Journal: bioRxiv

Article Title: Estrogen deprivation triggers an immunosuppressive phenotype in breast cancer cells

doi: 10.1101/715136

Figure Lengend Snippet: A, Overview of the phenotypic screen workflow. Briefly, A549 cells were seeded in 100ng/ml IFNγ 24hrs before addition of 4216 compounds at 10μM. After 24hrs of compound exposure, cells were stained with an anti-PD-L1 antibody conjugated to phycoerythrin and fixed with formaldehyde. Nuclei were stained with Hoechst and the immunofluorescences were analysed by high-throughput microscopy (HTM). B, Hit distribution of the screen described in A illustrating the enrichment of JAK inhibitors and corticosteroids among the compounds reducing PD-L1 signal in HTM. PD-L1 levels in wells only stimulating with IFNγor negative controls (DMSO) are also shown. C, Immunofluorescence of PD-L1 (red) in MCF7 cells grown in normal or steroid-free medium (SFM) for 15 days. DAPI (blue) was used to stain DNA. D, Scatterplot of PD-L1 intensity levels in SFM-grown MCF7 cells treated with ERαagonists or antagonists, screened at three concentrations, 0.1, 1.0 and 10 μ M.

Article Snippet: The following probes were used in this study: Hs01125301_m1 for CD274 , Hs99999901_s1 for 18S , Hs03929097_g1 for GAPDH , Hs01046817_m1 for ESR1 , Hs01100353_m1 for ESR2 , Hs00174128_m1 for TNFA , Hs00989291_m1 for IFNG .

Techniques: Staining, High Throughput Screening Assay, Microscopy, Immunofluorescence

Journal: bioRxiv

Article Title: Estrogen deprivation triggers an immunosuppressive phenotype in breast cancer cells

doi: 10.1101/715136

Figure Lengend Snippet: A, Flow cytometry mediated assessment of surface PD-L1 levels in MCF7 cells grown DMEM, SFM or SFM plus EE for 14 days. EE (10 nM) was added for the last 3 days. B, Flow cytometry mediated assessment of surface PD-L1 levels in MCF7 cells grown DMEM or fulvestrant (1μM) for 14 days. EE (10 nM) was added for the last 3 days. C,D Quantification from the data shown in A ( C ) and B ( D ). E, Immunofluorescence illustrating ERα (green) and PD-L1 (red) levels in MCF-7 cells cultured in DMEM, SFM or fulvestrant (1 μM) for 20 days. Where indicated, EE (10 nM) was supplemented during the last 4 days. Representative images for each condition are shown. F, Western blot illustrating the levels of PD-L1 and ERα in MCF-7 cells grown in DMEM, SFM or DMEM containing 1μM fulvestrant (Fulv) for 14 days. Where indicated EE (10nM) was added for the final 3 days. GAPDH levels are shown for loading control. G, H qRT-PCR analysis of PD-L1 ( CD274 ) expression in MCF-7 cells cultured in DMEM and SFM ( G ) or fulvestrant (1 μM, H ) for 18 days. Where indicated, media was supplemented with EE (10 nM) for the last 3 days. 18S RNA served as an internal control. * p <0,05; ** p <0,01

Article Snippet: The following probes were used in this study: Hs01125301_m1 for CD274 , Hs99999901_s1 for 18S , Hs03929097_g1 for GAPDH , Hs01046817_m1 for ESR1 , Hs01100353_m1 for ESR2 , Hs00174128_m1 for TNFA , Hs00989291_m1 for IFNG .

Techniques: Flow Cytometry, Immunofluorescence, Cell Culture, Western Blot, Control, Quantitative RT-PCR, Expressing

Journal: bioRxiv

Article Title: Estrogen deprivation triggers an immunosuppressive phenotype in breast cancer cells

