Journal: International Journal of Biological Sciences

Article Title: Integrative analysis reveals a clinicogenomic landscape associated with liver metastasis and poor prognosis in hepatoid adenocarcinoma of the stomach

doi: 10.7150/ijbs.71449

Figure Lengend Snippet: Clinicopathological characteristics of HAS and stage-matched non-HAS patients

Article Snippet: Following that, the sections were incubated with anti-ERBB2 monoclonal primary antibody (Cell Signaling Technology, #2165), followed by a 30-min incubation with secondary antibody.

Techniques:

Journal: International Journal of Biological Sciences

Article Title: Integrative analysis reveals a clinicogenomic landscape associated with liver metastasis and poor prognosis in hepatoid adenocarcinoma of the stomach

doi: 10.7150/ijbs.71449

Figure Lengend Snippet: Potential targets and therapy for HAS. (A) Frequently altered genomic targets in HAS compared with non-HAS. (B) Comparison of ERBB2 expression between HAS and non-HAS. (C) Comparison of TMB between HAS and non-HAS. (D) Assessment of serum AFP and CEA levels in HAS patients before and after treatment. Three HAS patients received anti-ERBB2 therapy and five HAS patients received anti-PD-1 therapy. The line charts represent serum AFP/CEA level and histograms represent the CP of serum AFP/CEA level; details are shown as follows: CP (%) = (serum AFP or CEA levels before treatment - serum AFP or CEA levels after treatment) / serum AFP or CEA levels before treatment *100%. The values of 20 ng/mL and 5 ng/mL are defined as the serum AFP and CEA levels threshold, respectively. For each patient with elevated AFP/CEA level before treatment, 65% was defined as the cutoff for response to anti-ERBB2/anti-PD-1 therapy. Blue and red represent response and no response at the serum tumor biomarker levels, respectively. Grey means that this evaluation criterion is inapplicable for a patient with a normal AFP/CEA level before treatment. (E) Representative CT images of abdomen showing tumor lesions of two patients before and after treatment; one received anti-ERBB2 therapy and the other received anti-PD-1 therapy. The dotted line represents the metastatic liver lesion, and the arrow represents the primary gastric lesion. IHC, immunohistochemistry; AFP, alpha-fetoprotein; CEA, carcinoembryonic antigen; CP, change percent; Pt, patient.

Article Snippet: Following that, the sections were incubated with anti-ERBB2 monoclonal primary antibody (Cell Signaling Technology, #2165), followed by a 30-min incubation with secondary antibody.

Techniques: Expressing, Biomarker Assay, Immunohistochemistry

Journal: Frontiers in Immunology

Article Title: Sequential Anti-PD1 Therapy Following Dendritic Cell Vaccination Improves Survival in a HER2 Mammary Carcinoma Model and Identifies a Critical Role for CD4 T Cells in Mediating the Response

doi: 10.3389/fimmu.2019.01939

Figure Lengend Snippet: Immunophenotyping and function of type I polarized DC1 from Balb/C mice. (A) DC collected after maturation with CpG and LPS were stained for MHC class II (IAd), CD80, CD86, and CD40 and data were acquired on LSRII flow cytometer and analyzed by FlowJo software. Flow gating strategy and representative flow dot plot of DC1 staining for MHC class II, CD80, CD86, and CD40. (B) Culture supernatants were collected before and after DC maturation and measured for IL-12 secretion using standard ELISA. (C) HER2 expression on TUBO cells using immunofluorescence staining. (D) Western blot analysis of HER2 protein expression on tumor cells. P -values were determined by Student t -test. *** p < 0.001.

Article Snippet: Cells were then incubated with monoclonal primary anti-HER2/ErbB2 antibody (Cell Signaling Technology, Beverly, MA, Cat. No. 2165) over night at 4°C followed by incubation with Alexa Fluor 594-conjugated anti-rabbit secondary antibody (Cell Signaling Technology, Beverly, MA, Cat. No. 8889) for 1 h at room temperature in the dark.

