Journal: bioRxiv

Article Title: Bivalent bispecific CD28 antibodies reinforce T-cell responsiveness and revert anergy/quiescence in patients treated with bispecific CD3 antibodies

doi: 10.64898/2026.03.25.714198

Figure Lengend Snippet: A PBMCs were collected from patients before and after TCE treatment with CC-1 (day 1 and day 8, respectively), incubated ex vivo with CC-1 alone or in combination with BiCos, and subsequently subjected to functional analysis. B Patient-derived PBMC (n=5) were cultured with LNCaP-E tumor cells at an E:T ratio of 1:1 in the presence of 1 nM CC-1 with or without 0.5 nM of BiCo-1, BiCo-2 or isotype control (BiCo-iso). After 72 h, T-cell proliferation was quantified by 3 H-thymidine incorporation, tumor cell killing was determined by flow cytometry. C PBMCs from three patients obtained before and after treatment (day 1 and day 8 of subsequent cycles; see scheme in ) were incubated with CC-1 (5 nM) alone or together with BiCo-1 (5 nM). After 72 h, T-cell proliferation was assessed by 3 H-thymidine incorporation. D Leiden-based subclustering of scRNAseq data visualized by UMAP, depicting T cell populations within patient PBMC samples collected after therapy (day 8), and incubated for 3 days in vitro with LNCaP-E and 1 nM CC-1 with or without 0.5 nM BiCos annotated using canonical lineage markers. UMAP visualization of T-cell subclustering, resolving distinct functional T-cell states. E Dot plots summarizing temporal expression changes of selected genes involved in T-cell activation, cytotoxicity, proliferation and quiescence. Dot size indicates the fraction of cells expressing the respective gene; and color intensity reflects the mean expression. F Density plots illustrating shifts in patient T-cell (day 8) state distributions of T-cells treated in vitro with CC-1 alone or CC-1+BiCos, demonstrating a pronounced transition toward proliferative phenotypes. G PBMCs (n=4) were cultured with LNCaP-E cells at an E:T ratio of 1:2 in the presence of CC-1 (1 nM); medium, target cells, and constructs were replenished on day 4 to mimic chronic exposure. At day 7, PBMCs were re-stimulated with LNCaP-E cells at an E:T ratio of 1:1 in the presence of CC-1 (1 nM) alone or in combination with the indicated BiCo constructs (0.5 nM). On day 10, T-cell proliferation was accessed by 3 H-thymidine incorporation and tumor cell killing was evaluated by flow cytometry. H PBMCs (n = 4) were cultured with LNCaP-E cells at an E:T ratio of 1:2 in the presence or absence of CC-1 (1 nM) alone or in combination with BiCo constructs (0.5 nM). Medium, target cells, and constructs were replenished on day 4 and day 7 with (LNCaP-E cells E:T ratio of 1:1 on day 7). At day 10, T-cell proliferation was assessed by 3 H-thymidine incorporation and tumor cell killing was evaluated by flow cytometry. I PBMCs (n=3) were cultured with SHP-77 cells at an E:T ratio of 1:2 in the presence of tarlatamab (1 nM). Medium and target cells were replenished on day 4. After the exposure until day 7 mimicking induction of hyporesponsiveness, PBMCs were re-stimulated with SHP-77 cells at an E:T ratio of 1:1 in the presence of tarlatamab (1 nM) or in combination with BiCo-2 (0.5 nM). Proliferation was measured on day 10 by 3 H-thymidine incorporation, and tumor cell killing was assessed by flow cytometry.

Article Snippet: The human prostate cancer cell line LNCaP was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) and SHP-77 cell line from American Type Culture Collection (ATCC).

Techniques: Incubation, Ex Vivo, Functional Assay, Derivative Assay, Cell Culture, Control, Flow Cytometry, In Vitro, Expressing, Activation Assay, Construct

Journal: Cancer Biology & Medicine

Article Title: Chidamide suppresses macrophage-mediated immune evasion and tumor progression in small cell lung cancer by targeting the STAT4/CCL2 signaling pathway

doi: 10.20892/j.issn.2095-3941.2024.0241

Figure Lengend Snippet: Chidamide inhibits malignancy proliferation in SCLC. (A-F) Effects of chidamide on the viability of various neuroendocrine SCLC cells in vitro (A-C), subcutaneous tumor growth, and toxicity in vivo (D-F). (G, H) Expression of Ki-67 in the control and chidamide groups. (I) Gene set enrichment analysis (GSEA) of apoptosis pathway transcriptomic differences in SCLC cells in the chidamide and control groups. (J) Quantification of apoptosis-associated gene expression in the chidamide and control groups according to qPCR and WB. (K-M) Flow cytometry analyses of SCLC cell apoptosis after treatment with various chidamide concentrations. Bars represent the mean ± SD values. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Student’s t -test (E, H, J), one-way ANOVA (M). These experiments were performed three times.

Article Snippet: Human SCLC cell lines (SHP77, NCI-H889, NCI-H446, and DMS53), the human mononuclear cell line, THP1, and the murine macrophage cell line, RAW264.7, were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA).

Techniques: In Vitro, In Vivo, Expressing, Control, Gene Expression, Flow Cytometry

Journal: Cancer Biology & Medicine

Article Title: Chidamide suppresses macrophage-mediated immune evasion and tumor progression in small cell lung cancer by targeting the STAT4/CCL2 signaling pathway

doi: 10.20892/j.issn.2095-3941.2024.0241

Figure Lengend Snippet: Chidamide promotes CCL2 expression in SCLC. (A) Differential gene expression changes in SHP77 and H889 cells before and after treatment with chidamide. KEGG signaling pathway enrichment in human (B) and murine SCLC cells (D) before and after chidamide treatment. Differential cytokine heatmap in cytokine-cytokine receptor interaction signaling pathways in human (C) and murine SCLC cells (E). (F) Venn diagram of the above-mentioned differentially expressed cytokines in the three SCLC cell lines. (G) Expression of three cytokines based on RNA sequencing. (H) Expression of three cytokines in SCLC cells verified by qPCR. (I) CCL2 expression between normal and SCLC tissues in the GSE43346 (43/25), GSE60052 (7/79), GSE15240 (3/38), and GSE149507 (18/18) datasets. (J) Survival curve of 81 SCLC patients with high or low CCL2 expression. (K-M) Changes in CCL2 expression in SCLC cells treated with chidamide for various times according to qPCR (K), ELISA (L), and WB (M). Bars represent the mean ± SD values. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Student’s t -test (G, H, I), one-way ANOVA (K, L). These experiments were performed three times (H, K, L, M).

Article Snippet: Human SCLC cell lines (SHP77, NCI-H889, NCI-H446, and DMS53), the human mononuclear cell line, THP1, and the murine macrophage cell line, RAW264.7, were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA).

Techniques: Expressing, Gene Expression, Protein-Protein interactions, RNA Sequencing, Enzyme-linked Immunosorbent Assay