Movel Three Dimensional and Dynamic (3DD) Cell Culture System for Optimization of molecularly Targeted Combination Therapy in Dual HER2 and mTOR Refractory Breast Cancer
Authors: Ande Anusha , Tran Bao Nguyen , Brown Ashley , Ait‐Oudhia Sihem 
Published: November 2023

Background

HER2‐positive (HER2+) breast cancer (BC) is more quickly‐growing and aggressive than other types of BC. The introduction of HER2 targeted therapy such as Trastuzumab (TmAb), a humanized monoclonal antibody directed against HER2 receptor markedly improved the prognosis associated with HER2+ BC. However, the occurrence of primary and acquired resistance to TmAb treatment poses a significant clinical challenge. One of the major mechanisms of resistance involves the PI3K/mTOR pathway, for which preclinical studies demonstrated the successful use of mTOR inhibitors such as Everolimus (EVE). However, counter‐regulation loops in this pathway also lead to resistance to EVE. Therefore, we hypothesized that Dasatinib (DAS), an Src inhibitor, while administered concomitantly with EVE and a cytotoxic agent such as paclitaxel (PAC) may be beneficial at overcoming the dual resistance to HER2 therapy and EVE.

Method

Cell culture studies on JIMT‐1 cells, a human HER2+ BC cell line refractory to both TmAb and EVE therapies were conducted in static (plate) and three dimensional dynamic (3DD) settings. The inhibitory concentrations resulting in 50% killing of cancer cells (IC50) were evaluated in static cell culture conditions for PAC, EVE, and DAS, by measuring JIMT‐1 cells viability using a CC‐8 proliferation assay kit. Upon testing the effect of the triple combination (PAC+DAS+EVE) in static studies, we conducted a pharmacokinetic‐pharmacodynamic (PK/PD) study in 3DD settings to examine the anticipated synergistic combinatorial effects of these three agents. The 3DD cell culture poses the advantages of: 1) mimicking human PK of each drug in the combination, 2) serial sampling of adherent cells, and 3) studies can be continued for several weeks after drug treatment.

Results

The IC50s for PAC, DAS, and EVE in JIMT‐1 cells are measured at 170, 50, and 10 nM. JIMT‐1 cells are found to be resistant to EVE achieving only 60% cytotoxic response that is saturated upon increased dose. However, the combination (PAC+DAS+EVE) significantly decreased cell viability compared to single agents EVE (p<0.0001), DAS (p<0.0001), PAC (p<0.001), and the dual combination EVE+DAS (p<0.0001). Furthermore, results from 3DD experiments to test the efficacy of the sequential triple combination (50 nM each agent), where PAC was infused for 3h one day prior to the continuous infusion of (EVE+DAS) over 4 days showed a significant decrease in total cell number (151 to 18.2 million) as well as cell viability compared to the control arm (155.7 to 274.26 million). More interestingly, we completely achieved the maximum efficacy of the combination without any regrowth of cancer cells observed for an additional 2 weeks upon cessation of the treatment.

Conclusion

A novel triple combination was successfully evaluated in both static and dynamic in vitro cell culture systems. The efficacy of this combination in overcoming the resistance of JIMT‐1 to HER2 and mTOR therapies is proven. Protein dynamics, PK, and PD data will help us develop a quantitative systems pharmacology model to simulate the clinical efficacy of this novel combination at overcoming resistance to HER2 and mTOR therapies in BC.