Journal: Nature Communications

Article Title: Antibody targeting intracellular oncogenic Ras mutants exerts anti-tumour effects after systemic administration

doi: 10.1038/ncomms15090

Figure Lengend Snippet: ( a ) Cellular internalization and cytosol localization of GFP11-SBP2-fused RT11 and TMab4 antibodies, as assessed by confocal microscopy measuring complemented GFP signals (green) in HeLa-SA-GFP1-10 cells after treatment with 1 μM of the antibodies for 6 h. Scale bar, 20 μm. ( b ) Cellular internalization and co-localization of RT11 (green), but not TMab4 (green), with the inner plasma membrane-anchored active Ras (red) in mCherry-KRas G12V -transformed NIH3T3 and KRas G12V -harbouring SW480 cells, analysed by confocal microscopy. The Ras WT -harbouring HT29 cells were also analysed as a control. The areas in the white boxes are shown at a higher magnification for better visualization. The arrow indicates the co-localization of RT11 with activated Ras. The cells were treated with 2 μM of antibody for 12 h. Scale bar, 5 μm. In a , b , nuclei are counterstained with Hoechst33342 (blue). ( c ) Immunoprecipitation (IP) of KRas mutant with RT11, but not TMab4, from endosome-depleted cell lysates of HA-KRas G12V -transformed NIH3T3 and SW480 cells, treated with 2 μM of antibody for 12 h before analysis. The endosome-depleted cell lysates were assessed by the absence of Rab5, an early endosome marker . In a – c , images are representative of at least two independent experiments.

Article Snippet: The human cell lines, cervix carcinoma HeLa cells, colorectal carcinoma SW480, LoVo, HT29, and Colo320DM cells, pancreatic carcinoma AsPC-1 and PANC-1 cells, soft tissue sarcoma HT1080 cells, lung carcinoma H1299, acute myeloid leukemia HL60 cells, chronic myeloid leukemia K562 cells, breast carcinoma MCF-7 cells and mouse embryonic fibroblast NIH3T3 cells were purchased from the American Type Culture Collection (ATCC).

Techniques: Confocal Microscopy, Clinical Proteomics, Membrane, Transformation Assay, Control, Immunoprecipitation, Mutagenesis, Marker

Journal: Nature Communications

Article Title: Antibody targeting intracellular oncogenic Ras mutants exerts anti-tumour effects after systemic administration

doi: 10.1038/ncomms15090

Figure Lengend Snippet: ( a ) Cellular proliferation assay, after cells were treated twice at 0 and 72 h with antibody at the indicated concentrations for 6 d. Error bars±s.d. ( n =3). * P <0.05, ** P <0.01, *** P <0.001 versus TMab4-treated cells; NS, not significant. ( b ) Intracellular distribution of eGFP-fused cRaf RBD protein (green) in eGFP-cRaf RBD -transformed SW480 cells, treated with antibody (2 μM) for 12 h before microscopic confocal analysis. Scale bar, 20 μm. ( c , d ) IP of endogenous Raf proteins (bRaf and cRaf) with HA-tagged KRas G12V from the endosome-depleted cell lysates of HA-KRas G12V -transformed NIH3T3 cells ( c ) and IP of endogenous KRas G12V with cRaf RBD from the endosome-depleted cell lysates of SW480 cells ( d ). The cells were treated with 2 μM of RT11 and TMab4 for 12 h before analysis. ( e , f ) Inhibitory effect of RT11 on the downstream signalling of KRas-effector PPIs in HA-KRas G12V -transformed NIH3T3 cells ( e ) and SW480 cells ( f ), analysed by western blotting. The cells were serum-starved for 6 h before treatment with antibody, Raf kinase inhibitor sorafenib, or PI3K-Akt inhibitor LY294002 for 6 h in serum-free growth medium. Cells were washed and then stimulated with 10% FBS ( e ) and EGF (50 ng ml −1 in serum free-media) ( f ) for 10 min before cell lysis. The number below the panel indicates relative value of band intensity of phosphorylated proteins compared to that in the PBS-treated control after normalization to the band intensity of respective total protein for each sample. * P <0.05, ** P <0.01, *** P <0.001 versus PBS-treated control cells. In b – f , images are representative of at least two independent experiments.

