical significance of different groups. Statistical significance is denoted as * P ＜0.05, ** P ＜0.01, ns not significant.

Article Snippet: Briefly, the slides were stained with anti-human HER2/ERBB2 (Cat #: 10004-T56, SinoBiological) or anti-human CD47 (ab218810, Abcam) for immunohistochemistry (IHC), and immunostained with anti-human CD3 (Cat #: ab11089, Abcam), anti-FLAG tag (Cat #: 637301, Biolegend) antibodies and DAPI for immunofluorescence, which performed by the immunoassay platform at the Shanghai Institute for Emerging and Re-emerging Infectious Diseases of SPHCC.

Techniques: Expressing, Quantitation Assay

Journal: eLife

Article Title: Unique integrated stress response sensors regulate cancer cell susceptibility when Hsp70 activity is compromised

doi: 10.7554/eLife.64977

Figure Lengend Snippet: ( A ) HER2 (diamonds), TNBC (circles), and luminal (squares and triangles) breast cancer lines were seeded into 96-well plates and treated with increasing doses of MAL3-101 for 72 hr. Viability is expressed as the average of three or more independent experiments, ± SEM. ( B ) MAL3-101-sensitive (MCF7 and MDA MB 231, denoted in blue) and resistant (MDA MB 453 and MDA MB 361, denoted in black) cells were treated with 12 µM MAL3-101 for the indicated times, and lysates were prepared and immunoblotted for cleaved caspase-3, caspase-7, and caspase-8. β-actin serves as a loading control. ( C ) The corresponding fold-increase of the indicated apoptotic markers relative to the DMSO control are plotted, ± SEM (n≥3 for cleaved caspase-3, n=3 for cleaved caspase-7, and n≥4 for cleaved caspase-8). Black asterisks correspond to statistical significance between MDA MB 231 cells (closed circle) and MDA MB 453 and MDA MB 361 (open circle and triangle, respectively), and the red asterisk represents statistical significance between MCF7 (closed triangle) and MDA MB 453 and MDA MB 361 (open circle and triangle) cells; * denotes p<0.05, ** denotes p<0.005. Figure 1—source data 1. Source data for cell viability assay and apoptotic marker accumulation in .

Article Snippet: For the remaining proteins aliquots from the same lysates were instead heated to 37°C for 30 min prior to SDS-PAGE using 4–20% Tris-Glycine gradient gels (XP04202Box, Thermo Fisher Scientific), and after transfer on nitrocellulose membranes (#84–874, Prometheus Laboratories Inc, California), the blots were incubated with anti-cleaved Caspase-8 (18C8, #9496; at 1:1000), anti-BiP (C50B12, #3177S; at 1:1000), anti-Hsp70 (smc-113, StressMarq Biosciences, Victoria, British Columbia; at 1:1000), anti-Hsc/Hsp70 (#4872S; at 1:1000), anti-HER2 (29D8, #2165; at 1:2000), anti-eIF2α (#9722S; at 1:2000), anti-phospho-eIF2α (#9721L; at 1:500), anti-PERK (D11A8, #5683S; at 1:2000), anti-PKR (D7F7, #12297S; at 1:2000), anti-Ire1α (14C10, #3294S; at 1:1000), anti-HRI (MBS2538114, MyBioSource, San Diego, CA; at 1:500) and anti-GCN2 and anti-GCN2 pT899 antibody (ab-134053 and ab-75836, Abcam, Cambtidge, UK; at 1:2000).

Techniques: Viability Assay, Marker

Journal: eLife

Article Title: Unique integrated stress response sensors regulate cancer cell susceptibility when Hsp70 activity is compromised

doi: 10.7554/eLife.64977

Figure Lengend Snippet: Breast cancer cells exhibit a range of sensitivities to MAL3-101, a specific Hsp70 inhibitor. The indicated breast cancer lines were seeded into 96-well plates and treated with increasing doses of the indicated compounds for 72 hr. Viability was measured using the CellTiter-Glo assay. IC 50 values were generate using a sigmoidal nonlinear regression with SigmaPlot 11.0. ND stands for an undetermined value. MAL3-101 sensitivities of Hsp70 inhibitor resistant cells are in bold.

