Journal: Acta Pharmaceutica Sinica. B

Article Title: LSD1 inhibition sensitizes anti-PD1 blockade immunotherapy by inhibiting the long-range attack of tumor-derived extracellular vesicles

doi: 10.1016/j.apsb.2025.10.030

Figure Lengend Snippet: The tumor microenvironment (TME)-targeted synergistic therapy system constructed by anti-PD1 and LSD1 inhibitor forsythiaside A (FA) exerts anti-tumor ability. Anti-PD1 is an immune checkpoint inhibitor; FA mediates the PD-L1 rearrangement mechanism on the surface of tumor cells by inhibiting the demethylation activity of LSD1, regulating the recovery of recycling endosomes and the efflux function of multivesicular bodies (MVBs), reducing the secretion of extracellular vesicles (EVs) and PD-L1 levels in the TME, and ultimately inhibiting the long-range attack of EVs. Meanwhile, CD4 + T cells have been found to directly exert tumor-killing function by inhibiting the cell G1/S cycle.

Article Snippet: After incubation, cells were collected and dyed using FITC-labeled PD1 flow cytometry antibody (E-AB-F1131C, Elabscience, Wuhan, China) for flow cytometry detection by CytoFLEX S Flow Cytometer (Beckman Coulter, Inc., Brea, CA, USA).

Techniques: Construct, Activity Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: LSD1 inhibition sensitizes anti-PD1 blockade immunotherapy by inhibiting the long-range attack of tumor-derived extracellular vesicles

doi: 10.1016/j.apsb.2025.10.030

Figure Lengend Snippet: Characterization of FPNs. (A) UV–Vis spectra of different formulations. (B) Encapsulation efficiency of different weight ratios of FA and anti-PD1. (C) The hydrodynamic diameter of FPNs was measured by NTA. (D) TEM images of FPNs and blank liposomes (negative control). Scale bar = 100 nm. (E) The size distribution of FPNs was determined by TEM imaging. (F) ζ -potentials of FPNs and control liposomes. (G) Particle size variation of FPNs in water, PBS, DMEM, and 10% FBS DMEM after 3 or 7 days of incubation. (H) FA release curve from FPNs in vitro . Data are presented as mean ± SD ( n = 3).

Article Snippet: After incubation, cells were collected and dyed using FITC-labeled PD1 flow cytometry antibody (E-AB-F1131C, Elabscience, Wuhan, China) for flow cytometry detection by CytoFLEX S Flow Cytometer (Beckman Coulter, Inc., Brea, CA, USA).

Techniques: Encapsulation, Liposomes, Negative Control, Imaging, Control, Incubation, In Vitro

Journal: Acta Pharmaceutica Sinica. B

Article Title: LSD1 inhibition sensitizes anti-PD1 blockade immunotherapy by inhibiting the long-range attack of tumor-derived extracellular vesicles

doi: 10.1016/j.apsb.2025.10.030

Figure Lengend Snippet: Targeting and in vivo biodistribution of FPNs in TME. (A) HCS fluorescence images of LLC cells incubated with FPNs for 1, 12, and 24 h. Scale bar = 50 μm. (B) Quantification of FPNs release experiments in A, C. (C) Flow cytometry analysis of LLC cells incubated with FPNs for 1, 6, 12, and 24 h. (D) In vivo fluorescence images of LLC tumor-bearing nude mice after intravenous injection of Cy5.5-FPNs or Cy5.5. Tumor regions were marked by the circles. (E) Quantitative plot of the average fluorescence intensity of tumors divided by regions of interest. (F) Fluorescence images of visceral organs and tumors harvested at 24 h post-injection. (G) Average fluorescence intensity of tumors and visceral organs. Data are presented as mean ± SD ( n = 3). ∗ P < 0.05, ∗∗ P < 0.01 and ∗∗∗ P < 0.001.

