Abstract
Abstract Background: The mechanism of induction of apoptosis has been one of the features to characterize and classify anti-tumor drugs. We have standardized a mechanism-based approach to identify apoptosis in live tumor cells by triple co-fluorescence (De et al., 2018; SCIENTIFIC REPORTS). Our protocol tests three crucial features of apoptosis; the enzymatic activity of executioner caspase3, cell surface presentation of caspase-dependent phosphatidylserine and mitochondrial function. Navigating the genomic landscape of tumors in patients with Triple Negative Breast Cancers (TNBC) at Avera Cancer Institute, we have identified algorithm for the rational combination of PI3K-pathway (PP) drugs. Aim: Here we present the results of testing the rational combinations of PP-targeted drugs on apoptosis using our mechanism-based laboratory friendly procedure. Methods: Our protocol co-stains live tumor cells in real time with NucView488 Casp3substrate (N), CF594AnnexinV (A), and MitoViewBlue (M). We have used carboplatin and targeted-drugs like olaparib, ABT888, BMN673, AZD2014, GDC0980, and AZD5363 in BRCA-WT (MDA-MB231, MDA-MB468) and BRCA-incompetent (SUM149) TNBC cells. Imaging of cells using simultaneous live/dead cell markers (Calcein AM green/EthD-1 red) was used for validation. Results: We retrospectively analyzed genomic data [FoundationOne] from 100 consecutive patients seen in our center from 2014. 137 genes were altered including 12 PP and 11 DNA-repair genes. We used AnnexinV-PE staining (Accuri C6), confocal IF imaging and WB expression of apoptotic signals and mitochondrial potential (TMRE-A) as validation criteria following different combinations. Treatment with the rational combination(s) like PARP inhibitor(s) plus carboplatin and PP inhibitor decreased proliferation, induced profound early apoptotic events, along with increased apoptotic signaling and increased mitochondrial depolarization in real time. Dual treatment also blocked cytoplasmic M staining while increasing both nuclear N substrate staining and membranous A staining. Control cells had higher Calcein AM positive (live) cells and fewer EthdD-1 stained red nuclei of dead cells than treated cells. Merged images (M+N+A) showed 100% mutual exclusivity between M and N or A stains in both control and treated cells as determined by overlap and colocalization coefficients. Conclusion: Our method of staining identified the apoptosis following treatment of drug-combination(s), which was based on the genomic alteration(s) present in TNBC patients. Citation Format: Nandini Dey, Jennifer Carlson Aske, Pradip De. Testing algorithmic basis for combinations of targeted drugs by a mechanism-based laboratory friendly identification of apoptosis in live breast cancer cells [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-03-13.
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