Article Snippet: The human cell lines, cervix carcinoma HeLa cells, colorectal carcinoma SW480, LoVo, HT29, and Colo320DM cells, pancreatic carcinoma AsPC-1 and PANC-1 cells, soft tissue sarcoma HT1080 cells, lung carcinoma H1299, acute myeloid leukemia HL60 cells, chronic myeloid leukemia K562 cells, breast carcinoma MCF-7 cells and mouse embryonic fibroblast NIH3T3 cells were purchased from the American Type Culture Collection (ATCC).

Techniques: Proliferation Assay, Transformation Assay, Western Blot, Lysis, Control

Journal: Nature Communications

Article Title: Antibody targeting intracellular oncogenic Ras mutants exerts anti-tumour effects after systemic administration

doi: 10.1038/ncomms15090

Figure Lengend Snippet: ( a ) Generation of integrin αvβ3/αvβ5-targeting RT11-i by genetic fusion of RGD10 peptide, using a (G 4 S) 2 linker, to the N-terminus of the LC of RT11. ( b , c ) RT11-i and TMab4-i bind to cell surface-expressed integrin ανβ3 and ανβ5. In b , flow cytometric analysis of the cell surface expression levels of integrin ανβ3 and ανβ5 on WT K562, integrin ανβ3-transformed K562, and human tumour cells, analysed by PE-conjugated anti-human integrin ανβ3 and ανβ5 antibodies. In c , flow cytometric analysis of cell surface binding levels of the indicated antibodies, co-incubated at 100 nM with 300 IU ml −1 heparin for 1 h at 4 °C with the indicated cells before analysis. ( d ) Cellular internalization and co-localization of RT11-i, but not TMab4-i, with the inner plasma membrane-anchored active Ras·GTP in KRas G12V -harbouring SW480 cells. The Ras WT -harbouring HT29 cells were also analysed as a control. The areas in the white boxes are shown at increased magnification for better visualization. The arrow indicates the co-localization of RT11-i with activated Ras. Nuclei were counterstained with Hoechst 33342 (blue). Scale bar, 5 μm. ( e ) IP of endogenous KRas G12V with RT11 or RT11-i, but not TMab4 and TMab4-i, from endosome-depleted cell lysates of SW480 cells. Images are representative of two independent experiments. In d , e , the cells were treated with 1 μM of antibodies for 12 h before analysis. ( f ) Inhibition of tumour cell soft agar colony formation by RT11-i compared to that with TMab4-i. Following treatment of cells with PBS, TMab4-i (2 μM), or RT11-i (2 μM) every 72 h for 2–3 weeks, the number of colonies (diameter>200 μm) was counted by BCIP/NBT staining, as shown in the pictures of representative soft agar plates . The results are presented as percentages compared to the PBS-treated control. Error bars represent the mean±s.d. ( n =3). **P <0.01, ***P <0.001; NS, not significant.

Article Snippet: The human cell lines, cervix carcinoma HeLa cells, colorectal carcinoma SW480, LoVo, HT29, and Colo320DM cells, pancreatic carcinoma AsPC-1 and PANC-1 cells, soft tissue sarcoma HT1080 cells, lung carcinoma H1299, acute myeloid leukemia HL60 cells, chronic myeloid leukemia K562 cells, breast carcinoma MCF-7 cells and mouse embryonic fibroblast NIH3T3 cells were purchased from the American Type Culture Collection (ATCC).

Techniques: Expressing, Transformation Assay, Binding Assay, Incubation, Clinical Proteomics, Membrane, Control, Inhibition, Staining

