Journal: Molecular Therapy Oncology

Article Title: CAR-T cells directed toward PD-L1 demonstrate potent, antigen-specific activity against cholangiocarcinoma: A proof of concept study

doi: 10.1016/j.omton.2026.201209

Figure Lengend Snippet: Generation of PD-L1 CAR-T and validation of PD-L1 expression in target cells (A) Schematic of the second-generation PD-L1 CAR construct containing an anti-PD-L1 scFv, CD4 transmembrane domain, and 4-1BB/CD3ζ signaling domains and tEGFR safety switch. (B) Flow cytometry results of PD-L1 expression in HuCCT1, HuCCT1-PD-L1 KO, and SNU1079 cells. (C) Characterization of non-CAR-T and CAR-T showing 98.8% and 98.6% CD3 expression and 1.03% and 28% EGFR expression, respectively.

Article Snippet: Cas9 was combined with multi-guide RNA targeting PD-L1 (Synthego, Redwood City, CA, USA) in NEB buffer (New England Biolabs) at a 12:1 ratio and incubated to form RNP complexes.

Techniques: Biomarker Discovery, Expressing, Construct, Flow Cytometry

Journal: Molecular Therapy Oncology

Article Title: CAR-T cells directed toward PD-L1 demonstrate potent, antigen-specific activity against cholangiocarcinoma: A proof of concept study

doi: 10.1016/j.omton.2026.201209

Figure Lengend Snippet: PD-L1 CAR-T delay tumor progression and reduce tumor burden in vivo (A) Longitudinal bioluminescent imaging of mice with orthotopic HuCCT1 tumors treated with PBS as a control, Non-CAR-T, or CAR-T at 7 and 14 days. (B) Quantification of total bioluminescent signal confirming significantly reduced tumor burden in CAR-T treated animals compared with both control groups. Results are reported as mean ± standard deviation (SD). Two-way ANOVA with Tukey’s multiple comparisons between tumor control and CAR-T are represented by ( p values: ∗∗ ≤0.01), and between non-CAR-T and CAR-T by ( p values: # # ≤ 0.01). n = 6 biological replicates at all days and time points with the exception of non-CAR-T week 9, where n = 5 biological replicates.

Article Snippet: Cas9 was combined with multi-guide RNA targeting PD-L1 (Synthego, Redwood City, CA, USA) in NEB buffer (New England Biolabs) at a 12:1 ratio and incubated to form RNP complexes.

Techniques: In Vivo, Imaging, Control, Standard Deviation

Journal: Molecular Therapy Oncology

Article Title: CAR-T cells directed toward PD-L1 demonstrate potent, antigen-specific activity against cholangiocarcinoma: A proof of concept study

doi: 10.1016/j.omton.2026.201209

Figure Lengend Snippet: Antigen-specific degranulation and granzyme B released by PD-L1 CAR-T (A) CD8 + T cell degranulation in response to HuCCT1 wild-type (WT) or PD-L1 knockout (KO) cells by flow cytometry after CAR-T co-culture at 6 h. (B) CD4 + T cell degranulation under the same conditions, showing CAR-T-mediated activity against WT but not KO cells. (C) Degranulation of CD8 + and CD4 + T cells in response to SNU1079 cells by flow cytometry after CART co-culture at 6 h compared with non-CAR-T controls. (D) Granzyme B release in HuCCT1 WT and KO cells following co-culture by ELISA after 72 h n = 3 technical replicates. Two-way ANOVA with Tukey’s multiple comparisons test. ∗∗∗∗ p value ≤ 0.0001. Results are reported as mean ± standard deviation (SD).

Article Snippet: Cas9 was combined with multi-guide RNA targeting PD-L1 (Synthego, Redwood City, CA, USA) in NEB buffer (New England Biolabs) at a 12:1 ratio and incubated to form RNP complexes.

Techniques: Knock-Out, Flow Cytometry, Co-Culture Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Molecular Therapy Oncology

Article Title: CAR-T cells directed toward PD-L1 demonstrate potent, antigen-specific activity against cholangiocarcinoma: A proof of concept study

doi: 10.1016/j.omton.2026.201209

Figure Lengend Snippet: CAR-T release cytotoxic effector molecules and reduce tumor cell viability in an antigen-dependent manner (A and B) Granzyme B and perforin release from CAR-T and non-transduced T cells co-cultured with HuCCT1 (A) or SNU1079 (B) cells at 1:1 and 2:1 effector-to-target (E:T) ratios. n = 3 technical replicates. One-way ANOVA with Tukey’s multiple comparisons test. (C) Luciferase viability assay of HuCCT1, PD-L1 knockout HuCCT1, or SNU1079 cells at 1:1 and 2:1 effector-to-target after 24 and 48 h. n = 6 technical replicates. One-way ANOVA with Tukey’s multiple comparisons test. ∗ p value ≤ 0.05, ∗∗∗∗ p value ≤ 0.0001. Results are reported as mean ± standard deviation (SD).