Techniques: Staining, Flow Cytometry, Software, Enzyme-linked Immunosorbent Assay, Expressing, Immunofluorescence, Western Blot

Journal: Frontiers in Immunology

Article Title: Sequential Anti-PD1 Therapy Following Dendritic Cell Vaccination Improves Survival in a HER2 Mammary Carcinoma Model and Identifies a Critical Role for CD4 T Cells in Mediating the Response

doi: 10.3389/fimmu.2019.01939

Figure Lengend Snippet: Vaccination with DC1 pulsed with class I or class II HER2 peptides delays tumor growth in TUBO bearing mice. (A) DC1 was generated as described in methods section and pulsed with class I rHER2 peptide p66 (class I HER2-DC1). Balb/C mice were injected with 2.5 × 10 5 TUBO cells subcutaneously on the right flank on day 0. When tumors were palpable on day 7, mice received class I HER2-DC1 vaccine subcutaneously on the left flank once, twice or three times a week for a total of six doses. N = 8 mice/group was used for these studies. Tumor area was measured 2–3 days a week. (B) Balb/C mice received tumor cells and HER2-DC1 vaccine as described above. DC1 were pulsed with 10 μg/ml of each peptides from the rat HER2 (rHER2) oncogene; p5 (ELAAWCRWGFLLALLPPGIAG), p435 (IRGRILHDGAYSLTLQGLGIH), and p1209 (SPPHPSPAFSPAFDNLYYWDQ) and p66 (TYVPANASL). (C) Balb/C mice received tumor cells as described above and received both class I and class II pulsed HER2-DC1 vaccine. Data shown are the representative from three independent experiments and are shown as mean number ± SEM *** p < 0.001, ** p < 0.01 using Student t -test.

Article Snippet: Cells were then incubated with monoclonal primary anti-HER2/ErbB2 antibody (Cell Signaling Technology, Beverly, MA, Cat. No. 2165) over night at 4°C followed by incubation with Alexa Fluor 594-conjugated anti-rabbit secondary antibody (Cell Signaling Technology, Beverly, MA, Cat. No. 8889) for 1 h at room temperature in the dark.

Techniques: Generated, Injection

Journal: Frontiers in Immunology

Article Title: Sequential Anti-PD1 Therapy Following Dendritic Cell Vaccination Improves Survival in a HER2 Mammary Carcinoma Model and Identifies a Critical Role for CD4 T Cells in Mediating the Response

doi: 10.3389/fimmu.2019.01939

Figure Lengend Snippet: Vaccination with HER2-DC1 enhances anti-HER2 Th1 immune response and increases T cell infiltration in TUBO bearing mice. (A) Splenocytes from class I HER2-DC1 vaccinated mice were re-stimulated with p66 rHER2 peptide. Culture supernatants were collected after 3 days and IFN-γ secretion was measured by standard IFN-γ ELISA. (B) Splenocytes from class II HER2-DC1 vaccinated mice were re-stimulated with (class II) rat HER2 peptides (rHER2); p5 (ELAAWCRWGFLLALLPPGIAG), p435 (IRGRILHDGAYSLTLQGLGIH), and p1209 (SPPHPSPAFSPAFDNLYYWDQ) individually and IFN-γ secretion was measured by standard IFN-γ ELISA. (C) Flow gating strategy and analysis of T cell infiltration in tumors by flow cytometry. Tumors were harvested on day 30 and processed as described in Materials and Methods section. Single cell suspension was stained for live/dead near IR, CD3, CD4, and CD8. Data were acquired on an LSRII flow cytometer and analyzed by FlowJo software. (D) Bar graphs represent T cell infiltration gated on live cells per mg of tumor and (E) percent T cells of all live cells within the tumor single cell suspension;. *** p < 0.001, ** p < 0.01, * p < 0.05 using Student t -test.

Article Snippet: Cells were then incubated with monoclonal primary anti-HER2/ErbB2 antibody (Cell Signaling Technology, Beverly, MA, Cat. No. 2165) over night at 4°C followed by incubation with Alexa Fluor 594-conjugated anti-rabbit secondary antibody (Cell Signaling Technology, Beverly, MA, Cat. No. 8889) for 1 h at room temperature in the dark.