Journal: Nature Communications

Article Title: Antibody targeting intracellular oncogenic Ras mutants exerts anti-tumour effects after systemic administration

doi: 10.1038/ncomms15090

Figure Lengend Snippet: ( a ) Pharmacokinetic profiles of RT11-i and TMab4-i in non-tumour bearing mice. Serum concentrations of TMab-i and RT11-i were determined by ELISA in female BALB/c nude mice following a single intravenous injection of 20 mg kg −1 in a total volume of 200 μl. Error bars represent the mean±s.d. ( n =3 per time point). The solid lines represent the fit of a two-compartment pharmacokinetic model to the data to estimate the initial rapid clearance phase (T 1/2 α) and later terminal serum clearance phase (T 1/2 β). The inset table shows the pharmacokinetic parameters. ( b ) Tumour-targeting ability of RT11-i and TMab4-i, evaluated by intravenously injecting Dylight755-labelled antibodies (20 μg per mouse) into SW480 xenograft tumour-bearing mice, followed by in vivo fluorescence imaging. Representative images are shown, which were acquired at the indicated times post-injection. Fluorescence intensities in the tumour tissue (T), as indicated by arrows, and normal tissues (N) were quantified by radiant efficiency (photons s −1 cm −2 steradian −1 μW −1 cm −2 ) using Living Image software. Error bars,±s.d. ( n =5 per group).

Article Snippet: The human cell lines, cervix carcinoma HeLa cells, colorectal carcinoma SW480, LoVo, HT29, and Colo320DM cells, pancreatic carcinoma AsPC-1 and PANC-1 cells, soft tissue sarcoma HT1080 cells, lung carcinoma H1299, acute myeloid leukemia HL60 cells, chronic myeloid leukemia K562 cells, breast carcinoma MCF-7 cells and mouse embryonic fibroblast NIH3T3 cells were purchased from the American Type Culture Collection (ATCC).

Techniques: Enzyme-linked Immunosorbent Assay, Injection, In Vivo, Fluorescence, Imaging, Software

Journal: Nature Communications

Article Title: Antibody targeting intracellular oncogenic Ras mutants exerts anti-tumour effects after systemic administration

doi: 10.1038/ncomms15090

Figure Lengend Snippet: ( a ) In vivo anti-tumour efficacy of RT11-i compared to that of vehicle and TMab4-i controls, analysed by measuring the tumour volume during treatment of female BALB/c nude mice harbouring the indicated tumour xenografts. Antibodies were intravenously dosed at 20 mg kg −1 every 2 d (indicated by the arrows). Error bars,±s.d. ( n =8 per group). ( b , c ) Immunohistochemical images showing the levels of p-ERK1/2 and p-Akt ( b ) or cellular penetration and co-localization of antibodies with the active Ras form ( c ) in SW480 tumour tissues excised from mice following treatment described in a . Images are representative of three independent experiments. Nuclei were counterstained with Hoechst33342 (blue). Scale bar, 100 μm in b or 10 μm in c . In b , the right panel shows the percent relative fluorescence intensity compared to that of vehicle-treated control. Error bars,±s.d. of five random fields for each tumour (two tumours per group). In c , the areas in the white boxes are shown at a higher magnification for better visualization. The arrows indicate the co-localization of RT11-i with activated Ras. In a , b , statistical analysis was performed using a one-way analysis of variance followed by the Newman–Keuls post-test. * P <0.05, ** P <0.01, *** P <0.001 versus TMab4-i; NS, not significant.

Article Snippet: The human cell lines, cervix carcinoma HeLa cells, colorectal carcinoma SW480, LoVo, HT29, and Colo320DM cells, pancreatic carcinoma AsPC-1 and PANC-1 cells, soft tissue sarcoma HT1080 cells, lung carcinoma H1299, acute myeloid leukemia HL60 cells, chronic myeloid leukemia K562 cells, breast carcinoma MCF-7 cells and mouse embryonic fibroblast NIH3T3 cells were purchased from the American Type Culture Collection (ATCC).

