Journal: STAR Protocols

Article Title: Protocol for 3D-guided sectioning and deep cell phenotyping via light sheet imaging and 2D spatial multiplexing

doi: 10.1016/j.xpro.2025.104296

Figure Lengend Snippet: 3D light sheet and 2D multi-cyclic imaging data comparison (Mouse Glioblastoma) (A) Imaris 3D surface rendering of autofluorescence (cyan) and glioblastoma target cells stained with anti-GFP-Alexa Fluor 647 nanobody (red). (B) Imaris 3D surface rendering of autofluorescence (cyan) and glioblastoma target cells stained with anti-GFP-Alexa Fluor 647 nanobody (red) with target plane in yellow. (C) Optical section of target plane of interest. (D) Fluorescence image of physical cryosection. (E) MICS image of section shown in D. (F) MICS image indicating anti-GFP-Alexa Fluor 647 nanobody (red) staining. (G) Magnified merged four color multiparameter MICS image with anti-EGFR (magenta), anti-GFAP (green), anti-NeuN (blue), anti-CD146 (yellow). (H–P) Nine exemplary MICS images with merges of anti-GFP-Alexa Fluor 647 nanobody staining (red) and antibody-conjugates against EGFR (H), Neurofilament (I), Nestin (J), GFAP (K), CD44 (L), CD146 (M), NeuN (N), EphA2 (O) and GLAST (P) (gray) (see “Antibodies”). Scale bars: (A–F) 500 μm; (G) 50 μm; (H–P) 500 μm.

Article Snippet: EphA2 antibody, anti-mouse, APC, REAfinity , Miltenyi Biotec B.V. & Co. KG , Cat# 130-109-187 RRID: AB_2651638.

Techniques: Imaging, Comparison, Staining, Fluorescence

Journal: Oncology Letters

Article Title: The combined use of EphA2/MMP-2 expression and MRI findings contributes to the determination of cerebral glioma grade

doi: 10.3892/ol.2019.10912

Figure Lengend Snippet: Expression of MMP-2 and EphA2 in cerebral glioma samples, according to the pathological grade of the tumor.

Article Snippet: One slide was routinely stained with hematoxylin and eosin in 25°C for 1–5 min. After blocking in BSA solution 37°C for 30 min (cat. no. P0260; Beyotime Institute of Biotechnology), the other slide was used for IHC staining using mouse anti-human MMP-2 and mouse anti-human EphA2 (Boster cat. no. M00286 and BM0833, both 1:200) monoclonal antibodies detected by streptavidin-peroxidase or 3′-diaminobenzidine visualization kits (OriGene Technologies, Inc.).

Techniques: Expressing

Journal: Oncology Letters

Article Title: The combined use of EphA2/MMP-2 expression and MRI findings contributes to the determination of cerebral glioma grade

doi: 10.3892/ol.2019.10912

Figure Lengend Snippet: Immunohistochemical staining of glioma tissues. (A) MMP-2 and (B) EphA2 expression was identified by immunofluorescence staining in the cytoplasm of glioma cells. Positive cells were stained brown. Percentages of stained cells are presented in the bar graphs. Data represent the mean ± standard deviation (n=5). ***P<0.001. Scale bar, 30 µm. EphA2, ephrin type-A receptor 2; MMP-2, metalloproteinase 2.

Article Snippet: One slide was routinely stained with hematoxylin and eosin in 25°C for 1–5 min. After blocking in BSA solution 37°C for 30 min (cat. no. P0260; Beyotime Institute of Biotechnology), the other slide was used for IHC staining using mouse anti-human MMP-2 and mouse anti-human EphA2 (Boster cat. no. M00286 and BM0833, both 1:200) monoclonal antibodies detected by streptavidin-peroxidase or 3′-diaminobenzidine visualization kits (OriGene Technologies, Inc.).