doi: 10.1101/715136

Figure Lengend Snippet: A, Whole-cell lysates from MCF-7 cells cultured in SFM for the specified days were analyzed by western blotting using the indicated antibodies. Where indicated, 10nM EE was added at 16 days for the final 4 days. Total p65 and β-ACTIN served as loading controls. B, Flow cytometry mediated evaluation of PD-L1 membrane levels in MCF-7 cells grown in DMEM or SFM for 21 days, alone or in combination with the NFkB inhibitor CAPE (10μM) or JAK2 inhibitor CEP-33779 (10μ) for the last 3 days. C,D qRT-PCR analysis of IFNγ ( IFNG ) ( C ) or TNFα ( TNFA ) ( D ) mRNA levels in MCF-7 cells cultured as in ( A ). 18S rRNA was used as an internal control. ** p <0,01 E, Levels of IL-6 in the supernatant of MCF7 cells cultured in DMEM or SFM for the specified days as analyzed by LegendPlex-FACS (see Methods). Where indicated EE (10 nM) was added at day 17 for the last 4 days. F, GSEA Hallmark gene sets ranked by normalised enrichment score (NES) comparing the transcriptional programs triggered by 3 week treatments with fulvestrant (1 μM) or SFM in MCF7 cells, both normalised to DMEM. Selected hallmarks are indicated in red. G,H,I Pre-ranked GSEA on the genes from the hallmarks “TNFA signaling via NF-κB” ( G ), “IFNG response” ( H ) and “Inflammatory response” ( I ) obtained from RNAseq analysis comparing the transcriptome of MCF7 cells grown in DMEM or SFM for 3 weeks. The heatmap representation illustrates the overall upregulation of these pathways in estrogen-deprived MCF7 cells.

Article Snippet: The following probes were used in this study: Hs01125301_m1 for CD274 , Hs99999901_s1 for 18S , Hs03929097_g1 for GAPDH , Hs01046817_m1 for ESR1 , Hs01100353_m1 for ESR2 , Hs00174128_m1 for TNFA , Hs00989291_m1 for IFNG .

Techniques: Cell Culture, Western Blot, Flow Cytometry, Membrane, Quantitative RT-PCR, Control

Journal: bioRxiv

Article Title: Estrogen deprivation triggers an immunosuppressive phenotype in breast cancer cells

doi: 10.1101/715136

Figure Lengend Snippet: A, CD274 mRNA expression levels in ERα + (n=1459) and ERα − (n=445) patient samples. Normalised Z-scores were extracted from the METABRIC breast cancer TCGA dataset . A factor was added to both populations in order to set the ERα+ group to 0. Data are presented as mean +SEM and two-tailed Student’s t-test was used to calculate the statistical significance, ***p<0.001. B, Western blot analysis of PD-L1 and ERαprotein in a panel of ERα + and ERα − BC cell lines. β-Actin served as a loading control. C, Schematics of the experimental procedure in mice. MMTV-PyVT mice that spontaneously develop mammary tumors were treated daily from week 5 until with 4-hydroxytamoxifen (4-OHT) or vehicle. D, Cd274 expression in tumors isolated from MMTV-PyVT mice treated with 4-OHT or vehicle as determined by RT-qPCR. Data were normalized to β- Actin mRNA levels. Graph represents the mean ±SD and two-tailed Student’s t-test was used to determine statistical significance, **p<0.01. E, Immunohistochemical staining of PD-L1 (brown) in paired samples of primary and metastatic human ER+ BC from two different patients. Sections were stained with Hematoxylin-Eosin to aid in the pathological analysis. F , Quantification of the percentage of PD-L1 positive cells in paired primary tumor and metastasis samples from 8 patients of ERα-positive BC.

Article Snippet: The following probes were used in this study: Hs01125301_m1 for CD274 , Hs99999901_s1 for 18S , Hs03929097_g1 for GAPDH , Hs01046817_m1 for ESR1 , Hs01100353_m1 for ESR2 , Hs00174128_m1 for TNFA , Hs00989291_m1 for IFNG .

Techniques: Expressing, Two Tailed Test, Western Blot, Control, Isolation, Quantitative RT-PCR, Immunohistochemical staining, Staining

Journal: Journal for Immunotherapy of Cancer

Article Title: Anti-TGF-β/PD-L1 bispecific antibody promotes T cell infiltration and exhibits enhanced antitumor activity in triple-negative breast cancer

doi: 10.1136/jitc-2022-005543

Figure Lengend Snippet: The structure and binding affinity of BiTP. (A) The structure of BiTP. BiTP is an IgG 1 /IgG 2 hybrid antibody, containing IgG 2 -derived CH2 domain and IgG 1 -derived CH3 domain. The VLa, CL, VHa, and CH1 are derived from the corresponding domains of anti-PD-L1. The VHb and VLb domains are derived from the corresponding domains of anti-TGF-β. (B) Non-reduced and reduced SDS-PAGE. (C) Non-reduced and reduced CE-SDS. (D–F) The binding affinity to TGF-β1. BiTP was captured by plate-coated TGF-β. The affinity was determined by ELISA. (G) The binding affinity to PD-L1. Antibodies were incubated with H358 cells. The binding affinity was measured by mean fluorescence intensity in flow cytometry assay. (H) The simultaneous binding to PD-L1 and TGF-β1. BiTP was captured by precoated TGF-β1. Then, PD-L1-HRP was added, and the simultaneous binding was detected by ELISA. CE-SDS, capillary electrophoresis-sodium dodecyl sulfate; MFI, mean fluorescence intensity; TGF-β, transforming growth factor-beta.

