Journal: Translational Oncology

Article Title: Tumor-stroma contributes to immunotherapeutic resistance in non-small cell lung cancer via SEMA3C-mediated immunosuppressive tumor microenvironment

doi: 10.1016/j.tranon.2026.102679

Figure Lengend Snippet: SEMA3C-targeting treatment inhibited tumor growth and restored T cell function. (A) Schematic of the treatment protocol in mice bearing Lewis cells. (B) Tumor growth curve of mice treated with PBS and the SEMA3C-targeting treatment. (C-D) Representative images showing the tumors harvested from mice bearing Lewis cells treated with PBS and the SEMA3C-targeting treatment, and weight of the harvested tumors. Data was presented as mean±SD. Significance was calculated with the Student’s t -test. **** P < 0.001. (E-G) Relative concentrations of PD-1 (E), GZMB (F), and IFN-γ (G) as measured by ELISA in Lewis cell-bearing mice treated with PBS or SEMA3C-targeting treatment. Data was presented as mean±SD. Significance was calculated with the Student’s t -test. * P < 0.05, ** P < 0.01.

Article Snippet: Additionally, mouse protein levels of programmed death-1 (PD-1, catalog EK2271), IFN-γ (catalog EK280), and granzyme B (GZMB, catalog EK2173) in peripheral blood from Lewis lung carcinoma-bearing mice were assessed using ELISA kits from MultiSciences (LIANKE) Biotech Co., Ltd (Hangzhou, China).

Techniques: Cell Function Assay, Enzyme-linked Immunosorbent Assay

Journal: Cell Reports Medicine

Article Title: CAR-T triggers TAM reeducation and adaptive anti-tumor response via TREM2 deficiency or CD40 agonist

doi: 10.1016/j.xcrm.2025.102539

Figure Lengend Snippet: The dual intervention of Trem2 -KO and GPC3-CAR-T induced profound remodeling of T cell ecosystems in both tumor and splenic microenvironments (A) UMAP clusters showed projection of cells onto a reference CD8 + T cell atlas (colored T cell subtypes) in tumor and spleen. (B) Proportion of the distinct CD8 + TILs and intrasplenic CD8 + T cell subtypes in tumor and spleen. (C) Violin plots revealed cell function based on each cell state scored by gene expressions of exhaustion, proliferation, cytotoxicity, and memory/naive phenotypes from CD8 + T subtypes in tumor, with medians and quartiles indicated. (D) Flow cytometry and quantification plots of IFN-γ + CD8 + T cells (effector CD8) in tumor and spleen. n = 8. (E) UMAP clusters showed projection of cells onto a reference CD4 + T cell atlas (colored T cell subtypes) in tumor and spleen. (F) Proportion of the distinct CD4 + TILs and intrasplenic CD4 + T cell subtypes in tumor and spleen. (G) Violin plots revealed cell function based on each cell state scored by gene expressions of exhaustion, proliferation, cytotoxicity, and memory/naive phenotypes from CD4 + T subtypes in tumor, with medians and quartiles indicated. (H) Flow cytometry and quantification plots of IFN-γ + CD4 + T cells in tumor and spleen. n = 8. (I and J) Multiple immunofluorescence images of tumor and spleen tissue between groups. Scale bars, 50 μm. Quantification of cells numbers from three random fields per mouse ( n = 8 mice/group). Data were represented by mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Mouse IFN-γ (Interferon Gamma) ELISA Kit , MultiSciences , Cat#E-HSEL-M0007.

Techniques: Cell Function Assay, Flow Cytometry, Immunofluorescence

Journal: Cell Reports Medicine

Article Title: CAR-T triggers TAM reeducation and adaptive anti-tumor response via TREM2 deficiency or CD40 agonist