Article Snippet: For the remaining proteins aliquots from the same lysates were instead heated to 37°C for 30 min prior to SDS-PAGE using 4–20% Tris-Glycine gradient gels (XP04202Box, Thermo Fisher Scientific), and after transfer on nitrocellulose membranes (#84–874, Prometheus Laboratories Inc, California), the blots were incubated with anti-cleaved Caspase-8 (18C8, #9496; at 1:1000), anti-BiP (C50B12, #3177S; at 1:1000), anti-Hsp70 (smc-113, StressMarq Biosciences, Victoria, British Columbia; at 1:1000), anti-Hsc/Hsp70 (#4872S; at 1:1000), anti-HER2 (29D8, #2165; at 1:2000), anti-eIF2α (#9722S; at 1:2000), anti-phospho-eIF2α (#9721L; at 1:500), anti-PERK (D11A8, #5683S; at 1:2000), anti-PKR (D7F7, #12297S; at 1:2000), anti-Ire1α (14C10, #3294S; at 1:1000), anti-HRI (MBS2538114, MyBioSource, San Diego, CA; at 1:500) and anti-GCN2 and anti-GCN2 pT899 antibody (ab-134053 and ab-75836, Abcam, Cambtidge, UK; at 1:2000).

Techniques:

Journal: eLife

Article Title: Unique integrated stress response sensors regulate cancer cell susceptibility when Hsp70 activity is compromised

doi: 10.7554/eLife.64977

Figure Lengend Snippet: The cell numbers and autophagy or proteasome inhibitor concentrations used for the cell viability assay in combination with increasing doses of MAL3-101 are shown. The concentrations of bortezomib, CQ, and bafilomycin to induce no greater than 30% of cell death in each line after 72 hr treatment are shown. ND stands for undetermined value.

Article Snippet: For the remaining proteins aliquots from the same lysates were instead heated to 37°C for 30 min prior to SDS-PAGE using 4–20% Tris-Glycine gradient gels (XP04202Box, Thermo Fisher Scientific), and after transfer on nitrocellulose membranes (#84–874, Prometheus Laboratories Inc, California), the blots were incubated with anti-cleaved Caspase-8 (18C8, #9496; at 1:1000), anti-BiP (C50B12, #3177S; at 1:1000), anti-Hsp70 (smc-113, StressMarq Biosciences, Victoria, British Columbia; at 1:1000), anti-Hsc/Hsp70 (#4872S; at 1:1000), anti-HER2 (29D8, #2165; at 1:2000), anti-eIF2α (#9722S; at 1:2000), anti-phospho-eIF2α (#9721L; at 1:500), anti-PERK (D11A8, #5683S; at 1:2000), anti-PKR (D7F7, #12297S; at 1:2000), anti-Ire1α (14C10, #3294S; at 1:1000), anti-HRI (MBS2538114, MyBioSource, San Diego, CA; at 1:500) and anti-GCN2 and anti-GCN2 pT899 antibody (ab-134053 and ab-75836, Abcam, Cambtidge, UK; at 1:2000).

Techniques: Viability Assay

Journal: eLife

Article Title: Unique integrated stress response sensors regulate cancer cell susceptibility when Hsp70 activity is compromised

doi: 10.7554/eLife.64977

Figure Lengend Snippet: Breast cancer cells exhibit a range of sensitivities to MAL3-101 in the presence of either autophagy or proteasome inhibitors. Cells were seeded into 96-well plates and treated with increasing doses of MAL3-101 in the presence or absence of subcritical doses of bortezomib (proteasome inhibitor), or CQ or bafilomycin (autophagy inhibitors) for 72 hr. Viability was measured using the CellTiter-Glo assay. IC 50 values were generate using a sigmoidal nonlinear regression with SigmaPlot 11.0. ND stands for undetermined value. MAL3-101 resistant cells are highlighted in yellow.

Article Snippet: For the remaining proteins aliquots from the same lysates were instead heated to 37°C for 30 min prior to SDS-PAGE using 4–20% Tris-Glycine gradient gels (XP04202Box, Thermo Fisher Scientific), and after transfer on nitrocellulose membranes (#84–874, Prometheus Laboratories Inc, California), the blots were incubated with anti-cleaved Caspase-8 (18C8, #9496; at 1:1000), anti-BiP (C50B12, #3177S; at 1:1000), anti-Hsp70 (smc-113, StressMarq Biosciences, Victoria, British Columbia; at 1:1000), anti-Hsc/Hsp70 (#4872S; at 1:1000), anti-HER2 (29D8, #2165; at 1:2000), anti-eIF2α (#9722S; at 1:2000), anti-phospho-eIF2α (#9721L; at 1:500), anti-PERK (D11A8, #5683S; at 1:2000), anti-PKR (D7F7, #12297S; at 1:2000), anti-Ire1α (14C10, #3294S; at 1:1000), anti-HRI (MBS2538114, MyBioSource, San Diego, CA; at 1:500) and anti-GCN2 and anti-GCN2 pT899 antibody (ab-134053 and ab-75836, Abcam, Cambtidge, UK; at 1:2000).

Techniques: