Journal: Molecular Oncology

Article Title: NKG2A and circulating extracellular vesicles are key regulators of natural killer cell activity in prostate cancer after prostatectomy

doi: 10.1002/1878-0261.13422

Figure Lengend Snippet: Removal of PCa altered NKA and NKG2A expression on NK cells. PBMCs were extracted from the blood of PCa patients before and 1 month after prostatectomy and stained with antibodies for analysis. Every parameter was compared in each patient before (pre) and after (post) surgery. The NK‐cell fraction (A) as well as NKA (B), NKG2D (C), and NKG2A (D) levels on NK cells were determined using flow cytometry. (A–D) Paired t ‐test.

Article Snippet: Small interfering RNAs (siRNAs; 50 ng) targeting nothing (negative control), NKG2A, and NKG2D (smart pools from Synbio Tech, Kaohsiung, Taiwan) were incubated with 3 μL polyfast transfection reagent (MedChemExpress, Princeton, NJ, USA) in medium without FBS for 15 min. Then, the mixture was added to medium containing 5 × 10 4 NK cells and incubated for 48 h. Forty microgram protein per 5 μL of circulating EVs from healthy donors and tumor patients were added to NK cells for 24 h. A total of 1 × 10 5 DiI‐labeled K562 cells were added to NK cells for 2 h, and the remaining DiI‐labeled K562 cells were measured by flow cytometry with an FL2‐H filter.

Techniques: Expressing, Staining, Flow Cytometry

Journal: Molecular Oncology

Article Title: NKG2A and circulating extracellular vesicles are key regulators of natural killer cell activity in prostate cancer after prostatectomy

doi: 10.1002/1878-0261.13422

Figure Lengend Snippet: Proportions of NK cells, NKA, NKG2D, NKG2A, and NKG2D/NKG2A increased after prostatectomy in four different stages.

Article Snippet: Small interfering RNAs (siRNAs; 50 ng) targeting nothing (negative control), NKG2A, and NKG2D (smart pools from Synbio Tech, Kaohsiung, Taiwan) were incubated with 3 μL polyfast transfection reagent (MedChemExpress, Princeton, NJ, USA) in medium without FBS for 15 min. Then, the mixture was added to medium containing 5 × 10 4 NK cells and incubated for 48 h. Forty microgram protein per 5 μL of circulating EVs from healthy donors and tumor patients were added to NK cells for 24 h. A total of 1 × 10 5 DiI‐labeled K562 cells were added to NK cells for 2 h, and the remaining DiI‐labeled K562 cells were measured by flow cytometry with an FL2‐H filter.

Techniques:

Journal: Molecular Oncology

Article Title: NKG2A and circulating extracellular vesicles are key regulators of natural killer cell activity in prostate cancer after prostatectomy

doi: 10.1002/1878-0261.13422

Figure Lengend Snippet: Proportions of NK cells, NKA, NKG2D, NKG2A, and NKG2D/NKG2A increased after prostatectomy in different stages.

Article Snippet: Small interfering RNAs (siRNAs; 50 ng) targeting nothing (negative control), NKG2A, and NKG2D (smart pools from Synbio Tech, Kaohsiung, Taiwan) were incubated with 3 μL polyfast transfection reagent (MedChemExpress, Princeton, NJ, USA) in medium without FBS for 15 min. Then, the mixture was added to medium containing 5 × 10 4 NK cells and incubated for 48 h. Forty microgram protein per 5 μL of circulating EVs from healthy donors and tumor patients were added to NK cells for 24 h. A total of 1 × 10 5 DiI‐labeled K562 cells were added to NK cells for 2 h, and the remaining DiI‐labeled K562 cells were measured by flow cytometry with an FL2‐H filter.

Techniques:

Journal: Molecular Oncology

Article Title: NKG2A and circulating extracellular vesicles are key regulators of natural killer cell activity in prostate cancer after prostatectomy

doi: 10.1002/1878-0261.13422

Figure Lengend Snippet: Extracellular vesicle number increased and the ligands for NKA regulatory receptors on EVs changed in the presence of tumors. (A) EVs were extracted from serum, and EV morphology was observed with TEM. (B) The size and number of EVs were measured by NTA. (C) A greater number of EVs was noted in tumor patients compared with healthy donors. The expression per EV (D–F) and total expression (G–I) of NKG2D ligands MICA and ULBP1 and the NKG2A ligand HLAE were analyzed based on tumor stages. (C) Paired t tests. (D–I) Mann–Whitney U test. Bar: mean ± SD.

