顶刊文献解读｜深度解析肿瘤微环境空间调控核心机制

Figure 2.

Synthesis, screening, and characterization of macrophage-specific mRNA delivery systems

(A) Construction of the ionizable lipid library.

(B) pLNP formulation library.

(C) Workflow for preparation and screening of EGFP-pLNPs.

(D) Heatmap of EGFP-positive percentages in RAW264.7 cells treated with EGFP-pLNPs.

(E) Chemical structure of DKP-2-O.

(F) Molar composition of the optimized pLNP formulation.

(G) Representative confocal images of RAW264.7 cells and BMDMs treated with free CRT mRNA, CRT-LNPs, or CRT-pLNPs. Cy5-labeled CRT mRNA (red) indicates the intracellular distribution of the delivered mRNA, while the cell membrane and nuclei were stained with DIO (green) and DAPI (blue), respectively. Cy5, Cyanine 5; DIO, 3,3′-dioctadecyloxacarbocyanine perchlorate; DAPI, 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride. Scale bars, 10 μm.

(H) Representative flow cytometry analysis of mRNA uptake.

(I) Quantification of fluorescence intensity by flow cytometry (n = 5). MFI, mean fluorescence intensity.

(J) Particle size distribution, PDI, and EE of CRT-pLNPs (n = 3).

(K) Zeta potential of CRT-pLNPs (n = 3).

(L) TEM images of CRT-pLNPs. The data are shown as the means ± SDs from biological replicates. Statistical analysis was carried out by two-way ANOVA.

Figure 3.

In vitro generation and functional assessment of CRT-Ms

(A) Representative flow cytometry analysis of CRT expression in TREM2-KO macrophages after 12-h treatment.

(B) Quantitative analysis of CRT expression levels (n = 5).

(C) Representative confocal images showing plasma membrane localization of myc-tagged CRT (green); cell membrane stained with DiD (red); nuclei stained with DAPI (blue). DiD, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt. Scale bars, 10 μm.

(D) Western blot analysis of CRT expression.

(E) CRT-pLNP-mediated delivery of CRT mRNA promotes macrophage efferocytosis. CRT-pLNPs enable efficient uptake of CRT mRNA, followed by lysosomal escape and subsequent translation into membrane-expressed CRT protein, which enhances the efferocytic activity of macrophages.

(F) Representative confocal images of macrophages coincubated with apoptotic cells. Apoptotic cells labeled with CellTracker Red; macrophages labeled with CellTracker Green. Scale bars, 50 μm.

(G) Quantification of efferocytosis based on confocal images (n = 5).

(H) Representative flow cytometry analysis of pHrodo-labeled apoptotic cell uptake.

(I) Quantitative analysis of apoptotic cell engulfment by flow cytometry (n = 5).

(J) Western blot analysis of sTREM2 in cell culture supernatants treated with IL-1β and PMA.

(K) Flow cytometry analysis of macrophage proliferation using the BeyoClick EdU-594 Cell Proliferation Detection Kit.

(L) Quantification of proliferating macrophages (n = 5).

(M) Cytotoxicity quantified by lactate dehydrogenase (LDH) released into the macrophage culture supernatant after overnight incubation (n = 5).

(N and O) Representative flow cytometry analysis of CD86 (N) and CD206 (O) expression in CRT-Ms after the indicated treatment.

(P and Q) Quantification of CD86-positive (P) and CD206-positive (Q) cells (n = 5).

(R) Quantification of macrophage polarization by flow cytometry. The proportions of CD86-positive and CD206-positive cells were used as markers to represent M1 and M2 macrophages, respectively (n = 5).

(S–U) IL-6 (S), IFN-γ (T), and TNF-α (U) levels in cell culture supernatants (n = 5). The data are shown as the means ± SDs from biological replicates. Statistical analysis was carried out by one-way ANOVA in (B), (P), (Q), (R), (S), (T), and (U); two-way ANOVA in (G) and (I); and an unpaired two-tailed Student’s t test in (L) and (M).

Figure 4.

Therapeutic effects of CRT-Ms mediated by sPirb/pLNPs in HFMCD-induced MASH model mice

(A) Schematic illustration of MASH model establishment and treatment strategy in C57BL/6 mice.

(B) Immunofluorescence images of macrophages (green) and apoptotic cells (red) in liver tissues after 2 weeks of treatment with the indicated reagents. The nuclei were stained with DAPI. TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. The white arrows indicate the colocalization of macrophages and apoptotic cells. Scale bars, 50 μm.

(C) Quantification of efferocytosis represented as the ratio of macrophage-associated apoptotic cells to total apoptotic cells per liver area (n = 5).

(D) Serum sTREM2 levels in mice after 2 weeks of treatment.

(E) Representative liver tissue sections stained with oil red O, H&E, and α-SMA after 4 weeks of treatment. H&E, hematoxylin-eosin staining; α-SMA, alpha-smooth muscle actin. Scale bars, 50 μm.

(F) Quantitative analysis of oil-red-O-positive areas (n = 5).

(G) Quantitative analysis of α-SMA-positive areas (n = 5).