doi: 10.1016/j.xcrm.2025.102539

Figure Lengend Snippet: Trem2 -KO combined with IFN-γ promotes metabolic reprogramming in TAMs (A) Heatmap of KEGG pathways and differential gene set analysis of glycolysis, TCA, and fatty acid metabolism. Results were applied separately to Cxcl9 + and Spp1 + subtypes. (B) Heatmap of differential gene set analysis of glycolysis, TCA, and fatty acid metabolism. Results were applied separately to each group of CAR-T-treated WT or Trem2 -KO mice. (C) Schematic representation of in vitro tumor-educated macrophage model. BMDMs isolated from Trem2 -KO or WT mice were educated for 24 h in a transwell system with cancer cells, with or without IFN-γ (40 ng/mL) intervention. (D) Relative mRNA expression levels of Cxcl9 and Spp1 in BMDMs were quantified by RT-qPCR, and the ratio of Cxcl9 / Spp1 was calculated. (E) AMP and ATP concentrations in BMDMs were measured using dedicated assay kits, and the AMP/ATP ratio was calculated from these values. Relative mRNA expression levels of Ldha, Idh2, and Cpt1 in BMDMs were quantified by RT-qPCR. (F) Relative protein expression levels of indicated molecules were assessed by western blot (WB) and normalized to β-actin. (G, H, and O) BMDMs were pretreated for 2 h with the AMPK inhibitor compound C (20 μM; G), mTOR inhibitor rapamycin (1 μM; H), or SYK inhibitor R406 (1 μM; O), followed by 24 h co-culture with tumor cells in the presence or absence of IFN-γ (40 ng/mL). Protein expression was analyzed by WB and normalized to β-actin. (I and J) The real-time changes of OCR of BMDMs were stimulated with or without IFN-γ in the basal state and following the additions of oligomycin (Oligo), fluorocarbonyl cyanide phenylhydrazone (FCCP), etomoxir (Eto), and rotenone + antimycin A (Rot/AA). The average of basal OCR, maximal OCR, and Eto-sensitive OCR were revealed. (K and L) The real-time measurement of ECAR of BMDMs was stimulated with or without IFN-γ in the basal state and following the additions of glucose (Gluc), oligomycin (Oligo) and 2-deoxy-D-glucose (2-DG). The average of basal ECAR, maximal ECAR, and glycolytic reserve were revealed. (M and N) The relative protein expression levels of indicated molecular were assessed by WB and normalized to β-actin. (P) The iBMDMs were transiently transfected to overexpress SOCS1 for 48 h, followed by 24-h stimulation with or without IFN-γ (40 ng/mL). Indicated molecular protein expression levels were tested by WB and normalized to β-actin. Data were represented by mean ± SEM. ns, no significance, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Mouse IFN-γ (Interferon Gamma) ELISA Kit , MultiSciences , Cat#E-HSEL-M0007.

Techniques: In Vitro, Isolation, Expressing, Quantitative RT-PCR, Western Blot, Co-Culture Assay, Transfection

Journal: Cell Reports Medicine

Article Title: CAR-T triggers TAM reeducation and adaptive anti-tumor response via TREM2 deficiency or CD40 agonist

doi: 10.1016/j.xcrm.2025.102539

Figure Lengend Snippet: CD40 agonism triggers a metabolic reprogram of TAMs similar to that of Trem2 -KO (A) Heatmap of differential gene set analysis in TAM subpopulations. Genes were enriched in Mac_Cxcl9 cluster. (B) The network diagram illustrated the highly expressed genes in Mac_Cxcl9 cluster and their interactions with shared transcription factors. (C) Correlation analysis between the ratio of Cd40 + TAMs and Cxcl9 + TAMs . Each dot represented a single-cell sequencing sample of tumor and spleen. (D) BMDMs isolated from Trem2 -KO or WT mice were educated for 24 h in a transwell system with cancer cells, with or without IFN-γ intervention. Relative protein expression levels of CD40 were assessed by WB and normalized to β-actin. (E) Schematic representation of in vitro tumor-educated macrophage model. BMDMs isolated from WT mice were educated for 24 h in a transwell system with cancer cells, with or without IFN-γ (40 ng/mL) and CD40 agonism (crosslinked FGK45, 20 ng/mL) intervention. (F) Relative mRNA expression levels of Cxcl9 and Spp1 in BMDMs were quantified by RT-qPCR, and the ratio of Cxcl9/Spp1 was calculated. n = 3. (G and H) The real-time changes of OCR of BMDMs were stimulated with or without IFN-γ and CD40 agonism in the basal state and following the additions of oligomycin (Oligo), FCCP, etomoxir (Eto), and rotenone + antimycin A (Rot/AA). The average of basal OCR, maximal OCR, and Eto-sensitive OCR were revealed. n = 3. (I and J) The real-time measurement of ECAR of BMDMs were stimulated with or without IFN-γ and CD40 agonism in the basal state and following the additions of glucose (Gluc), oligomycin (Oligo), and 2-deoxy-D-glucose (2-DG). The average of basal ECAR, maximal ECAR, and glycolytic reserve were revealed. n = 3. (K, L, O, and P) BMDMs from WT mice were co-cultured with tumor cells in transwells for 24 h with or without IFN-γ (40 ng/mL) and CD40 agonism (crosslinked FGK45, 20 ng/mL) (K and O). For inhibitor studies, BMDMs were pre-treated with AMPK inhibitor (20 μM) (L) or STAT1 inhibitor (100 μM) (P) for 2 h prior to co-culture. Protein levels were analyzed by western blot and normalized to β-actin. (M and N) AMP and ATP concentrations in BMDMs were measured using dedicated assay kits, and the AMP/ATP ratio was calculated from these values. n = 3. (Q) Mechanism schema diagram. Data were represented by mean ± SEM. ns, no significance, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Mouse IFN-γ (Interferon Gamma) ELISA Kit , MultiSciences , Cat#E-HSEL-M0007.

