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Background

Triple-negative breast cancer (TNBC) presents significant therapeutic limitations due to its aggressive heterogeneity and the rapid emergence of adaptive resistance to apoptosis-based regimens. Addressing these challenges requires polypharmacological strategies capable of modulating multiple signalling networks simultaneously. While the Cannabis sativa phytocomplex offers a vast chemical space for multi-target intervention, the quantitative pharmacological basis of its synergistic interactions remains largely uncharacterised.

Purpose

This study aimed to deconstruct the synergistic landscape of high-purity phytocannabinoids (CBD, CBG, CBD-A) in combination with the sesquiterpene β-caryophyllene (BCP) against TNBC, using MDA-MB-231 as a primary model and Hs578T as a validation line.

Methods

Growth Rate (GR) inhibition metrics and the SynergyFinder+ framework were used to map pharmacological interactions across four reference models. Subcellular dynamics and phenotypic transitions were characterised by high-resolution label-free holotomographic microscopy combined with live-cell kinetic imaging and single-cell fate mapping.

Results

Two highly potent synergistic clusters were identified for CBD-CBG-BCP combinations, with ZIP, HSA, and Bliss synergy scores exceeding 65. CBD-A exhibited minimal interaction potential and was excluded from ternary studies. GR-based quantification further revealed that these combinations produced net cytotoxicity (GR < 0) at sub-IC₅₀ concentrations of each component. Single-cell fate mapping by holotomographic microscopy identified a temporally ordered death programme: an initial phase of extensive cytoplasmic vacuolisation associated with focal perinuclear space swelling and progressive nuclear compression, morphological hallmarks of autosis, which is followed by a transition to apoptotic execution. The autotic nature of the primary death phase was confirmed by pharmacological rescue with digoxin, a selective inhibitor of the Na⁺,K⁺-ATPase. To the best of our knowledge, this sequential engagement of autosis followed by apoptotic execution represents the first documented instance of such a two-stage death programme in any cellular model.

Conclusion

These findings provide robust evidence that specific phytocannabinoid-terpene ratios engage a Na⁺,K⁺-ATPase-regulated autotic programme as an upstream commitment step, followed by apoptotic execution, effectively circumventing the caspase-independent resistance mechanisms characteristic of TNBC. This study establishes a rational, quantitatively validated framework for transitioning from empirical botanical use to evidence-based, multi-target cannabinoid polypharmacology in aggressive breast cancer.