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

Background: 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.

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

Methods: 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.

Results: 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.

Conclusions: 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.

CD40L; P‐selectin; cell adhesion; platelet–cancer cell interaction; shear accumulation; shear rate.