Techniques: Immunohistochemical staining, Staining, Expressing, Immunofluorescence, Standard Deviation

Journal: Oncology Letters

Article Title: The combined use of EphA2/MMP-2 expression and MRI findings contributes to the determination of cerebral glioma grade

doi: 10.3892/ol.2019.10912

Figure Lengend Snippet: Pearson's correlation analysis of the magnetic resonance imaging parameters and immunohistochemistry results. Scatterplots presenting the correlation between (A) MMP-2 and (B) EphA2 positivity with the EI; (C) MMP-2 and (D) EphA2 positivity with the EP; and (E) MMP-2 and (F) EphA2 positivity with maximum tumor diameter. EI, edema index; EP, enhancement percentage; EphA2, ephrin type-A receptor 2; MMP-2, metalloproteinase 2.

Article Snippet: One slide was routinely stained with hematoxylin and eosin in 25°C for 1–5 min. After blocking in BSA solution 37°C for 30 min (cat. no. P0260; Beyotime Institute of Biotechnology), the other slide was used for IHC staining using mouse anti-human MMP-2 and mouse anti-human EphA2 (Boster cat. no. M00286 and BM0833, both 1:200) monoclonal antibodies detected by streptavidin-peroxidase or 3′-diaminobenzidine visualization kits (OriGene Technologies, Inc.).

Techniques: Magnetic Resonance Imaging, Immunohistochemistry

Journal: Cancer Research

Article Title: Combination Therapy with HSP90 Inhibitor 17-DMAG Reconditions the Tumor Microenvironment to Improve Recruitment of Therapeutic T cells

doi: 10.1158/0008-5472.can-12-0538

Figure Lengend Snippet: Figure 1. 17-DMAG promotes proteasome-dependent EphA2 protein degradation in MCA205 sarcoma cells and the enhanced recognition of tumor cells by anti-EphA2 CD8þ T cells in vitro. A, MCA205 tumor cells were treated with various doses of 17-DMAG for 24 hours in vitro and then lysed, with EphA2 and control b-actin protein expression subsequently monitored by Western blotting as described in Materials and Methods. NC, negative control lysate from EphA2neg B16 melanoma cells. B, proteasome inhibitor (MG-132), but not lysosome inhibitor chloroquine (CLQ), blocks 17-DMAG (500 nmol/L)-induced degradation of EphA2 protein in MCA205 tumor cells. C, treatment of MCA205 cells with 17-DMAG at the indicated doses for 24 hours (or 48 hours, data not shown) did not affect MHC class I expression on tumor cells. D, 17-DMAG–treated EphA2þ MCA205 cells were better recognized versus control, untreated tumor cells by anti-EphA2 CD8þ T cells (developed from EphA2/ mice, per Supplementary Fig. S1 and Materials and Methods) in CD107 translocation assays as described in Materials and Methods. All data are representative of those obtained in 3 independent experiments.

Article Snippet: The following primary antibodies were used for staining sections: rat anti-mouse CD31 (BD Biosciences), rabbit anti-mouse EphA2 (Santa Cruz Biotechnology), rat antimouse VCAM-1, goat anti-mouse CXCL10 (R&D Systems).

Techniques: In Vitro, Control, Expressing, Western Blot, Negative Control, Translocation Assay

Journal: Cancer Research

Article Title: Combination Therapy with HSP90 Inhibitor 17-DMAG Reconditions the Tumor Microenvironment to Improve Recruitment of Therapeutic T cells

doi: 10.1158/0008-5472.can-12-0538

Figure Lengend Snippet: Figure 2. Treatment of mice bearing established MCA205 tumors with oral 17-DMAG transiently promotes a therapeutically preferred immunophenotype in the TME and is optimally effective in a 5-day regimen. A, C57BL/6 mice bearing established MCA205 tumors (day 18; 100 mm2 mean tumor size) were left untreated or they were administered 17-DMAG (10, 15, or 25 mg/kg/d for up to 10 days via oral gavage) and tumor size (mean SD, 5 animals per group) monitored longitudinally. , P < 0.05; , P < 0.01 (ANOVA) for 15 or 25 versus 10 mg/kg/d or untreated; not significant (ANOVA) for 15 versus 25 mg/kg/d. B, tumors were excised on the indicated day after initiating treatment, and single-cell suspensions of enzymatic tumor digests analyzed for immune cell infiltrates by flow cytometry as described in Materials and Methods. Tumor cells isolated from enzymatic digests (per B) were also analyzed as target cells for anti-EphA2 CD8þ T effector cells generated from EphA2/ mice (see Supplementary Fig. S1) as monitored using CD107 translocation (C) and IFN-g secretion (D) assays as described in Materials and Methods. All data are representative of those obtained in 3 independent experiments. For B–D, , P < 0.05; , P < 0.01 (ANOVA) versus all other determinations.

