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Smigiel et al. J Cancer Metastasis Treat 2019;5:47 I http://dx.doi.org/10.20517/2394-4722.2019.26 Page 3 of 20

tumors can be treated with antibodies or kinase inhibitors targeting HER2 signaling (i.e., Trastuzamab and
Lapatinib) and are often met with success provided the disease is caught early; the efficacy of these therapies
drastically decreases with late stage, metastatic HER2+ BC [20-23] . Conversely, TNBC currently lacks a targeted
therapy tailored to a specific driver oncogene and is most often treated with cytotoxic chemotherapies.
Patients with TNBC exhibit an increased risk of metastatic dissemination resulting in higher clinical stage
at diagnosis and lower disease-free survival compared to patients with non-TNBC cancers . Much like
[24]
metastatic HER2+ BC, metastatic TNBC is not effectively treated, highlighting the need for better therapies
targeting those cells which progress beyond the primary site and are ultimately responsible for patient
mortality and morbidity. Further challenges in treating BC involve the heterogeneous nature of the tumor
cells themselves, a phenomenon often referred to as intra-tumoral heterogeneity (ITH) . Evidence suggests
[25]
that across a panel of human cancers, including breast, increased ITH correlates with decreased overall
survival, and therapy resistance [26,27] . Furthermore, high ITH inversely correlates with low tumor infiltrating
lymphocytes, which are often associated with increased patient survival [28-36] .

The path to ITH is complex and involves a series of genetic and epigenetic events throughout the transformation
process which permit normal human mammary epithelial cells (HMEC) to develop into fully malignant
cancer cells [37-44] . Progress in RNA and DNA sequencing technologies have helped shape the evolutionary
picture of HMEC; losing tumor suppressor function (TP53 mutations or RB loss) and acquiring oncogenic
drivers (MYC, HER2, or CCND1 amplification or PIK3CA mutations) [45-49] . Genetic alterations lead to the
expansion of a pre-malignant population which progressively acquires additional genetic and epigenetic
changes until one or more cells become fully transformed [50,51] . These additional mutations are numerous, and
genomic profiling has found a wide variety of changes in copy number, chromatin alterations, chromosomal
rearrangements, and point mutations throughout the genome from single cell sequencing of bulk tumor
tissue in TNBC [36,52,53] . Not only does this dynamic process of transformation alter the cancer cell itself, but
transforming cells have a substantial impact on the surrounding environment. Evidence suggests that the
accumulation of mutations within epithelial cells can lead to a dysregulated secretory network, including a
number of inflammatory cytokines linked to poor prognosis, therapy failure, and disease recurrence (IL-6,
IL-8, TGF-β, CCL2, TNF-α, IL-17 and others) [54-59] . This dysregulated secretory network in turn, changes
the cellular composition of the TME, leading to a reciprocal cross-talk between non-cancerous stromal cells
and the transforming epithelial cells. Overall, this demonstrates the immense complexity of the tumor, as
the heterogeneity described above culminates in a highly diverse TME, with an array of cell types, secreted
factors, and structural make up.

Importantly, not all epithelial cells that begin the transformation process reach full malignancy. As a cell
senses aberrant activation of signaling pathways/gene induction, it may enact intact tumor suppressive
mechanisms, resulting in senescence [60-66] . Senescence is a major growth-inhibiting and tumor-suppressive
barrier that must be bypassed in vivo during transformation en-route to tumor development [63,67-73] . Large
senescent cell populations can be found at various stages of tumor development, further contributing to
tumor heterogeneity. Remarkably, an investigation by Cotarelo et al. was able to identify the presence
[74]
of senescent cells in approximately 83.7% of the human invasive breast carcinomas examined, suggesting
their involvement throughout the transformation process and as tumors evolve and progress. Since the 129
tumors surveyed in this study were from untreated patients, the origin of senescence within these invasive
BC is an in vivo physiological response.

Long thought inert, bystanders within the tumor, senescent cells have gained considerable interest for their
potential impact on the tumor as a whole. Despite being growth-arrested, senescent cells remain viable,
metabolically active, and play an important role in the developing TME [75-77] . A hallmark of senescent cells is
the secretion of a wide variety of growth factors, pro-inflammatory cytokines, chemokines, and proteinases,
a characteristic termed the senescence-associated secretory phenotype (SASP) [Figure 1] [78,79] . Under normal