Techniques: Enzyme-linked Immunosorbent Assay, Flow Cytometry, Staining, Software

Journal: Frontiers in Immunology

Article Title: Sequential Anti-PD1 Therapy Following Dendritic Cell Vaccination Improves Survival in a HER2 Mammary Carcinoma Model and Identifies a Critical Role for CD4 T Cells in Mediating the Response

doi: 10.3389/fimmu.2019.01939

Figure Lengend Snippet: PD-1 expression on TIL and PD-L1 expression on tumors of TUBO bearing mice. (A) Tumors were harvested 1 week after the last HER2-DC1 treatment and single suspensions were prepared as described in Materials and Methods section. Single cell tumor digest suspensions were stained for PD-L1 expression and data were acquired on a LSR-II and analyzed using Flowjo software. (B) Flow gating strategy of PD-1 expression on tumor infiltrating T cells. (C) Tumors were harvested as described in Materials and Methods section and flow staining was performed. Bar graphs show the percent PD-1 expression on CD4 and CD8+ TIL * p < 0.05, NS, Not significant using Student t -test.

Article Snippet: Cells were then incubated with monoclonal primary anti-HER2/ErbB2 antibody (Cell Signaling Technology, Beverly, MA, Cat. No. 2165) over night at 4°C followed by incubation with Alexa Fluor 594-conjugated anti-rabbit secondary antibody (Cell Signaling Technology, Beverly, MA, Cat. No. 8889) for 1 h at room temperature in the dark.

Techniques: Expressing, Staining, Software

Journal: Frontiers in Immunology

Article Title: Sequential Anti-PD1 Therapy Following Dendritic Cell Vaccination Improves Survival in a HER2 Mammary Carcinoma Model and Identifies a Critical Role for CD4 T Cells in Mediating the Response

doi: 10.3389/fimmu.2019.01939

Figure Lengend Snippet: Optimal scheduling of checkpoint antibodies is critical in improving the efficacy of class I HER2-DC1 vaccine in TUBO bearing mice. (A) Balb/C mice were injected with 2.5 × 10 5 TUBO cells subcutaneously on the right flank on day 0. When tumors were palpable on day 7, mice received class I HER2-DC1 vaccine subcutaneously on the left flank twice a week for a total of six doses concurrently with intraperitoneal injection of 150 μg/mouse/200 μl of anti-PD-1 or anti-PD-L1 antibody. (B) Balb/C mice were injected with 2.5 × 10 5 TUBO cells subcutaneously on the right flank on day 0. When tumors were palpable on day 7, mice received class I HER2-DC1 vaccine (1 × 10 6 DC1/mouse/100 μl) subcutaneously on the left flank twice a week for a total of six doses. Anti-PD-1 or anti-PD-L1 antibody therapy (150 mg/200 ml/mouse/intraperitoneally) twice a week was started after the last injection of HER2-DC1 and continued until the end point. N = 8 mice/group was used for these studies and the line graph shown is the representative of triplicate experiments. (C) Survival curve. Data shown are the representative from three independent experiments and are shown as the mean number ± SEM. P -values were determined by unpaired student t -test (A,B) or a log-rank test (C) . ** p < 0.01.

Article Snippet: Cells were then incubated with monoclonal primary anti-HER2/ErbB2 antibody (Cell Signaling Technology, Beverly, MA, Cat. No. 2165) over night at 4°C followed by incubation with Alexa Fluor 594-conjugated anti-rabbit secondary antibody (Cell Signaling Technology, Beverly, MA, Cat. No. 8889) for 1 h at room temperature in the dark.

Techniques: Injection

Journal: Frontiers in Immunology

Article Title: Sequential Anti-PD1 Therapy Following Dendritic Cell Vaccination Improves Survival in a HER2 Mammary Carcinoma Model and Identifies a Critical Role for CD4 T Cells in Mediating the Response

doi: 10.3389/fimmu.2019.01939

Figure Lengend Snippet: Anti-tumor efficacy of class II HER2 peptides pulsed DC1 in combination with anti-PD-1 antibody therapy. (A) Balb/C mice were injected with 2.5 × 10 5 TUBO cells subcutaneously on the right flank on day 0. When tumors were palpable on day 7, mice received class II HER2 peptides pulsed DC1 vaccine (1 × 10 6 DC1/mouse/100 μl) subcutaneously on the left flank twice a week for a total of six doses followed by intraperitoneal injection of 150 μg/mouse/200 μl of anti-PD-1 antibody twice a week until the end point. (B) Survival curve. Data shown are representative from three independent experiments and are shown as the mean number ± SEM. P -values were determined by unpaired student t- test (A) or a log-rank test (B) . *** p < 0.001, ** p < 0.01.