Article Snippet: The following primary antibodies were used for staining sections: rat anti-mouse CD31 (BD Biosciences), rabbit anti-mouse EphA2 (Santa Cruz Biotechnology), rat antimouse VCAM-1, goat anti-mouse CXCL10 (R&D Systems).

Techniques: Cytometry, Isolation, Generated, Translocation Assay

Journal: Cancer Research

Article Title: Combination Therapy with HSP90 Inhibitor 17-DMAG Reconditions the Tumor Microenvironment to Improve Recruitment of Therapeutic T cells

doi: 10.1158/0008-5472.can-12-0538

Figure Lengend Snippet: Figure 3. The impact of 17-DMAG–based therapy for 5 days persists after discontinuation of drug delivery. A, MCA205 tumor–bearing mice (5 mice/group) were left untreated or they were treated for 5 days with orally administered 17-DMAG (15 mg/kg/d), with tumor growth then monitored over a 4-week period. B, EphA2 protein expression in tumors harvested from 17-DMAG–treated versus untreated mice was analyzed longitudinally by Western blotting as outlined in Materials and Methods. C, tumor cells from untreated or 17-DMAG–treated mice were analyzed at the indicated time points for their ability to be recognized by anti-EphA2 CD8þ T cells generated from EphA2/ mice (see Supplementary Fig. S1) in CD107 translocation and IFN-g secretion assays, as described in Materials and Methods. D, single-cell suspensions from harvested tumor digests were analyzed by flow cytometry for the indicated T-cell, DC, and MDSC phenotypes. All data are representative of those obtained in 3 independent experiments. , P < 0.05; , P < 0.01 (t test) for treated versus untreated controls.

Article Snippet: The following primary antibodies were used for staining sections: rat anti-mouse CD31 (BD Biosciences), rabbit anti-mouse EphA2 (Santa Cruz Biotechnology), rat antimouse VCAM-1, goat anti-mouse CXCL10 (R&D Systems).

Techniques: Expressing, Western Blot, Generated, Translocation Assay, Cytometry

Journal: Cancer Research

Article Title: Combination Therapy with HSP90 Inhibitor 17-DMAG Reconditions the Tumor Microenvironment to Improve Recruitment of Therapeutic T cells

doi: 10.1158/0008-5472.can-12-0538

Figure Lengend Snippet: Figure 4. 17-DMAG administration improves the immunogenicity and antitumor efficacy of an EphA2 peptide–based vaccine in the MCA205 tumor model. A, C57BL/6 mice bearing established EphA2þ MCA205 sarcomas (s.c. right flank) remained untreated or they were treated with DC-based vaccines (s.c., left flank on days 0 and 7 of the treatment regimen) that contained or lacked EphA2 peptide epitopes, alone or in combination with 17-DMAG (15 mg/kg/d on the first 5 days of the treatment regimen by oral gavage). Tumor size (mean SD) is reported in mm2. All the mice in the DC/EphA2 þ 17-DMAG–treated group rendered tumor-free (80%) were rechallenged (s.c., right flank) with MCA205 tumor cells on day 30 of the experiment (as indicated by arrow with R inset) and monitored through day 60 after treatment initiation. B, CD8þ TILs recovered from tumors on day 14 after treatment initiation were assessed for their ability to recognize syngenic control DCs pulsed with no peptide or DC pulsed with the EphA2671–679 þ EphA2682–689 peptides. After 48-hour incubation, cell-free supernatants were analyzed for IFN-g content by ELISA. Response to DC (no peptide) was <50 pg/mL in all instances. C, single-cell suspensions of enzymatically digested day 14 (posttreatment initiation) tumors were analyzed by flow cytometry for the indicated T-cell, DC, and MDSC phenotypes as described in Materials and Methods. Each filled circle represents data from an individual animal in a given control or treatment cohort, with the mean of data indicated by a gray bar for each cohort. All data are representative of those obtained in 3independent experiments. , P < 0.05; , P < 0.01 (ANOVA) versus all other cohorts.