Techniques: Sequencing, Isolation, Expressing, In Vitro, Quantitative RT-PCR, Cell Culture, Co-Culture Assay, Western Blot

Journal: Cell Reports Medicine

Article Title: CAR-T triggers TAM reeducation and adaptive anti-tumor response via TREM2 deficiency or CD40 agonist

doi: 10.1016/j.xcrm.2025.102539

Figure Lengend Snippet: The combination of CD40 agonism with GPC3-CAR-T therapy held promising application prospects in the treatment of HCC (A) Schematic procedure of HCC model constructed by Hepa1-6 cell orthotopic injection. GPC3-CART and CD40 agonist (FGK45) were injected. (B) Tumor representative photographs of different groups were shown. (C) Dot plots showed liver weight of mice in different groups. n = 8. (D) Survival curves of each group of mice. n = 8. (E and F) Multiple immunofluorescence images of TAMs, CD8 + , and CD4 + T cells in tumor tissue between groups. Scale bar, 50 μm. Quantification of cells numbers from three random fields per mouse ( n = 8 mice/group). Data were represented by mean ± SEM. ∗∗∗ p < 0.001. (G and H) Flow cytometry and quantification plots of IFN-γ + CD8 + T cells and IFN-γ + CD4 + T cells, as well as IFN-γ − CD8 + T cells and IFN-γ - CD4 + T cells from TILs in vivo . N = 8. Data were represented by mean ± SEM. ∗ p < 0.01, ∗∗∗ p < 0.001. (I) Schematic procedure of a therapeutic breast cancer liver metastasis model constructed by 4T1 cell expressing hTrop2 protein. Murine hTrop2-CAR-T and CD40 agonist (FGK45) were injected. (J) Tumor representative photographs of different groups were shown. (K) Dot plots showed liver weight of mice in different groups. n = 8. Data were represented by mean ± SEM. ns, no significance, ∗∗∗ p < 0.001. (L) Survival curves of each group of mice. n = 8. (M) Schematic representation of the transendothelial migration assay. TAMs were isolated from human HCC tissue. Sotigalimab (20 ng/mL) and h-IFN-γ (40 ng/mL) were added. Flow cytometry quantification of migrated CD8 + T cells (CellTrace Far Red labeled, n = 5). Data were represented by mean ± SEM. ns, no significance, ∗∗ p < 0.01, ∗∗∗ p < 0.001. (N) Schematic representation of the transendothelial migration assay. DTS was isolated from human GPC3 + HCC tissue. GPC3-CAR-T was added at indicated ratio, with or without sotigalimab. Flow cytometry quantification of migrated CD8 + T cells (CellTrace Far Red labeled, n = 5). Data were represented by mean ± SEM. ns, no significance, ∗∗∗ p < 0.001.

Article Snippet: Mouse IFN-γ (Interferon Gamma) ELISA Kit , MultiSciences , Cat#E-HSEL-M0007.

Techniques: Construct, Injection, Immunofluorescence, Flow Cytometry, In Vivo, Expressing, Migration, Isolation, Labeling