(H) Representative TUNEL staining (red) of liver sections from mice treated for 4 weeks. The nuclei were stained with DAPI. The white arrows indicate TUNEL+ apoptotic cells. Scale bars, 50 μm.

(I) Quantitative analysis of apoptotic cells relative to the total number of hepatocytes (n = 5).

(J) Representative flow cytometry analysis of liver macrophages showing the expression of the M1 marker CD86 and the M2 marker CD206 expression in mice treated for 4 weeks.

(K) The ratio of M1 to M2 macrophages was quantified as the proportion of CD86-positive cells to CD206-positive cells (n = 5).

(L–N) Serum levels of inflammatory cytokines TNF-α (L), IL-1β (M), and IL-6 (N) after the indicated treatments (n = 5). The data are shown as the means ± SDs from biological replicates. Statistical analysis was carried out by one-way ANOVA.

Figure 5.

Therapeutic evaluation of steatosis, fibrosis, and inflammation in HFD-CCl4-induced MASH model mice treated with CRT-Ms mediated by sPirb/pLNPs

(A) Schematic illustration of MASH model induction and treatment regimen.

(B) Representative H&E, oil red O, and Sirius Red staining of liver sections from mice subjected to the indicated treatments.

(C) Quantitative analysis of NAS scoring based on H&E-stained sections (n = 8).

(D) Quantitative analysis of oil-red-O-positive areas (n = 8). Scale bars, 100 μm.

(E) Quantitative analysis of Sirius-Red-positive areas in liver sections (n = 8).

(F) Representative TUNEL staining (red) of liver sections from mice treated for 4 weeks. The nuclei were stained with DAPI. Scale bars, 100 μm.

(G) Quantitative analysis of apoptotic cells relative to the total number of hepatocytes (n = 8).

(H) Western blot analysis of SCD1, α-SMA, COL1A1, TNF-α, and IL-1β in liver tissues from mice receiving the indicated treatments.

(I–L) RT-qPCR quantification of Col1a1 (I), Scd1 (J), Tnf (K), and Il1b (L) expression levels in liver tissues.

(M) Representative flow cytometry analysis of the proportion of monocyte-derived macrophages within the hepatic macrophage population.

(N) Quantitative analysis of the proportion of monocyte-derived macrophages within the hepatic macrophage population (n = 8).

(O) Representative flow cytometry analysis of neutrophil populations in the liver.

(P) Quantitative analysis of neutrophil populations in the liver (n = 8). The data are shown as the means ± SDs from biological replicates. Statistical analysis was carried out by one-way ANOVA.

Figure 6.

In vivo therapeutic effects of CRT-Ms mediated by cRGDfk/pLNPs in atherosclerotic model mice

(A) Schematic illustration of atherosclerosis model establishment and treatment strategy in LDLr-KO mice.

(B) Representative immunofluorescence images of apoptotic cells (red) and macrophages (green) in the aortic root of mice after three administrations. The nuclei were stained with DAPI. The white arrows indicate the colocalization of macrophages and apoptotic cells. Scale bars, 50 μm.

(C) Quantification of macrophage engulfment capacity within plaques, represented as the ratio of macrophage-associated apoptotic cells to total apoptotic cells.

(D) Representative oil red O staining of frozen sections from the aortic root, aortic arch, and brachiocephalic artery of mice after 4 weeks of treatment. Scale bars, 200 μm.

(E–G) Quantitative analysis of oil-red-O-positive areas in sections of the aortic root (E), aortic arch (F), and brachiocephalic artery (G).

(H) Representative H&E staining of sections from the aortic root of mice after 4 weeks of treatment. The upper image shows the entire plaque area outlined by black dashed lines, while the lower image highlights the necrotic core area within the plaque. Scale bars, 200 μm.

(I) Quantification of the ratio of the necrotic core area to the plaque area in H&E-stained sections of the aortic root (n = 5).

(J) Representative images of Sirius-Red-stained frozen sections from the aortic root after 4 weeks of treatment. The lower image shows black dashed lines outlining the plaque area, and black solid lines indicate the fibrous cap thickness. Scale bars, 200 μm.

(K) Quantification of the fibrous cap area to plaque area ratio in Sirius-Red-stained sections of the aortic root.

(L) Representative TUNEL immunofluorescence staining (red) indicating apoptotic cells in frozen sections of the aortic root from mice after 4 weeks of treatment. The nuclei were labeled with DAPI. The white dashed lines in the lower image outline the plaque area. Scale bars, 200 μm.

(M) Quantification of the ratio of apoptotic cells to total cells in the aortic root from TUNEL-stained sections (n = 5).

(N–P) Plasma concentrations of TNF-α (N), IL-1β (O), and IL-6 (P) in mice after 4 weeks of treatment (n = 5). The data are shown as the means ± SDs from biological replicates. Statistical analysis was carried out by one-way ANOVA.

【免责声明】原文献《Cell Rep Med》（DOI：10.1016/j.xcrm.2025.102580），由 AI 解读整理;文中涉及的原文献图片、数据等知识产权归原期刊及研究团队所有。若存在侵权情形，敬请及时联系我方删除，我方将积极配合处理。