Article Snippet: The following primary antibodies were used for staining sections: rat anti-mouse CD31 (BD Biosciences), rabbit anti-mouse EphA2 (Santa Cruz Biotechnology), rat antimouse VCAM-1, goat anti-mouse CXCL10 (R&D Systems).

Techniques: Immunopeptidomics, Vaccines, Control, Incubation, Enzyme-linked Immunosorbent Assay, Cytometry

Journal: Cancer Research

Article Title: Combination Therapy with HSP90 Inhibitor 17-DMAG Reconditions the Tumor Microenvironment to Improve Recruitment of Therapeutic T cells

doi: 10.1158/0008-5472.can-12-0538

Figure Lengend Snippet: Figure 5. 17-DMAG improves the antitumor efficacy of an EphA2 peptide–based vaccine in the EphA2neg B16 melanoma model based on immune targeting of EphA2þ VEC. A, C57BL/6 mice bearing established s.c. B16 melanomas (right flank) were left untreated or treated as outlined, with tumor size (mean SD) reported in mm2 followed for up to 30 days. , P < 0.001 (ANOVA) versus all other cohorts. B, day 14 (posttreatment initiation) tumors were harvested and tissue sections analyzed by immunofluorescence microscopy and MetaMorph quantitation for coexpression of CD31 (i.e., VEC) and EphA2 proteins as described in Materials and Methods. , P < 0.05; , P < 0.01 (ANOVA) versus all other cohorts. Anti-EphA2 CD8þ T cells isolated from the spleens of immune EphA2/ mice (C; as outlined in Supplementary Fig. S1), or TIL from B16 tumor–bearing animals treated with combined DC/EphA2 peptide vaccination þ 17-DMAG (D; per Supplementary Fig. 5A) were analyzed for reactivity against flow-sorted CD31þ VECs isolated from the tumors of B16-bearing animals left untreated or treated for 6 days with DC/EphA2 vaccine only, 17-DMAG or DC/EphA2 vaccine þ 17-DMAG. CD31þ kidney VECs were also flow-sorted from animals treated for 6 days with DC/EphA2 vaccine þ 17-DMAG to discern autoimmunity of T cells against tumor-uninvolved VECs. C, the MHC class I–restricted nature of VEC recognition by CD8þ T cells was assessed by inclusion of anti-class I or isotype control mAb per culture well, as described in Materials and Methods. C and D, , P < 0.05; , P < 0.01 (t test) versus control antibody treatment or untreated controls, respectively. All data are representative of those obtained in 3 independent experiments. HPF, high-power field.

Article Snippet: The following primary antibodies were used for staining sections: rat anti-mouse CD31 (BD Biosciences), rabbit anti-mouse EphA2 (Santa Cruz Biotechnology), rat antimouse VCAM-1, goat anti-mouse CXCL10 (R&D Systems).

Techniques: Microscopy, Quantitation Assay, Isolation, Control

Journal: Cancer Research

Article Title: Combination Therapy with HSP90 Inhibitor 17-DMAG Reconditions the Tumor Microenvironment to Improve Recruitment of Therapeutic T cells

doi: 10.1158/0008-5472.can-12-0538

Figure Lengend Snippet: Figure 6. 17-DMAG improves the antitumor efficacy of adoptively transferred anti-EphA2 CD8þ T cells in a combination therapy. A, C57BL/6 mice bearing established s.c. MCA205 sarcomas (right flank) were left untreated or they were treated with 17-DMAG (15 mg/kg/d provided orally on the first 5 days of the treatment regimen) adoptive transfer (i.v. tail vein on day 4 of the treatment regimen) of 5 106 CD8þ T cells isolated from EphA2/

Article Snippet: The following primary antibodies were used for staining sections: rat anti-mouse CD31 (BD Biosciences), rabbit anti-mouse EphA2 (Santa Cruz Biotechnology), rat antimouse VCAM-1, goat anti-mouse CXCL10 (R&D Systems).

