Aggregation of Platelets and Human EGFR Mutant Lung Adenocarcinoma Cells Under Flow Is Governed by Shear Accumulation

Circulating tumor cells (CTCs) utilize platelets to withstand hemodynamic stress and evade immune clearance; however, the mechanisms driving platelet-cancer cell aggregation under flow remain unclear.

We investigated which physical quantity-shear rate, exposure time, or an integrated metric-governs the aggregation and identified the key adhesion molecules involved.

Human platelets and EGFR-mutant lung adenocarcinoma PC-9 cells were fluorescently labeled, mixed at a 10:1 ratio, and subjected to mechanical stimulation using a vortex mixer or a TA ARES G2 rheometer across shear rates 0-4050 s^-1 and durations of 0-4860 s. Aggregates were quantified by flow cytometry via dual-positive gating. The functional roles of platelet P-selectin and CD40 ligand (CD40L) were assessed using antibodies Inclacumab and 5c8, respectively. Shear significantly promoted platelet-PC-9 aggregation compared with static conditions, while blocking P-selectin or CD40L markedly suppressed this effect.

Aggregation exhibited a biphasic dependence on both shear rate and exposure time. Notably, when plotting aggregation against shear accumulation (rate × time), all data converged onto a unified biphasic curve with an optimum of ~27 000.

These results indicate that platelet-PC-9 aggregation is governed by a shear accumulation-dependent mechanism, optimized under moderate shear and finite exposure, and regulated by P-selectin and CD40L. These findings provide new biophysical insights into transient platelet-PC-9 interactions in circulation and suggest that targeting platelet activation pathways or modulating hemodynamics may prevent hematogenous metastasis.