Article Snippet: PD-L1-HIS (1 µg/mL, 10084-H08H, Sino Biological) and serially diluted BiTP were used in this assay.

Techniques: Binding Assay, Derivative Assay, SDS Page, Enzyme-linked Immunosorbent Assay, Incubation, Fluorescence, Flow Cytometry, Electrophoresis

Journal: Journal for Immunotherapy of Cancer

Article Title: Anti-TGF-β/PD-L1 bispecific antibody promotes T cell infiltration and exhibits enhanced antitumor activity in triple-negative breast cancer

doi: 10.1136/jitc-2022-005543

Figure Lengend Snippet: Competitive binding experiments and pharmacokinetic study. (A–C) BiTP competitively inhibited the binding of TGFBR2 to TGF-β. TGFBR2-HIS and BiTP were incubated with precoated TGF-β. The competitive inhibition capability was evaluated with anti-HIS ELISA. (D) BiTP competitively inhibited the binding of PD-1 to PD-L1. PD-L1-HIS and BiTP were incubated with precoated PD-L1. The competitive inhibition capability was evaluated with anti-HIS ELISA. (E) CD-1 mice received 3 mg/kg, 9 mg/kg, 27 mg/kg BiTP treatment by intravenous injection. Then, 5 min, 2 hours, 8 hours, 24 hours, 3 days, 7 days, 14 days, 21 days, and 28 days later, 0.6 mL peripheral blood was collected to measure the concentration of BiTP in plasma. (F–H) The influence of BiTP on the concentration of TGF-β in plasma. B-cell-depleted (μMT) mice received 9 mg/kg BiTP and 6.6 mg/kg anti-PD-L1 treatment. Before treatment, as well as 2 hours, 6 hours, 24 hours, 24 hours, 72 hours, 120 hours, and 168 hours after treatment, peripheral blood was collected to measure the concentration of TGF-β. TGF-β, transforming growth factor-beta.

Article Snippet: PD-L1-HIS (1 µg/mL, 10084-H08H, Sino Biological) and serially diluted BiTP were used in this assay.

Techniques: Binding Assay, Incubation, Inhibition, Enzyme-linked Immunosorbent Assay, Injection, Concentration Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Anti-TGF-β/PD-L1 bispecific antibody promotes T cell infiltration and exhibits enhanced antitumor activity in triple-negative breast cancer

doi: 10.1136/jitc-2022-005543

Figure Lengend Snippet: The in vitro bioactivity of BiTP. (A–C) CCK-8 assays to detect the capability of BiTP against TGF-β-inhibited proliferation of TF-1 cells. (D) Luciferase reporter experiments to evaluate Smad-mediated transcriptional activity. Smad-Luc-transfected A549 cells were treated with 20 ng/mL TGF-β1 and antibodies for 24 hours. Then, luminescence was detected. (E) The diagram showing NFAT luciferase reporter system. In the system, Jurkat-1-PD-1-NFAT-Luc and CHO-K1-PD-L1-CD3L were used. The activity of NFAT-Luc could be hampered by PD-1-PD-L1 axis. (F) NFAT luciferase reporter experiments to detect PD-1 signaling. Jurkat-1-PD-1-NFAT-Luc and BiTP were incubated with CHO-K1-PD-L1-CD3L for 6 hours. Then, luminescence was detected. (G) Superantigen stimulation assay assessing the activity of the anti-PD-L1 moiety of BiTP. PBMCs were mixed with antibodies and staphylococcal enterotoxin A (SEA). Four days later, IL-2 concentration in the supernatants was detected. PBMCs, peripheral blood mononuclear cell; TGF-β, transforming growth factor-beta.

Article Snippet: PD-L1-HIS (1 µg/mL, 10084-H08H, Sino Biological) and serially diluted BiTP were used in this assay.

Techniques: In Vitro, CCK-8 Assay, Luciferase, Activity Assay, Transfection, Incubation, Concentration Assay