Article Snippet: Small interfering RNAs (siRNAs; 50 ng) targeting nothing (negative control), NKG2A, and NKG2D (smart pools from Synbio Tech, Kaohsiung, Taiwan) were incubated with 3 μL polyfast transfection reagent (MedChemExpress, Princeton, NJ, USA) in medium without FBS for 15 min. Then, the mixture was added to medium containing 5 × 10 4 NK cells and incubated for 48 h. Forty microgram protein per 5 μL of circulating EVs from healthy donors and tumor patients were added to NK cells for 24 h. A total of 1 × 10 5 DiI‐labeled K562 cells were added to NK cells for 2 h, and the remaining DiI‐labeled K562 cells were measured by flow cytometry with an FL2‐H filter.

Techniques: Expressing, MANN-WHITNEY

Journal: Molecular Oncology

Article Title: NKG2A and circulating extracellular vesicles are key regulators of natural killer cell activity in prostate cancer after prostatectomy

doi: 10.1002/1878-0261.13422

Figure Lengend Snippet: Extracellular vesicle number and ligands for NKGAD on EVs increased after PCa removal. (A, B) The EV number in patients before and after prostatectomy was measured based on CD63 expression, and the CD63 expression level was significantly altered in patients after prostatectomy. (C) Compared with EVs obtained from patients before the removal of PCa (pre), the EVs extracted from patients after prostatectomy (post) exhibited dramatically increased NK cytotoxicity. (D) The cell survival of DU145 under treatment of EVs‐treated NK92 cells. (E–J) Total expression levels of NKG2D ligands MICA (E) and ULBP1 (F) and NKG2A ligand HLAE (G) on EVs were analyzed before (pre) and after (post) prostatectomy. The expression levels per EV of MICA (H), ULBP1 (I), and HLAE (J) were also analyzed before (pre) and after (post) prostatectomy. The expression levels of ligands on EVs in the patients before prostatectomy (pre) were set as 100%. Red dashed line shows the level of 100%. (B–J) Paired t tests.

Article Snippet: Small interfering RNAs (siRNAs; 50 ng) targeting nothing (negative control), NKG2A, and NKG2D (smart pools from Synbio Tech, Kaohsiung, Taiwan) were incubated with 3 μL polyfast transfection reagent (MedChemExpress, Princeton, NJ, USA) in medium without FBS for 15 min. Then, the mixture was added to medium containing 5 × 10 4 NK cells and incubated for 48 h. Forty microgram protein per 5 μL of circulating EVs from healthy donors and tumor patients were added to NK cells for 24 h. A total of 1 × 10 5 DiI‐labeled K562 cells were added to NK cells for 2 h, and the remaining DiI‐labeled K562 cells were measured by flow cytometry with an FL2‐H filter.

Techniques: Expressing

Journal: Molecular Oncology

Article Title: NKG2A and circulating extracellular vesicles are key regulators of natural killer cell activity in prostate cancer after prostatectomy

doi: 10.1002/1878-0261.13422

Figure Lengend Snippet: Extracellular vesicles interacted with NK cells, and NKG2A was the key regulator of NK cytotoxicity in tumor patients. (A) EVs labeled with deep red fluorescence were incubated with NK92 cells for 24 h. Cells were observed under fluorescence microscopy. The cell nuclei of NK92 cells were labeled with Hoechst 33342. EVs were attached to NK92 cells or located in the cytoplasm of NK cells. (B) Deep red fluorescence inside NK cells was analyzed using flow cytometry. The deep red FI was significantly increased in NK cells + EVs compared with NK92 cells alone. (C–I) Equal amounts of EVs were added to NK92 cells and incubated for 24 h. The cytotoxic effects of NK cells toward K562 cells, which were labeled with CM‐DiI, were measured using flow cytometry. (C) EVs extracted from patients promoted significantly lower NK cytotoxicity than those extracted from healthy donors. (D, E) NKG2D inhibitory antibody decreased NK cytotoxicity in healthy donors (8 of 8), whereas NK cytotoxicity was only decreased in half of the patients (7 of 15). (F, G) NKG2A inhibitory antibody increased NK cytotoxicity in patients (13 of 15) but only in a few of the healthy donors (1 of 8). (H, I) HLAE increased NK cytotoxicity in half of the healthy donors (4 of 8) and PCa patients (10 of 20). (J–L) NK cytotoxicity in cells with siRNAs targeting nothing (Neg), NKG2A and NKG2D was measured with treatment of medium only (J) and EVs from healthy donors (K) and tumor patients (L). (B, C) Paired t tests. (J–L) Mann–Whitney U test. Scale bar: 25 μm. Bar: mean ± SD.