Article Snippet: Cells were then incubated with monoclonal primary anti-HER2/ErbB2 antibody (Cell Signaling Technology, Beverly, MA, Cat. No. 2165) over night at 4°C followed by incubation with Alexa Fluor 594-conjugated anti-rabbit secondary antibody (Cell Signaling Technology, Beverly, MA, Cat. No. 8889) for 1 h at room temperature in the dark.

Techniques: Injection

Journal: Frontiers in Immunology

Article Title: Sequential Anti-PD1 Therapy Following Dendritic Cell Vaccination Improves Survival in a HER2 Mammary Carcinoma Model and Identifies a Critical Role for CD4 T Cells in Mediating the Response

doi: 10.3389/fimmu.2019.01939

Figure Lengend Snippet: HER2-DC1 vaccine in combination with anti-PD-1 antibody therapy improves T cell infiltration, function, and specificity. (A,B) Tumors were collected and single cell suspensions were prepared and stained for CD3, CD4, and CD8 as described in methods. Bar graph shows T cell infiltration per mg of tumor. (C) Percent T cells of all live cells within the tumor single cell suspension. (D,E) Splenocytes were re-stimulated with class I, class II HER2 peptides or irrelevant OT-I or OT-II peptides. Culture supernatants were collected after 72 h and IFN-γ was measured by standard ELISA. P -values were determined by unpaired student t -test *** p < 0.001, ** p < 0.01, * p < 0.05.

Article Snippet: Cells were then incubated with monoclonal primary anti-HER2/ErbB2 antibody (Cell Signaling Technology, Beverly, MA, Cat. No. 2165) over night at 4°C followed by incubation with Alexa Fluor 594-conjugated anti-rabbit secondary antibody (Cell Signaling Technology, Beverly, MA, Cat. No. 8889) for 1 h at room temperature in the dark.

Techniques: Staining, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Sequential Anti-PD1 Therapy Following Dendritic Cell Vaccination Improves Survival in a HER2 Mammary Carcinoma Model and Identifies a Critical Role for CD4 T Cells in Mediating the Response

doi: 10.3389/fimmu.2019.01939

Figure Lengend Snippet: Depletion of CD4+ T cells abrogates the anti-tumor efficacy of HER2-DC1 vaccine and anti-PD1 antibody therapy. (A) Balb/c mice were injected s.c. with TUBO cells and treated with class II HER2-DC1 vaccine (1 × 10 6 DC1/mouse/100 μl, twice a week for 3 weeks) with or without anti-PD1 antibody (150 mg/200 ml/mouse/intraperitoneally twice a week until the end point). CD4 depleting antibody (clone GK1.5) was administered 3 days prior to TUBO induction and continued twice a week until the end point. Tumor growth was monitored periodically. (B) Survival curve. Data shown are representative from three independent experiments and are shown as the mean number ± SEM. P -values were determined by unpaired student t- test (A) or a log-rank test (B) . *** p < 0.001, ** p < 0.01, * p < 0.05.

Article Snippet: Cells were then incubated with monoclonal primary anti-HER2/ErbB2 antibody (Cell Signaling Technology, Beverly, MA, Cat. No. 2165) over night at 4°C followed by incubation with Alexa Fluor 594-conjugated anti-rabbit secondary antibody (Cell Signaling Technology, Beverly, MA, Cat. No. 8889) for 1 h at room temperature in the dark.

Techniques: Injection

Journal: Frontiers in Immunology

Article Title: Sequential Anti-PD1 Therapy Following Dendritic Cell Vaccination Improves Survival in a HER2 Mammary Carcinoma Model and Identifies a Critical Role for CD4 T Cells in Mediating the Response

doi: 10.3389/fimmu.2019.01939

Figure Lengend Snippet: HER2 targeted therapy in combination with HER2-DC1 vaccine and anti-PD-1 therapy delays tumor growth and improves survival. (A) The treatment schema. (B) Balb/C mice were injected with 3 × 10 4 TUBO cells into the mammary fat pad to create orthotopic primary tumors. TUBO bearing mice with orthotopic primary tumor were treated with class II HER2 peptide pulsed DC1 vaccine (1 × 10 6 DC1/mouse/100 μl, twice a week for 3 weeks), anti-PD-1 antibody (two times per week) or the combination of HER2 targeted monoclonal antibodies 7.16.4 and 7.6.5 (once in a week) and class II HER2 peptide pulsed DC1 vaccine followed up with anti-PD-1 antibody twice a week. (C) Survival curve. Data shown are representative from three independent experiments and are shown as the mean number ± SEM. P -values were determined by unpaired student t -test (B) or a log-rank test (C) . *** p < 0.001, ** p < 0.01.