Techniques: In Vivo, Immunohistochemical staining, Fluorescence, Control

Journal: Frontiers in Immunology

Article Title: Plasminogen activator inhibitor-1 promotes immune evasion in tumors by facilitating the expression of programmed cell death-ligand 1

doi: 10.3389/fimmu.2024.1365894

Figure Lengend Snippet: PAI-1 induces the expression of PD-L1 in human tumor cells. (A) Representative flow cytometric profiles of PD-L1 expression and MFI in the human cell lines HEK293T-WT, HEK293T-PAI-1 KO, ES2-WT, ES2-PAI-1 KO, MOLM14, K562-WT cells, and K562-PAI-1 OE cells treated with PAI-1 OE supernatant overnight. The experiments were repeated five times. (B) Representative flow cytometric profiles of PD-L1 expression and MFI ( n = 5/group) in HEK293T, ES2, MOLM14, and K562 cells after incubated with 100 nM rPAI-1 with or without 100 μM PAI-1 inhibitor TM5614. (C) Relative expression of PD-L1 mRNA in HEK293T, ES2, MOLM14, and K562 cells ( n = 5/cell type) after incubation with 100 nM rPAI-1 with or without 100 μM of the PAI-1 inhibitor TM5614. (D) Pearson’s correlation analysis between MFI of PAI-1 and MFI of PD-L1 in murine tumor cell lines (B16F1, B16F10, 32Dp210-WT, and 32Dp210-PAI-1 OE) and human tumor cell lines (HEK293T-WT, HEK293T-PAI-1 OE, ES2-WT, ES2-PAI-1 OE, K562-WT, and K562-PAI-1 OE). r: Pearson’s correlation coefficient. Bars are expressed as means ± SD of three independent experiments. ** p < 0.01, NS, non-significant.

Article Snippet: B16 (murine melanoma; F1 (parental), F10 (metastatic)), HEK293T (human embryonic kidney), ES2 (human clear cell ovarian carcinoma), and MOLM14 (human acute myeloid leukemia) cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Incubation

Journal: Frontiers in Immunology

Article Title: Plasminogen activator inhibitor-1 promotes immune evasion in tumors by facilitating the expression of programmed cell death-ligand 1

doi: 10.3389/fimmu.2024.1365894

Figure Lengend Snippet: PAI-1 promotes the production of soluble PD-L1, whereas blockade of PAI-1 suppresses the expression of PD-L1 expression both in vitro and in vivo. (A) Production of soluble PD-L1 in culture supernatants from MC38, B16F10, ES2, MOLM14, and K562 cells ( n = 5/cell type) after 4 days of incubation with 100 nM rPAI-1 with or without 100 μM of the PAI-1 inhibitor TM5614. (B) Representative flow cytometry graphs of PD-L1 expression and MFI were obtained from 32Dp210 cells engrafted in the spleen ( n = 6) and ES2 cells ( n = 6) engrafted in subcutaneous tissue at 14 days post-inoculation. Bars are expressed as means ± SD of three independent experiments. ** p < 0.01.

Article Snippet: B16 (murine melanoma; F1 (parental), F10 (metastatic)), HEK293T (human embryonic kidney), ES2 (human clear cell ovarian carcinoma), and MOLM14 (human acute myeloid leukemia) cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, In Vitro, In Vivo, Incubation, Flow Cytometry

Journal: Scientific Reports

Article Title: Antitumor effects of the GM3(Neu5Gc) ganglioside-specific humanized antibody 14F7hT against Cmah -transfected cancer cells

doi: 10.1038/s41598-019-46148-1

Figure Lengend Snippet: Expression of GM3(Neu5Gc) under hypoxic conditions on the SKOV3 human ovarian carcinoma cell line. SKOV3 cells were cultured under normoxic and hypoxic conditions with fetal bovine serum (FBS) for 7 days. ( A ) Expression of carbonic anhydrase IX (CAIX) as assessed by Western blot in cell lysates from SKOV3 cultures under normoxic or hypoxic conditions. GAPDH was used as loading control. Full-length blots are presented in Suppl. Fig. . ( B ) Cells were stained with 10 µg/mL of 14F7hT, 7C1 or isotype-matched control (itolizumab) followed by a fluorescein isothiocyanate (FITC)-conjugated anti-human IgG F(ab’) . Numbers represent percentage of positive cells and mean fluorescence intensity (MFI) value of 14F7hT staining. ( C ) Quantitative analysis of the expression of GM3(Neu5Gc) (left panel) or GM3(Neu5Gc/Ac) (right panel) under hypoxic conditions (n = 7). Statistical analysis was performed using Student’s t test ( p = 0.0006). ( D ) SKOV3 cells were cultured under normoxic or hypoxic conditions with FBS or HS for seven days. Cells were stained with 10 µg/mL of 14F7hT (filled histogram), 7C1 (black line) and isotype control (dotted line) followed by an FITC-conjugated anti-human IgG F(ab’) . ( E ) Quantitative analysis of four independent experiments. Normoxia: white bar; hypoxia: grey bar. Statistical analysis was performed using Mann-Whitney U test.