Article Snippet: Small interfering RNAs (siRNAs; 50 ng) targeting nothing (negative control), NKG2A, and NKG2D (smart pools from Synbio Tech, Kaohsiung, Taiwan) were incubated with 3 μL polyfast transfection reagent (MedChemExpress, Princeton, NJ, USA) in medium without FBS for 15 min. Then, the mixture was added to medium containing 5 × 10 4 NK cells and incubated for 48 h. Forty microgram protein per 5 μL of circulating EVs from healthy donors and tumor patients were added to NK cells for 24 h. A total of 1 × 10 5 DiI‐labeled K562 cells were added to NK cells for 2 h, and the remaining DiI‐labeled K562 cells were measured by flow cytometry with an FL2‐H filter.

Techniques: Labeling, Fluorescence, Incubation, Microscopy, Flow Cytometry, MANN-WHITNEY

Journal: Molecular Oncology

Article Title: NKG2A and circulating extracellular vesicles are key regulators of natural killer cell activity in prostate cancer after prostatectomy

doi: 10.1002/1878-0261.13422

Figure Lengend Snippet: Natural killer‐cell infiltration and the binding of HLAE and NKG2A in PCas and adjacent normal tissues varied at different stages. (A) Prostate tumor tissues from PCa patients at various stages were stained with NKG2D (green), CD56 (red), and DAPI (blue). NK cells with the colocalization of NKG2D and CD56 are indicated in yellow. The NK‐cell number was decreased in adjacent normal (B) and tumor regions (C) in higher stages. (D) Tissues were stained with HLAE (green), NKG2A (red), and DAPI (blue). The binding of HLAE and NKG2A is indicated in yellow, and the colocalization of green and red fluorescence in adjacent normal (N) and tumor tissues (T) is shown (E). (F) The colocalization of NKG2A and HLAE inside NK cells in adjacent normal tissues was calculated. Scale bar: 50 μm. G, Gleason Score; SI, stage 1; SII, stage 2; SIII, stage 3; SIV, stage 4. (B–F) Mann–Whitney U test. Bar: mean ± SD.

Article Snippet: Small interfering RNAs (siRNAs; 50 ng) targeting nothing (negative control), NKG2A, and NKG2D (smart pools from Synbio Tech, Kaohsiung, Taiwan) were incubated with 3 μL polyfast transfection reagent (MedChemExpress, Princeton, NJ, USA) in medium without FBS for 15 min. Then, the mixture was added to medium containing 5 × 10 4 NK cells and incubated for 48 h. Forty microgram protein per 5 μL of circulating EVs from healthy donors and tumor patients were added to NK cells for 24 h. A total of 1 × 10 5 DiI‐labeled K562 cells were added to NK cells for 2 h, and the remaining DiI‐labeled K562 cells were measured by flow cytometry with an FL2‐H filter.

Techniques: Binding Assay, Staining, Fluorescence, MANN-WHITNEY

Journal: Molecular Oncology

Article Title: NKG2A and circulating extracellular vesicles are key regulators of natural killer cell activity in prostate cancer after prostatectomy

doi: 10.1002/1878-0261.13422

Figure Lengend Snippet: Schematic diagram of this study. More circulating EVs were found in the blood of prostate tumor patients. Two types of receptors mainly regulate NKA: NKG2D, the activating receptor that leads to NK activation or exhaustion, and NKG2A, the inhibiting receptor that leads to NK dysfunction. After treatment with an inhibitory antibody or siRNA, NKG2A was shown to be the main receptor regulating NKA through EV binding. After prostatectomy, the number of circulating EVs increased, which subsequently increased NKA by decreasing NKG2A and increasing total NKG2D ligands. In conclusion, NKG2A is the key regulator of NKA in PCas, and blocking NKG2A could represent a potential therapy for inhibiting the effects of tumor‐dependent EVs on NKA.

Article Snippet: Small interfering RNAs (siRNAs; 50 ng) targeting nothing (negative control), NKG2A, and NKG2D (smart pools from Synbio Tech, Kaohsiung, Taiwan) were incubated with 3 μL polyfast transfection reagent (MedChemExpress, Princeton, NJ, USA) in medium without FBS for 15 min. Then, the mixture was added to medium containing 5 × 10 4 NK cells and incubated for 48 h. Forty microgram protein per 5 μL of circulating EVs from healthy donors and tumor patients were added to NK cells for 24 h. A total of 1 × 10 5 DiI‐labeled K562 cells were added to NK cells for 2 h, and the remaining DiI‐labeled K562 cells were measured by flow cytometry with an FL2‐H filter.