Article Snippet: After incubation, cells were collected and dyed using FITC-labeled PD1 flow cytometry antibody (E-AB-F1131C, Elabscience, Wuhan, China) for flow cytometry detection by CytoFLEX S Flow Cytometer (Beckman Coulter, Inc., Brea, CA, USA).

Techniques: In Vivo, Fluorescence, Incubation, Flow Cytometry, Injection

Journal: Acta Pharmaceutica Sinica. B

Article Title: LSD1 inhibition sensitizes anti-PD1 blockade immunotherapy by inhibiting the long-range attack of tumor-derived extracellular vesicles

doi: 10.1016/j.apsb.2025.10.030

Figure Lengend Snippet: FPNs affect tumor growth by participating in the immune response. (A–D) After treatment with FPNs, FA + anti-PD1, anti-PD1, FA, Liposome, and PBS, flow cytometry was performed to detect the infiltration levels of Treg cells (CD25 + Foxp3 + ), activated DC cells (CD11C + MHC II + ), M1 TAMs (CD86 + )/M2 TAMs (CD206 + ) ratio and CD4 + T cells (CD4 + CD8 - ) in LLC and LA795 tumor tissues. (E) Pictures of immunohistochemistry of CD4 in LLC and LA795 tumor tissues following treatments. Scale bar = 100 μm. (F) CD4 + T trafficking and biodistribution of DiI-labeled CD4 + T cells in LLC tumor with PBS, anti-PD1, or FPNs treatment administered intravenously. Scale bar = 100 μm. (G) In vitro transwell migration assay. (Image display used ImageJ to transform the bright field view into black-and-white (B&W) high-contrast mode). Scale bar = 100 μm. (H) Quantitative results for F and G. (I) Changes of IFN- γ , TNF- α , and IL-2 levels in tumor tissues of LLC mice after different drug treatments. Data are presented as mean ± SD ( n = 3). ∗ P < 0.05, ∗∗ P < 0.01 and ∗∗∗ P < 0.001.

Article Snippet: After incubation, cells were collected and dyed using FITC-labeled PD1 flow cytometry antibody (E-AB-F1131C, Elabscience, Wuhan, China) for flow cytometry detection by CytoFLEX S Flow Cytometer (Beckman Coulter, Inc., Brea, CA, USA).

Techniques: Flow Cytometry, Immunohistochemistry, Labeling, In Vitro, Transwell Migration Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: LSD1 inhibition sensitizes anti-PD1 blockade immunotherapy by inhibiting the long-range attack of tumor-derived extracellular vesicles

doi: 10.1016/j.apsb.2025.10.030

Figure Lengend Snippet: CD4 + T cells can directly kill tumor cells through G1/S phase arrest. (A) CCK-8 analysis of cells treated with different concentrations of FPNs in the presence and absence of CD4 + T cells. (B) Cell survival of A549 and LLC cells under different conditions after treatment with anti-CD3/CD28-activated CD4 + T cells. Scale bar = 200 μm. (C) Activated CD4 + T cells induced cell cycle arrest at G1/S phase under different conditions determined by flow cytometry. (D) Expression of the marker proteins of G1/S phase arrest, CDK4, CDK6, and cyclinD1, in A549 and LLC cells under different conditions after treatment with anti-CD3/CD28 activated CD4 + T cells. Data are presented as mean ± SD ( n = 3). ∗ P < 0.05, ∗∗ P < 0.01 and ∗∗∗ P < 0.001.

Article Snippet: After incubation, cells were collected and dyed using FITC-labeled PD1 flow cytometry antibody (E-AB-F1131C, Elabscience, Wuhan, China) for flow cytometry detection by CytoFLEX S Flow Cytometer (Beckman Coulter, Inc., Brea, CA, USA).

