Fig 2: Bioluminescence analysis of cellular uptake of THP-engineered PalmReNL-EVs. (A) Bioluminescence analysis of the recipient MDA-MB-231 cells cultured with two different concentrations (6.0 × 108 and 6.0 × 109 EVs/mL) of THP-engineered PalmReNL-EVs using furimazine. (B) Bioluminescence analysis of cellular uptake of PalmReNL-EVs (6.0 × 109 EVs/mL) engineered with the EGFR-binding peptide #2, together with PalmReNL-EVs bearing PDL1-, uPAR-binding peptides, or DOPE-lipid nanoprobes only. (A,B) Error bars, SD (n = 5); two-sided Student’s t-tests was used for comparison.

Fig 3: Engineered PalmReNL-EVs with tumor-homing peptides (THPs). (A) Schematic diagram of EV membrane labeling with PalmReNL BRET probes and functionalization with THP-coupled lipid nanoprobes. (B) Transmission electron microscopy of HEK293FT cell-derived PalmReNL-EVs with or without uPAR-, PDL1-, and EGFR-binding peptides. Scale bar, 200 nm. (C) A phase contrast image of MDA-MB-231 cells. Scale bar = 100 µm. (D) Western blot analysis of the target membrane protein expression in MDA-MB-231 cells under the non-reducing condition. GAPDH is a loading control.

Fig 4: Inhibition of FOXQ1 induces downregulation of angiogenic factors and upregulation of angiogenic inhibitors. (A) Matrix distribution of 60 well-established angiogenic proteins on a Quantibody Human Angiogenesis Array (QAH-ANG-2 and QAH-ANG-3); each protein is distributed in quadruplicate horizontally. (B) Fluorescence detection of protein arrays for either cell lysate of HUVECs or conditioned medium (CM) of colorectal cancer (CRC) cells. CM was collected from either DLD1-shFOXQ1 or DLD1-shControl and added to HUVECs. After 48 h, HUVECs were harvested and lysed. Both the cell lysates and CM were collected, and the secretion of 60 angiogenic proteins were measured by hybridization with QAH-ANG-2 and QAH-ANG-3. The 17 green and 6 red rectangles on (A, B) show 17 angiogenesis factors and 6 angiogenic inhibitors within the detection range of the protein array. (C) Quantitative concentration of the 17 angiogenic factors and 6 angiogenic factors in cell lysates of HUVECs (pg/ml). (D) Quantitative concentration of the 17 angiogenic factors and 6 angiogenic factors in CM collected from either DLD1-shFOXQ1 or DLD1-shControl (pg/ml). The values above each bar on (C, D) represent quantitative concentration of corresponding protein, is the average of quadruplicates (n = 1 per group). (E) Western blot analyses for ANG, PDGF, PDGFRB, ANGPT1, PLAUR, tPA, VEGF, EGFR, HB-EGF, and Twist1 were performed with DLD1-shFOXQ1 and DLD1-shControl cell lysates. ß-actin was used as the loading control. (F) Inhibition of FOXQ1 prevented the autocrine secretion of CCL2, whereas overexpression of FOXQ1 enhanced the secretion of CCL2, as determined by ELISA analysis. ** P<0.01, *** P<0.001 signifies a significant difference between the indicated groups (two-tailed, unpaired Student’s t-test). Bars represent mean ± S.E. of three independent experiments. HUVEC, human umbilical vein endothelial cell; CM, conditioned media.

Fig 5: Fluorescence analysis of THP-engineered PalmReNL-EV uptake by MDA-MB-231 cells. MDA-MB-231 cells cultured with PalmReNL-EVs bearing DOPE-lipid nanoprobes only (A,E,I), EGFR- (B,F,J), PDL1- (C,G,K), or uPAR-binding peptides (D,H,L) for 8, 16, and 24 h. Punctate fluorescence signals of tdTomato (red) were merged with nuclei stained with Hoechst 33342 (blue). Scale bar = 10 µm.