Article Snippet: Cells were then incubated with monoclonal primary anti-HER2/ErbB2 antibody (Cell Signaling Technology, Beverly, MA, Cat. No. 2165) over night at 4°C followed by incubation with Alexa Fluor 594-conjugated anti-rabbit secondary antibody (Cell Signaling Technology, Beverly, MA, Cat. No. 8889) for 1 h at room temperature in the dark.

Techniques: Injection

Journal: Journal of medicinal chemistry

Article Title: Multiplexed Targeting of Barrett’s Neoplasia with a Heterobivalent Ligand: Imaging Study on Mouse Xenograft In Vivo and Human Specimens Ex Vivo

doi: 10.1021/acs.jmedchem.8b00405

Figure Lengend Snippet: On confocal microscopy, fluorescence from binding of candidate heterodimer with linkers A) E2, B) Hex, C) E3, D) E6, and E) E10 to the surface (arrow) of SKBr3 cells can be seen. F) Quantified results show that the E3 linker provides the highest mean fluorescence intensity. P-values were determined using unpaired t-test. Measurements are an average of 10 randomly chosen cells from 4 images collected independently. G) Western blot shows EGFR and ErbB2 expression in SKBr3 and QhTERT cells.

Article Snippet: The sections were then washed for 3 min with PBS 3X and further incubated sequentially with 1:200 dilution of EGFR monoclonal antibody (H11, #MA5–13070, Invitrogen) and 1:500 goat anti-mouse IgG (H+L) AF-568-labeled secondary antibody (#A-11004, Thermo Fisher Scientific), 1:500 dilution of ErbB2 primary antibody (#29D8, rabbit monoclonal antibody #2165S, Cell Signaling Inc) and 1:1000 goat anti-rabbit IgG (H+L) AF-488-labeled secondary antibody (# {"type":"entrez-nucleotide","attrs":{"text":"A11008","term_id":"492390","term_text":"A11008"}} A11008 , Life Technologies Corp).

Techniques: Confocal Microscopy, Fluorescence, Binding Assay, Western Blot, Expressing

Journal: Journal of medicinal chemistry

Article Title: Multiplexed Targeting of Barrett’s Neoplasia with a Heterobivalent Ligand: Imaging Study on Mouse Xenograft In Vivo and Human Specimens Ex Vivo

doi: 10.1021/acs.jmedchem.8b00405

Figure Lengend Snippet: On representative confocal microscopy images of human esophageal specimens ex vivo, QRH*-KSP*-E3-Cy5.5 (red) shows minimal staining to A) squamous (SQ) and B) Barrett’s esophagus (BE), and increased intensity with C) high-grade dysplasia (HGD) and D) esophageal adenocarcinoma (EAC). Similar results were found with AF568-labeled anti-EGFR antibody (yellow) and for AF488-labeled anti-ErbB2 antibody (green). Merged images show co-localization of peptide and antibody binding.

Article Snippet: The sections were then washed for 3 min with PBS 3X and further incubated sequentially with 1:200 dilution of EGFR monoclonal antibody (H11, #MA5–13070, Invitrogen) and 1:500 goat anti-mouse IgG (H+L) AF-568-labeled secondary antibody (#A-11004, Thermo Fisher Scientific), 1:500 dilution of ErbB2 primary antibody (#29D8, rabbit monoclonal antibody #2165S, Cell Signaling Inc) and 1:1000 goat anti-rabbit IgG (H+L) AF-488-labeled secondary antibody (# {"type":"entrez-nucleotide","attrs":{"text":"A11008","term_id":"492390","term_text":"A11008"}} A11008 , Life Technologies Corp).