Techniques: CCK-8 Assay, Flow Cytometry, Expressing, Marker

Journal: Acta Pharmaceutica Sinica. B

Article Title: LSD1 inhibition sensitizes anti-PD1 blockade immunotherapy by inhibiting the long-range attack of tumor-derived extracellular vesicles

doi: 10.1016/j.apsb.2025.10.030

Figure Lengend Snippet: Inhibition of LSD1 hinders tumor growth by participating in CD4 + T cell responses. (A) Expression of LSD1 in LLC and A549 cells with or without LSD1 knocked out. (B) Changes of tumor volume in BALB/c and BALB/c nude mice bearing LLC cells, whether LSD1 was abrogated or not ( n = 5). (C) Images of tumors of BALB/c and BALB/c nude mice bearing LLC cells ( n = 5). (D) Tumor weight of BALB/c mice and BALB/c nude mice, whether LSD1 was abrogated or not ( n = 5). (E, F) Percentage of CD4 + T cells in CD3 + infiltration cells isolated from tumors of BALB/c mice bearing LLC cells in the presence or absence of LSD1. (G) Cell survival of LLC and A549 cells in the presence or absence of LSD1, treated with anti-CD3/CD28-activated T cells. Scale bar = 200 μm. (H) Activated CD4 + T cells induced cell cycle arrest in the G1/S phase, and knockout of LSD1 had a facilitating effect. (I) Flow cytometry quantification of cycles in A549 cells and LLC cells. (J) Expression of the marker proteins of G1/S phase arrest in LLC and A549 cells under anti-CD3/CD28 activated CD4 + T cells and with or without LSD1. Data are presented as mean ± SD ( n = 3), unless otherwise noted. ∗ P < 0.05, ∗∗ P < 0.01 and ∗∗∗ P < 0.001.

Article Snippet: After incubation, cells were collected and dyed using FITC-labeled PD1 flow cytometry antibody (E-AB-F1131C, Elabscience, Wuhan, China) for flow cytometry detection by CytoFLEX S Flow Cytometer (Beckman Coulter, Inc., Brea, CA, USA).

Techniques: Inhibition, Expressing, Isolation, Knock-Out, Flow Cytometry, Marker

Journal: Acta Pharmaceutica Sinica. B

Article Title: LSD1 inhibition sensitizes anti-PD1 blockade immunotherapy by inhibiting the long-range attack of tumor-derived extracellular vesicles

doi: 10.1016/j.apsb.2025.10.030

Figure Lengend Snippet: Inhibition of LSD1 reduces the expression of EVs and EVs-PD-L1. (A) TEM images of purified EVs from A549 cells in the presence or absence of LSD1 (A-EVs and KO-EVs, for short). Scale bar = 100 nm. (B) Expression of total PD-L1 in A549 cells treated with GW4869 and quantitative results. (C) Expression of membrane PD-L1 in A549 cells treated with GW4869 and quantitative results. (D) Expression of PD-L1, ALIX, TSG101, and CD9 in EVs, from the same number of cells in the presence of 10 μmol/L GW4869 or not. (E) Expression of PD-L1 in A-EVs and KO-EVs. (F) Concentration of A-EVs and KO-EVs from the same number of cells. (G) Expression of membrane PD-L1 in A549 or A549 KO cells treated with or without 10 μmol/L GW4869. (H) Representative HCS images of Dil-stained A-EVs and KO-EVs with wells coated with recombinant PD1. Scale bar = 200 μm. (I) HCS images of the binding of T cells to A-EVs and KO-EVs. Scale bar = 20 μm. Data are presented as mean ± SD ( n = 3). ∗ P < 0.05, ∗∗ P < 0.01 and ∗∗∗ P < 0.001.

Article Snippet: After incubation, cells were collected and dyed using FITC-labeled PD1 flow cytometry antibody (E-AB-F1131C, Elabscience, Wuhan, China) for flow cytometry detection by CytoFLEX S Flow Cytometer (Beckman Coulter, Inc., Brea, CA, USA).

Techniques: Inhibition, Expressing, Purification, Membrane, Concentration Assay, Staining, Recombinant, Binding Assay