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David. J Cancer Metastasis Treat 2022;8:32 https://dx.doi.org/10.20517/2394-4722.2022.71 Page 5 of 12

cells with classical gene expression, while adherent culture as cell lines tended to favor basal-like. Given the
distinct media composition between the two culture systems, one possibility this suggested is that
differential exposure to specific cytokines may underlie differences in transcriptional phenotype. For
example, PDA organoid culture routinely calls for the inclusion of a TGF-β inhibitor in the culture medium.
Adding TGF-β to the organoid culture medium was sufficient to shift the transcriptional profile back
towards the basal expression state, suggesting that transcriptional states in vivo may be strongly influenced
by microenvironment-derived TGF-β signaling.

In support of this idea, xenografted organoids displayed distinct transcriptional profiles depending on
where they were implanted in the pancreas. To complement existing orthotopic xenograft models that
involve an injection of cells into the pancreatic interstitial space, Tuveson and colleagues developed an
[32]
intraductal organoid transplantation model . Interestingly, the tumors formed by individual organoid lines
could differ in transcriptional subtype depending on the location of organoid implantation. Cancer cells
implanted in the interstitial space of the pancreas were more prone to adopt a basal-like transcriptional
profile than those implanted within pancreatic ducts. Tumors within the interstitial space frequently
contained reactive desmoplastic stroma, while intraductal transplants were often only exposed to the
normal murine ducts, suggesting that stromal-derived signals indeed play an important role in driving the
adoption of the basal-like transcriptional phenotype. Importantly, depletion of macrophages from the PDA
TME resulted in a switch away from the squamous/basal-like subtype in cancer cells, suggesting that
[33]
macrophages may supply important signals enforcing that transcriptional phenotype . A recent study
[34]
suggests this may occur through tumor necrosis factor (TNF)-α secretion . Additional work will be
necessary to fully elucidate the function of stromal cell populations in driving classical versus basal-like
differentiation.

Cell-autonomous mechanisms may also contribute to the evolution of PDA transcriptional phenotypes.
Gains of mutant KRAS signaling have been implicated in PDA metastasis in mouse models, and cells
derived from these models exhibit a transcriptional phenotype consistent with basal-like cells . Later work
[27]
in human PDA confirmed a correlation between basal-like gene expression and gains in KRAS signaling.
Basal-like tumors were significantly more likely than classical tumors to harbor genomic imbalances
[23]
favoring the mutant allele of KRAS . Genomic imbalances usually involve loss of the wild-type KRAS allele
coupled with genome duplication. In one patient, a classical primary tumor contained only a minor
imbalance favoring the mutant KRAS allele (loss of the wild-type allele), while a basal-like metastasis that
[20]
occurred later had gained copies of mutant KRAS . This data suggests that subtype switching, driven by
amplification of KRAS signaling, can occur during pancreatic cancer progression. Direct evidence for such
an idea has been provided in organoids engineered to express tamoxifen-inducible mutant KRAS.
Activation of KRAS using this system was shown to push implanted tumors towards a more basal-like
phenotype both histologically and transcriptionally . Given that mutations in KRAS initiate and drive all
[32]
stages of pancreatic tumorigenesis [35,36] , it will be of great interest to identify the mechanisms by which a
quantitative change in KRAS output can contribute to such dramatic phenotypic and transcriptional
differences during tumor progression.

In addition to KRAS gains, additional genetic correlates of tumor subtypes have been uncovered.
Amplifications of the TF GATA6 and loss of SMAD4 are enriched in classical subtype tumors . GATA6 is
[23]
an endodermal TF associated with normal pancreatic development , so its association with well-
[37]
differentiated pancreatic tumors of the classical subtypes appears to fit. As discussed above, TGF-β signaling
appears to play a role in the emergence of the basal-like phenotype, so a paucity of SMAD4 alterations in the
basal-like subtype is consistent with those observations. In the basal-like subtype, loss of function of the