Techniques: Confocal Microscopy, Ex Vivo, Staining, Labeling, Binding Assay

Journal: Journal of medicinal chemistry

Article Title: Multiplexed Targeting of Barrett’s Neoplasia with a Heterobivalent Ligand: Imaging Study on Mouse Xenograft In Vivo and Human Specimens Ex Vivo

doi: 10.1021/acs.jmedchem.8b00405

Figure Lengend Snippet: In siRNA knockdown experiments, QRH*-KSP*-E3-Cy5.5 (red) shows significantly greater binding to the surface (arrows) of A) siCL (control) SkBr3 cells compared with that for B) siEGFR (knockdown) cells. Similar results were found for C) siCL and D) siErbB2 (knockdown) cells. E) Quantified results show significantly greater intensity for siCL versus siEGFR and siCL versus siErbB2, P=3.6×10−4 and P=7.8×10−3, respectively, by unpaired t-test. The mean value was calculated from 5 cells chosen randomly from 3 images collected independently. F) Western blot shows EGFR and ErbB2 expression in control and knockdown cells. G) The apparent dissociation constant (binding affinity) for QRH*-KSP*-E3-Cy5.5 was found to be kd = 23 versus 98 and 54 nM for QRH*-Cy5.5 and KSP*-Cy5.5. H) The apparent association time constant for QRH*-KSP*-E3-Cy5.5 was found to be k = 0.22 min−1 (4.5 min) versus 0.21 min−1 (4.8 min) and 0.35 min−1 (2.9 min) for QRH*-Cy5.5 and KSP*-Cy5.5. Results for each measurement are representative of 3 independent experiments.

Article Snippet: The sections were then washed for 3 min with PBS 3X and further incubated sequentially with 1:200 dilution of EGFR monoclonal antibody (H11, #MA5–13070, Invitrogen) and 1:500 goat anti-mouse IgG (H+L) AF-568-labeled secondary antibody (#A-11004, Thermo Fisher Scientific), 1:500 dilution of ErbB2 primary antibody (#29D8, rabbit monoclonal antibody #2165S, Cell Signaling Inc) and 1:1000 goat anti-rabbit IgG (H+L) AF-488-labeled secondary antibody (# {"type":"entrez-nucleotide","attrs":{"text":"A11008","term_id":"492390","term_text":"A11008"}} A11008 , Life Technologies Corp).

Techniques: Binding Assay, Western Blot, Expressing

Journal: Journal of medicinal chemistry

Article Title: Multiplexed Targeting of Barrett’s Neoplasia with a Heterobivalent Ligand: Imaging Study on Mouse Xenograft In Vivo and Human Specimens Ex Vivo

doi: 10.1021/acs.jmedchem.8b00405

Figure Lengend Snippet: We evaluated the effect of the QRH*-KSP*-E3-Cy5.5 on downstream cell signaling after binding to SKBr3 cells. On Western blot, we observed no change in phosphorylation of EGFR (p-EGFR), ErbB2 (p-ErbB2) or of downstream AKT (p-AKT) and ERK (p-ERK) with incubation of heterodimer at 1, 5, and 20 μM. By comparison, the addition of EGF, an endogenous ligand for EGFR, showed increased expression of p-AKT and p-ERK. The addition of 100 nM of lapatinib, a tyrosine kinase inhibitor known to interrupt EGFR/ErbB2 signaling in solid tumors, showed reduced expression of p-EGFR, p-ErbB2 and p-AKT. Cells treated with 1% DMSO and untreated cells showed no suppression of EGFR and ErbB2 mediated signaling. β-tubulin is used as loading control.

Article Snippet: The sections were then washed for 3 min with PBS 3X and further incubated sequentially with 1:200 dilution of EGFR monoclonal antibody (H11, #MA5–13070, Invitrogen) and 1:500 goat anti-mouse IgG (H+L) AF-568-labeled secondary antibody (#A-11004, Thermo Fisher Scientific), 1:500 dilution of ErbB2 primary antibody (#29D8, rabbit monoclonal antibody #2165S, Cell Signaling Inc) and 1:1000 goat anti-rabbit IgG (H+L) AF-488-labeled secondary antibody (# {"type":"entrez-nucleotide","attrs":{"text":"A11008","term_id":"492390","term_text":"A11008"}} A11008 , Life Technologies Corp).