Techniques: Adoptive Transfer Assay, Isolation

Journal: PLoS ONE

Article Title: Aptamers provide superior stainings of cellular receptors studied under super-resolution microscopy

doi: 10.1371/journal.pone.0173050

Figure Lengend Snippet: ( A ) Simple scheme describing the procedure to stain living cells using aptamers or antibodies (probes). Cells are incubated with aptamer or antibodies allowing the internalization of the probes by the endocytosis of the receptor. ( B ) Aptamers against EGFR, ErbB2 and Epha2 were used to live-stain human cell lines expressing these receptors (A431, SKB3 and HeLa cells respectively). A control aptamer with randomized sequence of equivalent length and coupled to the same fluorophore was used to stain these cell lines. Additionally, cell lines that do not express such receptors were used as negative control cells (MCF7 cells are negative for EGFR and ErbB2; HEK293 cells are negative for Epha2). Both controls, the cell lines not expressing the receptor and the randomized aptamer show virtually no aptamer signal (nuclei were DAPI stained, displayed in blue). All images were acquired using an epifluorescence microscope with the same settings and they are equally scaled to allow a direct comparison. Scale bar represents 5 μm.

Article Snippet: Epha2 , Ab13770 , Abcam , Rabbit, polyclonal , Ab1.

Techniques: Staining, Incubation, Expressing, Sequencing, Negative Control, Microscopy

Journal: PLoS ONE

Article Title: Aptamers provide superior stainings of cellular receptors studied under super-resolution microscopy

doi: 10.1371/journal.pone.0173050

Figure Lengend Snippet: List of antibodies used.

Article Snippet: Epha2 , Ab13770 , Abcam , Rabbit, polyclonal , Ab1.

Techniques:

Journal: PLoS ONE

Article Title: Aptamers provide superior stainings of cellular receptors studied under super-resolution microscopy

doi: 10.1371/journal.pone.0173050

Figure Lengend Snippet: Figure legend is equivalent to legend on , but for Epha2.

Article Snippet: Epha2 , Ab13770 , Abcam , Rabbit, polyclonal , Ab1.

Techniques:

Journal: JCI Insight

Article Title: MyD88/CD40 signaling retains CAR T cells in a less differentiated state

doi: 10.1172/jci.insight.136093

Figure Lengend Snippet: ( A ) Scheme of EphA2-CAR constructs. ( B ) Summary plot of %tCD19 + T cells ( n = 5, mean ± SEM, 1-way ANOVA with Tukey’s test for multiple comparisons). ( C ) Summary plot of %F(ab′) 2 -positive T cells ( n = 5, mean ± SEM, 1-way ANOVA with Tukey’s test for multiple comparisons). ( D ) CAR T cell production of Th1 (IFN-γ and IL-2) and Th2 (IL-4 and IL-10) cytokines after 24-hour coculture at a 2:1 ratio against EphA2-positive (LM7, U373 WT) and EphA2-negative (BV173, U373 EphA2 KO) cell lines or in media alone ( n = 5, mean ± SEM, 2-way ANOVA with Dunnett’s test for multiple comparisons, all statistical analysis is in comparison with NT cells). Dot colors: black, media; light gray, BV173 (EphA2 negative); dark gray, U373 EphA2 KO (EphA2 negative); dark blue: U373 (EphA2 positive); light blue, LM7 (EphA2 positive). ( E ) Summary plots of Th1 and Th2 cytokine production against EphA2-positive cell lines U373 and LM7 ( n = 5, mean ± SEM, values were log transformed before 2-way ANOVA with Tukey’s test for multiple comparisons). ( F ) CAR T cells were incubated with increasing amounts of tumor cells for 24 hours, and the remaining live tumor cells were quantified with an MTS assay (2-way ANOVA with Tukey’s test for multiple comparisons, mean ± SEM, LM7: n = 4, U373 WT and U373 EphA2 KO: n = 9). For LM7 and U373, asterisks refer to statistical comparison of MC-CAR with NT and CD28-CAR with MC-CAR. For U373 EphA2 KO, asterisks refer to statistical comparison of 41BB-CAR with NT and CD28-CAR with NT. # P < 0.1; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: After SDS-PAGE and wet transfer, the membrane was blocked with 5% milk in TBS-Tween (TBST), then incubated with primary antibodies: goat anti-human EphA2 (R&D Systems, Bio-Techne, AF3035), rabbit anti-human Bcl-2 (clone D55G8, Cell Signaling Technology), rabbit anti-human Myb (clone D2R4Y, Cell Signaling Technology), or mouse anti-human GAPDH (clone 0411, Santa Cruz Biotechnology).