Article Snippet: Mouse P3X63-Ag8.653 (P3X63, non-immunoglobulin-producing myeloma, CRL-8375), B16-F10 (melanoma, CRL-6475), 4T1 (breast carcinoma, CRL-2539), human SKOV3 (ovarian adenocarcinoma, HTB-77), K562 (chronic myeloid leukemia, CCL-243), HeLa (cervix adenocarcinoma, CCL-2), A431 (epidermoid carcinoma, CRL-1555), MDA-MB-468 (mammary carcinoma, HTB-132), WERI-Rb-1 (retinoblastoma, HTB-169) and Y79 (retinoblastoma, HTB-18) and colorectal cell lines LS174T (CL-188), HCT116 (CCL-247), HT29 (HTB-38), SW620 (CCL-227) and SW116 (CCL-233) were purchased from the American Type Culture Collection (ATCC, Rockville, MD).

Techniques: Expressing, Cell Culture, Western Blot, Control, Staining, Fluorescence, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Antitumor effects of the GM3(Neu5Gc) ganglioside-specific humanized antibody 14F7hT against Cmah -transfected cancer cells

doi: 10.1038/s41598-019-46148-1

Figure Lengend Snippet: GM3(Neu5Gc) expression after transfection of the mouse Cmah gene in 3LL and SKOV3 cells. ( A ) Expression of the mouse CMAH enzyme as determined in cell lysates by Western Blot. GAPDH was used as loading control. Full-length blots are presented in Suppl. Fig. . ( B ) GM3(Neu5Gc) content in the transfected 3LL and SKOV3 cell lines. Total lipids were extracted and monosialogangliosides were purified by ion exchange chromatography and visualized by orcinol staining (upper panel). For immunostaining, the plates were incubated with mouse 14F7 and binding was revealed with an alkaline phosphatase-conjugated goat anti-mouse IgG antibody (bottom panel). Full-length blots are presented in Suppl. Fig. . ( C) Total Neu5Gc expression in the surface of transfected and wild type 3LL and SKOV3 cell lines. A chicken anti-Neu5Gc antiserum (filled histogram) was used, followed by an FITC-conjugated goat anti-IgY antiserum. Chicken polyclonal IgY was used as negative control (black line). ( D) 14F7hT binding to transfected and wild type 3LL and SKOV3 cell lines. Cells were stained with 10 µg/mL of 14F7hT (filled histogram), 7C1 (black line) or isotype-matched control antibody (itolizumab, dotted line) followed by a PE-conjugated anti-human IgG + IgM antiserum. P3X63 cells were used as positive control of GM3(Neu5Gc) expression. Data are representative of three independent experiments.

Article Snippet: Mouse P3X63-Ag8.653 (P3X63, non-immunoglobulin-producing myeloma, CRL-8375), B16-F10 (melanoma, CRL-6475), 4T1 (breast carcinoma, CRL-2539), human SKOV3 (ovarian adenocarcinoma, HTB-77), K562 (chronic myeloid leukemia, CCL-243), HeLa (cervix adenocarcinoma, CCL-2), A431 (epidermoid carcinoma, CRL-1555), MDA-MB-468 (mammary carcinoma, HTB-132), WERI-Rb-1 (retinoblastoma, HTB-169) and Y79 (retinoblastoma, HTB-18) and colorectal cell lines LS174T (CL-188), HCT116 (CCL-247), HT29 (HTB-38), SW620 (CCL-227) and SW116 (CCL-233) were purchased from the American Type Culture Collection (ATCC, Rockville, MD).