Techniques: Binding Assay, Western Blot, Incubation, Expressing

Journal: Journal of medicinal chemistry

Article Title: Multiplexed Targeting of Barrett’s Neoplasia with a Heterobivalent Ligand: Imaging Study on Mouse Xenograft In Vivo and Human Specimens Ex Vivo

doi: 10.1021/acs.jmedchem.8b00405

Figure Lengend Snippet: A) On confocal microscopy, serial sections of HGD in human esophageal specimens are shown following staining with QRH*-KSP*-E3-Cy5.5 (red), anti-EGFR antibody labeled with AF568 (yellow) and anti-ErbB2 antibody labeled with AF488 (green). Fluorescence intensities were quantified from the mean of a set of 3 boxes with dimensions of 20×20 μm2 placed over random crypts. Co-localization of binding can be appreciated on the merged image. B) High-magnification images are shown from dashed boxes. On the merged image, Pearson’s correlation coefficient of ρ = 0.60 and 0.75 was measured for EGFR and ErbB2, respectively. C) From n = 31, 8, 23, and 12 specimens of SQ, BE, HGD, and EAC, respectively, we found significantly greater mean fluorescence intensity from HGD and EAC compared with that for BE and SQ with QRH*-KSP*-E3-Cy5.5, the P-value for difference are calculated by Tukey’s multiple comparisons. A similar result was found for anti-EGFR-AF568 and anti-ErbB2-AF488. D) ROC curve shows 88% sensitivity, 87% specificity and 0.95 AUC with QRH*-KSP*-E3-Cy5.5; 74% sensitivity, 69% specificity, and 0.79 AUC with QRH*-Cy5.5, and 85% sensitivity, 79% specificity, and 0.91 AUC with KSP*-Cy5.5.

Article Snippet: The sections were then washed for 3 min with PBS 3X and further incubated sequentially with 1:200 dilution of EGFR monoclonal antibody (H11, #MA5–13070, Invitrogen) and 1:500 goat anti-mouse IgG (H+L) AF-568-labeled secondary antibody (#A-11004, Thermo Fisher Scientific), 1:500 dilution of ErbB2 primary antibody (#29D8, rabbit monoclonal antibody #2165S, Cell Signaling Inc) and 1:1000 goat anti-rabbit IgG (H+L) AF-488-labeled secondary antibody (# {"type":"entrez-nucleotide","attrs":{"text":"A11008","term_id":"492390","term_text":"A11008"}} A11008 , Life Technologies Corp).

Techniques: Confocal Microscopy, Staining, Labeling, Fluorescence, Binding Assay

Journal: Science Advances

Article Title: A versatile 3D tissue matrix scaffold system for tumor modeling and drug screening

doi: 10.1126/sciadv.1700764

Figure Lengend Snippet: ( A ) The workflow of the porous TMS fabrication. (1) Collection of breast tissues from 8- to 12-week-old mice. (2) Decellularization of the native tissues to produce ECM. (3) Lyophilization of the ECM at −50°C. (4) Enzymatic digestion of the ground ECM in acidic solution. (5) Neutralization of the acidic ECM solution–generated hydrogel. (6) Loading the hydrogel into the spherical molds. (7) Formation of the pre-scaffolds in the molds at −80°C. (8) Lyophilization of the pre-scaffolds. (9) Formation of the porous scaffolds in the molds. (10) Treatment of the scaffolds with absolute ethanol and cross-linking the ECM proteins under UV light. (11) Lyophilization of the scaffolds to remove the ethanol. (12) Characterization of the finished TMS scaffolds. A microscopic view of the TMS cross sections after H&E staining is shown. Scale bars, 100 μm. ( B ) Comparison of the composition of the decellularized tissues with that of the native tissues at DNA and major ECM protein levels. Error bars represent the SD of the measurements of three independent batches of the ECM samples. ( C ) Characterization of the TMS porosity under SEM. Different amounts of the lyophilized ECM powder were used to generate TMSs at different pore sizes. ( D ) Histological comparison of the cross sections of the blank and the cell-laden DBT-TMS with the decellularized and the native mouse breast tissue. ( E ) Comparison of the occupancies of the cells grown inside the DBT-TMS with that of the native cells that lived in mouse breast tissues. Left: The closeup views of the H&E-stained cross sections of the fibroblast-laden TMS and the mammary fat pad tissues. Top right: An SEM image showing the occupancies of the MM231 cells on the surface and within the porous TMS. Bottom right: Distribution patterns of the MM231 cells and stromal cells immunostained with Ki-67 (green) and HER2 (red), respectively, on the cross sections of mouse breast tumors that originated from the MM231 cell–laden TMS. DAPI (4′,6-diamidino-2-phenylindole) was used to stain the nuclei of the cells. The red and the yellow arrows indicate stromal and MM231 cells, respectively. Scale bars, 100 μm (C to E).

