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Tumor Discovery Sorafenib induces MVPs in NSCLC

and 15 µL of 2 M magnesium chloride. H1299 cells were assessed using the NanoSight NS300 instrument (Malvern
grown in RPMI-1640 medium containing 10% FBS, 2.5 mL Instruments, UK). MVP counts were normalized with the
penicillin-streptomycin, 2.5 mL antibiotic-antimycotic, cell number as per previous reports. 14,18,19
2.25 mL of 40% glucose, and 5 mL of 100 mM sodium
pyruvate. All cell cultures were maintained at 37°C with 2.5. Statistical analysis
95% humidity and 5% CO . All statistical analyses were conducted using GraphPad
2
Prism software version 10 (GraphPad Software, San Diego,
2.3. Cell survival assay CA, USA). Each in vitro experiment was performed
As per our previous reports, sulforhodamine-B (SRB) assay independently at least three times using biological
was used to assess cell survival. 14,15 H1299 and A549 cells replicates. Data were analyzed by unpaired Student’s t-test
were plated into 96-well plates at a seeding density of 5 × or one-way analysis of variance (ANOVA) with post hoc
10 cells per well and treated with 0.1% dimethyl sulfoxide Dunnet’s multiple comparison tests. The p<0.05 was
3
(DMSO) as control, or with sorafenib at concentrations considered statistically significant.
ranging from 1 to 16 µM. In separate experiments,
sorafenib was used at a concentration of 4 µM, imipramine 3. Results
at 20 µM, and their co-treatment at given concentrations.
After 24, 48, and 72 h, cells were fixed with 100 µL of 10% 3.1. Sorafenib inhibits the survival of NSCLC cell
trichloroacetic acid followed by incubation at 4°C for 1 h. lines in a time- and dose-dependent manner
Fixed cells were gently rinsed with distilled water three Our first studies tested the dose- and time-response effects
times and stained using 100 µL 0.4% (w/v) SRB (prepared of sorafenib treatment on the survival of A549 and H1299
in 1% acetic acid), followed by 15-min incubation at room NSCLC cell lines through the SRB assay. These cell lines
temperature in the dark. Excess dye was removed by triple have been widely used as NSCLC models to determine the
rinsing with distilled water containing 1% glacial acetic acid mechanisms and cellular responses of sorafenib alone or its
and then allowed to air dry. Bound SRB dye was solubilized combination with other agents. 39,41-44 It was observed that
using 150 µL of 10 mM Tris base (tris(hydroxymethyl) the survival of A549 and H1299 cell lines was inhibited
aminomethane) while placing the plates on a shaker for by sorafenib in a dose- and time-dependent manner
10 min. Absorbance was measured at 570 nm using a (Figure 1A and B).
Synergy H1Mf plate reader. Cell viability for each group Interestingly, despite both A549 and H1299 cells
was normalized to its respective vehicle-treated control lacking EGFR mutations and being inherently resistant to
(0.1% DMSO). EGFR-TKIs, A549 cells demonstrated greater sensitivity
45
2.4. MVP isolation and quantification to sorafenib compared to H1299 cells. This observation is
consistent with previous report showing that A549 cells,
Isolation and quantification of MVP were performed using which harbor a KRAS G12S mutation, are more susceptible
methods previously described by our group. 14,18,19 Briefly, to sorafenib’s effects, likely due to its inhibition of RAF-
A549 and H1299 cells were grown to approximately dependent signaling. Given that sorafenib also targets
46
80–90% confluency, after which cultures were rinsed three vascular endothelial growth factor receptor (VEGFR)
times with serum-free Hanks’ Balanced Salt Solution and platelet-derived growth factor receptor (PDGFR), its
(HBSS, Cytiva, USA). Cells were then incubated with anti-proliferative effect on A549 cells may also involve
0.1% DMSO for negative control, or 100 nM CPAF and angiogenic signaling pathways. 47
phorbol myristate acetate (PMA) for positive controls,
and sorafenib at various concentrations (4, 8, and 16 Despite being p53-null and KRAS wild-type, H1299 cells
µM) in HBSS containing 1% free fatty acid. Similarly, also exhibited a significant reduction in cell viability with
combination experiments used pre-treatments with the sorafenib treatment. However, the degree of inhibition was
PAFR antagonist, WEB2086 (10 µM), and imipramine lower than in A549 cells at comparable doses. This suggests
(20 µM) for 1 h, followed by treatment with or without that sorafenib’s mechanism of action may be more effective
sorafenib (8 µM). After 4 h of incubation, the conditioned in KRAS-mutant NSCLC models, aligning with findings
medium was centrifuged at 2,000 ×g for 20 min at 4°C to from previous studies utilizing A549 and PC-9 cells. 38
remove residual cells and debris. The clarified supernatant
was centrifuged at 20,000 ×g for 70 min at 4°C to pellet 3.2. PAFR and aSMase pathways mediate sorafenib-
MVP. Pellets were then resuspended with 100 µL of sterile- induced MVP release
14
filtered phosphate-buffered saline (PBS) to prepare for Given that exposure to EGFR-TKIs induces MVP release,
nanoparticle tracking analysis. MVP concentration was which have been shown to carry PAF agonists and serve

Volume 4 Issue 3 (2025) 83 doi: 10.36922/TD025110019