PROJECT SUMMARY
Ploidy altering mechanisms such as cell-cell fusion and whole genome doubling (WGD) can drive intratumoral
heterogeneity and alter chemotherapeutic response. Understanding the causes and consequences of these
mechanisms is especially important in triple negative breast cancer (TNBC) which accounts for 15-20% of
diagnosed breast cancers and does not have targeted treatment options. Broad spectrum chemotherapies
remain the standard of care for TNBC, but their efficacy is dampened by high cellular heterogeneity and 40% of
treated TNBC patients will relapse. We hypothesize that ploidy altering mechanisms are a driving force behind
intratumoral heterogeneity and can function as active escape or damage recovery mechanisms to allow cells to
resist chemotherapy. An updated version of the ClonMapper barcoding system which includes nucleotide-
indexed expressed DNA barcodes on GFP and mCherry will be used to enable parallel tracking of cell-cell fusion
events in fluorescent live-cell imaging and determination of detailed transcriptomic and karyotypic clonal
trajectories in ploidy-separated longitudinal single-cell sequencing. The ClonMapper expressed DNA barcodes
will be added to TNBC cell lines and patient-derived cell cultures. In a series of controlled experiments we will
(1) determine the rates and distinguish the effects of pre-existing aneuploidy and therapy-induced aneuploidy in
clinically relevant TNBC models across different chemotherapeutic treatments, (2) follow subclonal
transcriptomic and karyotypic trajectories in scRNA-seq and scDNA-seq under normal and chemotherapeutic
conditions to determine if different chemotherapies select for or generate specific transcriptomic or karyotypic
patterns, and (3) elucidate the molecular factors which activate cell-cell fusion, WGD, or other ploidy altering
mechanisms. The results of these experiments will be rich in longitudinal single cell data on transcriptomic and
chromosomal transitions utilized by TNBC cells across chemotherapeutic perturbation and recovery. As the
degree of tumor aneuploidy can be easily determined from patient samples, we will experimentally parameterize
and validate an agent-based model which includes aneuploidy fraction, spontaneous and stress-induced
mechanisms of ploidy alteration, and genomic stability to predict tumor evolution under different therapeutic
schedules. This study will be the first to systematically investigate spontaneous and chemotherapy-induced
mechanisms of ploidy alteration in a longitudinal single-cell framework. In elucidating the cellular states which
predispose cancer cells for ploidy alteration, identifying the phenotypic and chromosomal transitions induced by
these events, and quantifying the effects of pre-existing and de novo generated aneuploidy on chemotherapeutic
resistance, the causes and consequences of cell-cell fusion and WGD may be revealed as primary contributors
to chemoresistance and inspire novel treatment strategies to improve TNBC patient outcomes.