Techniques: Expressing, Transfection, Western Blot, Control, Purification, Ion Exchange Chromatography, Staining, Immunostaining, Incubation, Binding Assay, Negative Control, Positive Control

Journal: Scientific Reports

Article Title: Antitumor effects of the GM3(Neu5Gc) ganglioside-specific humanized antibody 14F7hT against Cmah -transfected cancer cells

doi: 10.1038/s41598-019-46148-1

Figure Lengend Snippet: 14F7hT-mediated antibody-dependent cell-mediated cytotoxicity of Cmah -transfected cell lines. (A) Induction of 14F7hT-mediated ADCC with human PBMC in mouse Cmah -transfected cell lines. Cytotoxicity was measured by a lactate dehydrogenase (LDH)-release assay. PBMC from human healthy donors were used as effector cells. ( B) Cytotoxicity was evaluated at different effector:target cell ratios on 3LL- Cmah (left panel) and SKOV3- Cmah (right panel) cells. Isotype-matched antibodies (infliximab in left panels and rituximab in right panels) were used as negative controls. Statistical analysis was performed using Mann-Whitney U test. Data are representative of three independent experiments.

Article Snippet: Mouse P3X63-Ag8.653 (P3X63, non-immunoglobulin-producing myeloma, CRL-8375), B16-F10 (melanoma, CRL-6475), 4T1 (breast carcinoma, CRL-2539), human SKOV3 (ovarian adenocarcinoma, HTB-77), K562 (chronic myeloid leukemia, CCL-243), HeLa (cervix adenocarcinoma, CCL-2), A431 (epidermoid carcinoma, CRL-1555), MDA-MB-468 (mammary carcinoma, HTB-132), WERI-Rb-1 (retinoblastoma, HTB-169) and Y79 (retinoblastoma, HTB-18) and colorectal cell lines LS174T (CL-188), HCT116 (CCL-247), HT29 (HTB-38), SW620 (CCL-227) and SW116 (CCL-233) were purchased from the American Type Culture Collection (ATCC, Rockville, MD).

Techniques: Transfection, Lactate Dehydrogenase Assay, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Antitumor effects of the GM3(Neu5Gc) ganglioside-specific humanized antibody 14F7hT against Cmah -transfected cancer cells

doi: 10.1038/s41598-019-46148-1

Figure Lengend Snippet: Antitumor effects of 14F7hT against Cmah -transfected cell lines in vivo . ( A,B) Anti-metastatic effect of 14F7hT in the 3LL- Cmah model. Cells (0.25 × 10 6 ) were injected into the lateral tail vein of C57BL/6 mice (n = 7–8) and 14FhT was administered intravenously in six doses until day 13. Weight ( A ) and representative macroscopic pictures ( B ) of the lungs upon euthanasia at day 21. Statistical analysis was performed using Mann-Whitney U test ( p = 0.0012). ( C) Antitumor effect of 14F7hT against SKOV3- Cmah cells. Cells (1 × 10 6 ) were subcutaneously inoculated to BALB/c nu/nu mice and 14F7hT administered intraperitoneally three times a week for three weeks. Tumor diameters were measured with a caliper and tumor volume was calculated. Data represent mean ± standard error of the mean, n = 6–7. Statistical analysis was performed using the two-way ANOVA test with Sidak’s post-test ( p = 0.0004). An isotype-matched antibody (itolizumab) was used as negative control. Data are representative of two independent experiments.

Article Snippet: Mouse P3X63-Ag8.653 (P3X63, non-immunoglobulin-producing myeloma, CRL-8375), B16-F10 (melanoma, CRL-6475), 4T1 (breast carcinoma, CRL-2539), human SKOV3 (ovarian adenocarcinoma, HTB-77), K562 (chronic myeloid leukemia, CCL-243), HeLa (cervix adenocarcinoma, CCL-2), A431 (epidermoid carcinoma, CRL-1555), MDA-MB-468 (mammary carcinoma, HTB-132), WERI-Rb-1 (retinoblastoma, HTB-169) and Y79 (retinoblastoma, HTB-18) and colorectal cell lines LS174T (CL-188), HCT116 (CCL-247), HT29 (HTB-38), SW620 (CCL-227) and SW116 (CCL-233) were purchased from the American Type Culture Collection (ATCC, Rockville, MD).

Techniques: Transfection, In Vivo, Injection, MANN-WHITNEY, Negative Control