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Page 4 of 12 David. J Cancer Metastasis Treat 2022;8:32 https://dx.doi.org/10.20517/2394-4722.2022.71

While the classical vs. basal-like dichotomy in PDA has become widely accepted by the field, additional
questions remain to be answered. For one, the role of EMT in the acquisition of basal-like gene expression is
unclear. Basal-like tumors usually show enrichment of markers of the epithelial-to-mesenchymal transition
(EMT), although adenosquamous cancers that occur in the pancreas and is associated with the basal-like
subtype are epithelial. PDA cell lines of epithelial and mesenchymal morphology can be obtained,
[25]
although cell lines expressing the basal-like or squamous transcriptional program are often epithelial in
morphology (unpublished observations). Are basal-like tumors simply more prone to undergo EMT in
response to the correct signals, or is there a direct connection between EMT or EMT-promoting signals in
driving cells towards basal-like gene expression? Second, while mouse models have shown an ability to
recapitulate classical vs. basal-like gene expression, mice with pancreas-specific Cdkn2a deletion combined
[26]
with mutant KRAS activation develop sarcomatoid tumors that are not seen in human PDA . These
tumors are completely devoid of epithelial features and have been suggested to be underrepresented in
human surgical specimens due to their highly invasive nature, precluding their surgical resection . Do
[27]
these tumors represent the extreme end of an EMT spectrum that includes basal-like tumors?

DETERMINANTS OF TRANSCRIPTIONAL SUBTYPES
What explains the tendency of a given tumor towards either of the two subtypes? One potential explanation
is epigenetic, based on PDA cell of origin. This hinges on the fact that the introduction of appropriate
genetic lesions in both ductal and acinar cells in mouse models can give rise to PDA [12,13,28] . PDA derived
from acinar cells is associated with PanIN lesions, both before and after progression to invasive carcinoma,
while duct-derived carcinomas display far fewer attendant PanIN lesions . Importantly, duct-derived
[13]
carcinoma cells show a tendency towards basal-like gene expression, while acinar-derived tumors more
[29]
closely resemble the classical subtype identified in human cohorts . Definitively ascribing a cell of origin to
advanced human malignancies is impossible, meaning the role of the cell of origin in human PDA subtypes
will remain undetermined. That said, human ducts have been recently shown to harbor cells expressing the
squamous lineage-determining transcription factor (TF) ΔNp63, and displaying the full spectrum of basal
[30]
markers in addition to the ductal marker KRT19 . While mouse ducts did not contain basal cells, normal
mouse ductal cells could be induced to express ΔNp63 under organoid growth conditions, suggesting ductal
cells can easily transdifferentiate into a basal-like phenotype .
[30]
While these studies suggest that cell of origin may skew individual tumors towards one of the two major
subtypes, additional data suggest that the transcriptional subtype is not predetermined, but is an evolving
tumor phenotype. Importantly, there is evidence in at least one patient harboring a classical or hybrid
primary tumor in which a metastasis that emerged later had switched to the basal subtype . Given the
[23]
difficulty of obtaining tumor material at multiple stages of progression in individual patients, the data
supporting the generality of this phenomenon are still limited, although several additional observations
provide further support. An important recent study showed that basal and classical cells coexist within
individual tumors, a phenomenon observed in 13/15 tumors examined , and which was recapitulated in
[23]
[31]
organoid culture . This suggests that basal vs. classical tumor designations using bulk RNA-seq data reflect
only tipping of the balance towards one or the other differentiation state. This implies that, given the correct
circumstances, the balance could be tipped in the other direction.

What might drive tumor cell plasticity? One important contributor appears to be the tumor
microenvironment (TME), which has recently been shown to determine the transcriptional phenotype of
PDA cells . This conclusion is partly based on observations that in vitro culture conditions played a role in
[24]
determining transcriptional phenotype, often causing in vitro models to deviate significantly from the
tumors from which they were derived. Growth in tumor organoid conditions favored outgrowth of tumor