Fig 1: Uptake and lysosomal co-localization of protein-LNP complexes in HepG2 cells.a HepG2 cells internalizing LNPs loaded with Cy5-mRNA pre-incubated with high-binding corona proteins were visualized by confocal microscopy. Representative image of LNP + VTN incubations showing LNPs (Cy5; red), cell membrane (CellBrite membrane dye; green), and nuclei (Hoechst; blue). b Inset showing a magnified view of the region outlined by the red box in (a). c Quantification of Cy5 (LNP) signal per cell demonstrates differences in cell Cy5-mRNA uptake between select corona protein incubations (n = 4 technical replicates, n = 3 biological replicates). Each dot represents an individual field-of-view-level measurement, color-coded by biological replicate; larger, black-outlined dots indicate the mean value for each biological replicate. Statistical analysis was performed by a nested one-way ANOVA test followed by Dunnett’s multiple comparisons test. d, e Cy5 signal from HepG2 cells internalizing LNPs loaded with Cy5-mRNA pre-incubated with high-binding corona proteins was also quantified by flow cytometry. d Percentage of Cy5-positive cells and e mean fluorescence intensity show uptake trends consistent with microscopy. Data points shown are biological replicates (n = 3 technical replicates, n = 3 biological replicates). Error bars denote standard deviation. Statistical analysis was performed by repeated measures one-way ANOVA test with Geisser-Greenhouse correction, followed by Dunnett’s multiple comparisons test. To compare endosome entrapment for select protein incubations, co-localization of the Cy5 signal (LNP) and fluorescently labeled lysosomes (green) was analyzed. Representative image of LNP + ApoE incubation shows f LNPs (red), lysosomes (green) and nuclei (blue) fluorescently labeled. g Quantification of overlapping Cy5 (LNP) and lysosome signal per cell (n = 4 technical replicates, n = 4 biological replicates). Each dot represents an individual field-of-view-level measurement, color-coded by biological replicate; larger, black-outlined dots indicate the mean value for each biological replicate. Statistical analysis was performed by a nested one-way ANOVA test followed by Dunnett’s multiple comparisons test. For all statistical analyses performed, *, **, and *** represent p ≤ 0.05, 0.01, and 0.001, respectively. All image thresholding was applied uniformly across samples, and image channels were adjusted solely for visualization purposes.
Fig 2: In vitro mRNA expression with delivery by protein-LNP complexes.a LNPs loaded with mRNA encoding luciferase were incubated with selected high-binding corona proteins (0.05 ng mRNA: 1 ng protein) prior to introduction to HepG2 cells seeded at 4.7 × 104 cells per cm2 (100 ng mRNA per well). The luminescence was measured as a proxy for mRNA expression to understand the effect of proteins on LNP delivery efficiency. Luminescence was normalized to the average of each no-corona LNP biological control for all in vitro studies. Created in BioRender. Voke, E. (2025) https://BioRender.com/58u4s4t. b Resulting luminescence of pre-incubations of individual proteins with LNPs showed no significant change in luminescence (mRNA expression) for ApoE, A2M, or a mixture of the proteins, while showing a significant decrease for VTN or CR, each relative to the no-corona LNP control. c Dose–response of protein concentrations for VTN incubated LNPs showed a significant decrease in mRNA expression compared to the no-corona LNP control. d Cell viability showed no statistical difference for protein incubations. N = 4 technical replicates, n = 3 biological replicates. Data points shown are biological replicates. All data are presented as mean values with standard deviation. Statistical analysis was performed by repeated measures one-way ANOVA test with Geisser–Greenhouse correction, followed by Dunnett’s multiple comparisons test where * and ** represent p ≤ 0.05 and p ≤ 0.01, respectively.