Techniques: Construct, Comparison, Transformation Assay, Incubation, MTS Assay

Journal: JCI Insight

Article Title: MyD88/CD40 signaling retains CAR T cells in a less differentiated state

doi: 10.1172/jci.insight.136093

Figure Lengend Snippet: T cells were cocultured with tumor cells at a 2:1 ratio with weekly restimulation against fresh tumor cells until they lost their effector function and no longer killed all the tumor cells. ( A ) Average expansion of CAR T cells against EphA2-positive (U373 and LM7) and U373 EphA2 KO cell line (mean ± SEM, LM7: n = 4; U373: n = 8 [NT, CD28, MC], n = 4 [41BB]; U373 KO: n = 8 [NT, CD28, MC], n = 6 [41BB]). ( B ) Summary of the maximum expansion CAR T cells from individual donors achieved against EphA2-positive tumor cells and the maximum number of times CAR T cells were able to kill fresh EphA2-positive tumor cells ( n = 12 [NT, CD28, and MC], n = 8 [41BB]; median and quartiles, 1-way ANOVA with Tukey’s test for multiple comparisons). T cells were phenotyped 7 days after stimulation with U373. ( C ) Summary plot of CD4/CD8 composition after stimulation with U373 ( n = 3, mean ± SEM). ( D ) Scheme for phenotyping T cells and representative flow cytometry plots of CCR7 and CD45RA expression on CAR T cells after stimulation with U373. ( E ) Summary plot of T cell phenotype after stimulation with U373 ( n = 4, mean ± SEM, 2-way ANOVA with Tukey’s test for multiple comparisons). All statistical tests were performed in comparison with MC-CAR T cells (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001).

Article Snippet: After SDS-PAGE and wet transfer, the membrane was blocked with 5% milk in TBS-Tween (TBST), then incubated with primary antibodies: goat anti-human EphA2 (R&D Systems, Bio-Techne, AF3035), rabbit anti-human Bcl-2 (clone D55G8, Cell Signaling Technology), rabbit anti-human Myb (clone D2R4Y, Cell Signaling Technology), or mouse anti-human GAPDH (clone 0411, Santa Cruz Biotechnology).

Techniques: Flow Cytometry, Expressing, Comparison

Journal: JCI Insight

Article Title: MyD88/CD40 signaling retains CAR T cells in a less differentiated state

doi: 10.1172/jci.insight.136093

Figure Lengend Snippet: ( A – D ) NSG mice were injected with 1 × 10 6 LM7-ffLuc i.p. One week later, mice were injected with 1 × 10 4 or 1 × 10 5 CD28-, 41BB-, or MC-CAR T cells. PBS and 1 × 10 5 Delta-CAR T cells were used as controls. ( A ) Total flux from tumor cells in all mice treated with 1 × 10 4 EphA2 CAR T cells (PBS: n = 5; CD28, 41BB, MC: n = 10). ( B ) Total flux from tumor cells in all mice treated with 1 × 10 5 EphA2 CAR T cells (Delta: n = 5, CD28: n = 8, 41BB: n = 9, MC: n = 10). ( C and D ) Kaplan-Meier survival analysis of mice treated with 1 × 10 4 ( C ) or 1 × 10 5 ( D ) EphA2 CAR T cells (log-rank Mantel-Cox test with Bonferroni’s correction for multiple comparisons; * P < 0.05; ** P < 0.01; *** P < 0.001). Experiments were repeated twice with CAR T cells generated from 2 different healthy donors.

Article Snippet: After SDS-PAGE and wet transfer, the membrane was blocked with 5% milk in TBS-Tween (TBST), then incubated with primary antibodies: goat anti-human EphA2 (R&D Systems, Bio-Techne, AF3035), rabbit anti-human Bcl-2 (clone D55G8, Cell Signaling Technology), rabbit anti-human Myb (clone D2R4Y, Cell Signaling Technology), or mouse anti-human GAPDH (clone 0411, Santa Cruz Biotechnology).

Techniques: Injection, Generated