EUROPEAN BREAST CANCER CONFERENCE
BACKGROUD: Molecular profiling of patient tumors today uses high
throughput genome and transcriptome technologies, yet proteins and
especially their functions, on which drugs can act, are poorly predicted.
Proteomic immunoassay approaches like Reverse Phase Protein Arrays
(RPPA) have demonstrated that signaling pathway profiling of up to hundreds
of samples and proteins in parallel can add valuable information (i) providing
multiple marker patterns from small amounts of tissue specimen, and (ii)
providing biological answers based on a functional protein level: Are
pathways active downstream of a driver mutation? Will drug treatment be
effective? What are the underlying mechanisms? Can we identify predictive
marker proteins? We combine RPPA with cell line and patient-derived
model systems as efficient “close-to-in-vivo” in vitro systems to generate
phenotype/pathway signature information to be used for drug testing and
personalized medicine applications.
MATERIAL AND METHODS: Protein lysates were prepared and analyzed from
various tumor model systems treated with different chemotherapeutic and
targeted drugs and compounds. Sample sources included patient-derived
Xenograft models, homo and heterotypic 3D InsightTM tumor microtissues
(Insphero) and 3D microtumor cultures derived from fresh patient tumor
material, treated at different doses and times. Protein lysates were analyzed
via RPPA applying sensitive fluorescence immunoassays with up-front wellvalidated
antibodies against 100+ focused protein markers (total, phospho
forms) of key oncology pathways (e.g. MAPK, PI3K/Akt/mTOR).
RESULTS: Phenotype/pathway signatures were generated from the RPPA
data. Changes upon treatment of the individual pathways – their activities
and associate protein markers – are used for correlations to mechanisms
of drug action e.g. efficacy/resistance, and for treatment response prediction.
Treatment responses of 3D tumor models were benchmarked with
conventional 2D cell culture models as controls.
CONCLUSION: Multiplex protein and pathway profiling using RPPA can
provide meaningful biological information from functional protein and
pathway signatures generated from of up to hundreds of proteins and
samples – measured in parallel and from minute amounts of starting
materials. Leveraging several advanced patient-derived tumor models, our
results demonstrate the power of phenotype profiling for gaining conclusive
drug mode-of-action information, identification of early response treatment
markers and development of precision therapies.
No conflict of interest