Fig 3: The adhesion effects of VTN are robust across assay conditions and biological sources. A, Representative microscopy of prostate cancer cells plated on polyHEMA-coated plates depicting the abrogation of cell attachment and successful spheroid formation. B, Adhesion assays were performed with polyHEMA-coated plates and serum-free media with or without 5 μg/mL VTN. Bars show three biological replicates, and significance was calculated with two-tailed Welch t tests. ns, not statistically significant. C, Cells were subjected to a time-course adhesion assay in serum-free media with or without 5 μg/mL VTN. Bars show three biological replicates, and significance was calculated for the 24-hour timepoint with two-tailed Welch t tests. D, Images were taken of well centers from the 24-hour timepoint, and cells within captured fields of view were counted. Results are for three biological replicates, and two-tailed Welch t tests were used for statistical determinations. E, Adhesion assays were performed in serum-free media with 5 μg/mL rVTN, 5 μg/mL human plasma–purified VTN (hVTN), or no VTN. Complete, use of complete media as a positive control. Bars show three biological replicates, and significance was calculated with two-tailed Welch t tests. F, Conditioned media was recovered from 5-donor human plateable hepatocytes (first collection period/day 1). An adhesion assay was performed with 22Rv1 and 100% conditioned media serially diluted 1:1 for seven timepoints. Images show representative regions of adhered or suspended 22Rv1 cells following exposure to 100%, 50%, 25%, or 0% conditioned media. G, Conditioned media (24 μL) was directly mixed with 8 μL of 4× SDS-PAGE and immunoblotted for VTN as specified in “Materials and Methods.” As media was collected and replenished every 24 hours, lane labels indicate the period of media collection since hepatocyte plating (day 1 corresponds to collection on day 1 since plating). Uncond. media, unconditioned hepatocyte maintenance media. H, An adhesion assay was performed with basal hepatocyte maintenance media, 50% hepatocyte-conditioned maintenance media, or complete culture media as a positive control (RPMI-1640 with 10% FBS). Complete, use of complete culture media as a positive control; maintenance, use of maintenance media as the basal serum-free media (rather than RPMI-1640). Bars show three biological replicates, and significance was calculated with two-tailed Welch t tests. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.
Fig 4: VTN is associated with greater liver metastatic burden, and its phenotypic effects may be mitigated by defactinib. A, Schematic of the in vivo experimental plan wherein mice undergo orthotopic 22Rv1 injection to the anterior prostate, tail-vein injections of VTN or PBS every other day, and necropsy to diagnose metastatic spread. B, Tumor volume, tumor mass, and liver mass were measured at necropsy. Results are plotted for two biological replicates. C, Whole livers were excised and examined for metastatic foci. Livers were chunked, formalin-fixed, paraffin-embedded, and hematoxylin and eosin (H&E)–stained before re-examination for micrometastases. Whole tissue with grossly visible metastatic foci highlighted in green and H&E of tissue regions with metastases and corresponding AR IHC are shown. D, Cells were plated and serum-starved overnight with or without 1 μmol/L defactinib. Then, cells were exposed to VTN and DMSO or VTN and defactinib for the specified amounts of time before cells were lysed, and immunoblotting was performed. Red triangles indicate the pERK bands. E, Adhesion assays were performed in serum-free media with the specified compounds and either 5 μg/mL VTN in dH2O or dH2O alone. Bars show three biological replicates, and significance was calculated with two-tailed Welch t tests. Complete, complete media used as a positive control; def., defactinib; ns, not statistically significant. F, Cells were plated in 6-well plates in serum-free media with either 5 μg/mL VTN in dH2O or dH2O alone, in the context of the MEK and PI3K inhibitors trametinib and alpelisib in combination or the FAK inhibitor defactinib, and allowed to adhere for 24 hours. Cells were harvested for PI staining followed by flow cytometry, and data were gated to exclude debris and doublets. Hypodiploid cell fractions were calculated as described in “Materials and Methods,” and statistical significance was determined from individual experiments with a two-tailed Fisher exact test. alp., alpelisib; tram., trametinib. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.
Fig 5: Schematic of the study findings. VTN accumulates along the hepatic sinusoid lumen, where current models of metastasis suggest it would encounter circulating tumor cells. Our results indicate that VTN promotes adhesion and spreading, inhibits hypodiploid accumulation in a manner consistent with prosurvival effects, and activates MAPK and PI3K in prostate cancer cells. [Created in BioRender. Egelberg, J. (2026) https://BioRender.com/xlzui2w.].
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