Article Snippet: Cas9 was combined with multi-guide RNA targeting PD-L1 (Synthego, Redwood City, CA, USA) in NEB buffer (New England Biolabs) at a 12:1 ratio and incubated to form RNP complexes.

Techniques: Cell Culture, Luciferase, Viability Assay, Knock-Out, Standard Deviation

Journal: Molecular Therapy Oncology

Article Title: CAR-T cells directed toward PD-L1 demonstrate potent, antigen-specific activity against cholangiocarcinoma: A proof of concept study

doi: 10.1016/j.omton.2026.201209

Figure Lengend Snippet: PD-L1 CAR-T disrupt and kill tumor cells in multicellular CSFE spheroids (A) Brightfield images of HuCCT1 and SNU1079 CSFE spheroids following 24 h co-culture with non-CAR-T or CAR-T at 1:1 or 2:1 effector-to-target (E:T) ratios. Quantification of spheroid area is shown. n = 3 technical replicates. One-way ANOVA with Tukey’s multiple comparisons test. Scale bar is 300 µm (B) Live/dead staining (calcein-AM/propidium iodide) and luciferase viability assays of spheroids under the same conditions. n = 3 technical replicates. One-way ANOVA with Tukey’s multiple comparisons test. ∗ p value ≤ 0.05, ∗∗ p value ≤ 0.01, ∗∗∗ p value ≤ 0.001, ∗∗∗∗ p value ≤ 0.0001. Results are reported as mean ± standard deviation (SD). Scale bar is 100 µm.

Article Snippet: Cas9 was combined with multi-guide RNA targeting PD-L1 (Synthego, Redwood City, CA, USA) in NEB buffer (New England Biolabs) at a 12:1 ratio and incubated to form RNP complexes.

Techniques: Co-Culture Assay, Staining, Luciferase, Standard Deviation

Journal: Molecular Therapy Oncology

Article Title: CAR-T cells directed toward PD-L1 demonstrate potent, antigen-specific activity against cholangiocarcinoma: A proof of concept study

doi: 10.1016/j.omton.2026.201209

Figure Lengend Snippet: Gemcitabine upregulates PD-L1 and enhances CAR-T cytotoxicity in HuCCT1 cells (A) Schematic of experimental design for gemcitabine pretreatment. (B) Flow cytometry showing increased PD-L1 surface expression in HuCCT1 cells after Gem treatment, with maximal induction at 0.2 μM for 48 h. n = 3 technical replicates. One-way ANOVA with Tukey’s multiple comparisons test. (C) Luciferase-based viability assays at both effector-to-target (E:T) ratios and at 24 and 48 h time points. n = 6 technical replicates, two-way ANOVA with Šídák’s multiple comparisons test. (D) Representative live/dead staining of HuCCT1 CSFE spheroids under the same conditions. ∗∗ p value ≤ 0.01, ∗∗∗ p value ≤ 0.001, ∗∗∗∗ p value ≤ 0.0001. Results are reported as mean ± standard deviation (SD). Scale bar is 50 µm.

Article Snippet: Cas9 was combined with multi-guide RNA targeting PD-L1 (Synthego, Redwood City, CA, USA) in NEB buffer (New England Biolabs) at a 12:1 ratio and incubated to form RNP complexes.

Techniques: Flow Cytometry, Expressing, Luciferase, Staining, Standard Deviation

Journal: Molecular Therapy Oncology

Article Title: CAR-T cells directed toward PD-L1 demonstrate potent, antigen-specific activity against cholangiocarcinoma: A proof of concept study

doi: 10.1016/j.omton.2026.201209

Figure Lengend Snippet: Gemcitabine upregulates PD-L1 and enhances CAR-T cytotoxicity in SNU1079 cells (A) Schematic depicting Gemcitabine pretreatment. (B) Flow cytometry showing Gemcitabine-induced PD-L1 upregulation in SNU1079 cells, with maximal effect at 0.2 μM for 48 h. n = 3 technical replicates. One-way ANOVA with Tukey’s multiple comparisons test. (C) Luciferase-based viability assays at both effector-to-target (E:T) ratios and at 24 and 48 h time points. n = 6 technical replicates. Two-way ANOVA with Šídák’s multiple comparisons test. (D) Representative live/dead staining of SNU1079 CSFE spheroids under the same conditions. p value∗ ≤ 0.05, p value∗∗ ≤ 0.01, p value∗∗∗∗ ≤ 0.0001. Results are reported as mean ± standard deviation (SD). Scale bar is 50 µm.

Article Snippet: Cas9 was combined with multi-guide RNA targeting PD-L1 (Synthego, Redwood City, CA, USA) in NEB buffer (New England Biolabs) at a 12:1 ratio and incubated to form RNP complexes.

Techniques: Flow Cytometry, Luciferase, Staining, Standard Deviation