Article Snippet: Primary rabbit antibody for HER2 (#2165) and mouse antibody for Ki-67 (#9449) were purchased from Cell Signaling Technology.

Techniques: Neutralization, Generated, Staining

Journal: Science Advances

Article Title: A versatile 3D tissue matrix scaffold system for tumor modeling and drug screening

doi: 10.1126/sciadv.1700764

Figure Lengend Snippet: ( A ) Generation of the multilayered/compartmentalized TMS culture system. MM231 cells were cultured on the porous DBT-TMS followed by either covering them with a layer of blank TMS hydrogel or directly placing them into culture for in vitro or in vivo experiments. Hydrogel premixed with another type of cells different from those coated on the porous TMS was applied outside the first layer and enzymatically cross-linked, forming a second gel layer. The multilayered TMS assembly was then subjected to culture and/or implantation into animals for further analysis or applications. ( B ) H&E staining of the cross sections of a TMS coated with MM231 cells and a layer of hydrogel. ( C ) H&E staining of the cross sections of a multilayered TMS containing the porous TMS core coated with MM231 cells and two hydrogel layers with the second gel layer containing the human GM637 fibroblasts. The middle region outlined by dotted lines was a blank hydrogel layer. ( D ) DAPI staining of the cross sections of the compartmentally cultured cells grown in the multilayered TMS after 3 days of culture, as shown in (C). ( E ) IF microscopic view of Ki-67 (green, MM231 cells) and HER2 (red, GM637 cells) staining on the cross sections of the compartmental TMS samples. Selected regional blowups of the Ki-67 and HER2 staining are shown as insets. ( F to I ) Live/Dead Cell staining of the cross sections of the compartmentally cultured MM231 cells (on the porous TMS, right side of the blank hydrogel layer) and the human GM637 fibroblasts (within the second hydrogel layer, left side of the blank hydrogel layer) at different time points of the cultures. Scale bars, 100 μm.

Article Snippet: Primary rabbit antibody for HER2 (#2165) and mouse antibody for Ki-67 (#9449) were purchased from Cell Signaling Technology.

Techniques: Cell Culture, In Vitro, In Vivo, Staining

Journal: Science Advances

Article Title: A versatile 3D tissue matrix scaffold system for tumor modeling and drug screening

doi: 10.1126/sciadv.1700764

Figure Lengend Snippet: ( A ) Evaluation of the biodegradability of the scaffolds and their supports on the MM231 cell–originated tumor development (dissection microscopy images). Scale bars, 4 mm. ( B ) Quantification of the sizes of the tumors formed from the different MM231 cell–laden scaffolds. The plotted values reflect the ex vivo measurements of the tumors. The error bars represent the SD of the sizes of three individual tumors of the same implantation background. * P < 0.05; ** P < 0.01, significance of the comparison between the indicated sample groups. ( C ) (i to iv) H&E staining of the cross sections of the tumors that originated from the MM231 cell–laden DBT-TMS and DMM231 scaffolds with or without hydrogel coverage. The tumor ECM structure, cell distribution, and capillaries (containing the stained red blood cells) are demonstrated. (v to viii) IF staining of Ki-67 (green) and HER2 (red) on the tumor cross sections. The cell nuclei were stained with DAPI (blue). Scale bars, 100 μm (C, i to viii).

Article Snippet: Primary rabbit antibody for HER2 (#2165) and mouse antibody for Ki-67 (#9449) were purchased from Cell Signaling Technology.

Techniques: Dissection, Microscopy, Ex Vivo, Staining