Techniques: Activation Assay, Binding Assay, Blocking Assay

Journal: Advanced Science

Article Title: FAPα + Macrophages Orchestrate Immune Evasion in Multiple Myeloma by Dual Regulation of PD‐L1 and T Cell Senescence

doi: 10.1002/advs.202506239

Figure Lengend Snippet: FAPα + Macrophages Are Enriched in the Myeloma Microenvironment and Correlate with Disease Progression. (A) Heatmap of gene expression in macrophages from patients with MM (n = 3) or healthy donors (HDs, n = 3). (B,C) Cell percentages of FAPα + macrophages (CD11b + CD14 + ) and FAPα + BMSC (CD45 − CD38 − CD29 + ) in PBMCs or BMMCs from patients with MM (n = 9). (D) Reprehensive immunofluorescence staining (IF) images of CD138, CD68, and FAPα in the BM of a NDMM patient (Magnification ×400. Scale bar, 50 µm). (E–G) Flow cytometry analysis of the cell percentages of FAPα + macrophages and FAPα + BMSCs in BMMCs from patients with MM at different disease stages (n = 27) or HDs (n = 4). (H) Correlation analysis of the cell percentages between CD138 + MM cells and FAPα + macrophages or FAPα + BMSCs in patients with NDMM (n = 15). (I) Reprehensive immunohistochemical staining (IHC) images of CD138 and FAPα in patients with NDMM or MM‐CR (Magnification ×200. Scale bar, 50 µm). (J,K) Western blot (J) and flow cytometry (K) analysis of FAPα expression in macrophages co‐cultured with MM cells (n = 3). (L) TGFβ1 expression in different MM cell lines. (M‐N) TGFβ1‐induced FAPα protein expression in macrophages (Magnification ×400. Scale bar, 50 µm). (O) M‐CSF, TGFβ1, and FAPα levels in BM supernatants from patients with MM at different disease stages (n = 37) using ELISA assay. (P) Correlation analysis of FAPα and TGFβ1 or M‐CSF in BM supernatant from patients with NDMM (n = 17). (Q) Reprehensive IHC images of bone marrow samples from patients with MM (n = 18) (Magnification ×200. Scale bar, 50 µm). (R) IOD values of CD138, CD68, and FAPα in patients with NDMM or RRMM (n = 18). (S) Correlation analysis between CD138 and FAPα in patients with NDMM. (T) Kaplan–Meier curves of PFS and overall OS in the set of patients with NDMM based on FAP protein expression level detected in tumor tissues. The median value of FAP RNA expression in the was 88.26 (IOD). The expression value of the FAP high group (n = 9) was >88.26(IOD) and the FAP low group (n = 9) was <88.26(IOD). Data are presented as mean ± SD. Each dot means independent samples. ns, no significant difference. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Statistical analysis was performed using a 2‐tailed Student's t ‐test in C, E, F, G, K, O, and R, a Pearson correlation in H, P, and S, a log‐rank test in T. MM, multiple myeloma; HD, healthy donors; BMSCs, bone marrow mesenchymal stem cells; PBMCs, peripheral blood mononuclear cells; BMMCs, bone marrow mononuclear cells; BM, bone marrow; NDMM, newly diagnosed MM; RRMM, relapsed or refractory MM; CR, complete response; TGFβ1, Transforming growth factor beta 1; M‐CSF, macrophage colony stimulating factor; IHC, Immunohistochemistry; IOD, Integrated Optical Density; RRMM, Relapsed/Refractory MM; PFS, Progression‐free survival; OS, overall survival.

Article Snippet: The bone marrow supernatants of patients with different MM stages were collected, and different cytokines were detected as described by the respective manufacturers: Human M‐CSF AccuSignal ELISA Kit (Cat #KOA0253, Rockland, USA), Human Seprase/FAP AccuSignal ELISA Kit (Cat #KOA0627, Rockland, USA), Human TGFβ1 ELISA Kit (Cat #1117102) (All from Dakewe Bioengineering Co., Ltd, China).

Techniques: Biomarker Discovery, Gene Expression, Immunofluorescence, Staining, Flow Cytometry, Immunohistochemical staining, Western Blot, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, RNA Expression, Immunohistochemistry