Ungefroren Hendrik, Thürling Isabel, Färber Benedikt, Kowalke Tanja, Fischer Tanja, De Assis Leonardo Vinícius Monteiro, Braun Rüdiger, Castven Darko, Oster Henrik, Konukiewitz Björn, Wellner Ulrich Friedrich, Lehnert Hendrik, Marquardt Jens-Uwe
First Department of Medicine, University Hospital Schleswig-Holstein, Campus Lübeck, Center of Brain, Behavior and Metabolism (CBBM), University of Lübeck, D-23538 Lübeck, Germany.
Clinic for Surgery, University Hospital Schleswig-Holstein, Campus Lübeck, University of Lübeck, D-23538 Lübeck, Germany.
Cancers (Basel). 2022 Apr 19;14(9):2057. doi: 10.3390/cancers14092057.
Intratumoral heterogeneity (ITH) is an intrinsic feature of malignant tumors that eventually allows a subfraction of resistant cancer cells to clonally evolve and cause therapy failure or relapse. ITH, cellular plasticity and tumor progression are driven by epithelial-mesenchymal transition (EMT) and the reverse process, MET. During these developmental programs, epithelial (E) cells are successively converted to invasive mesenchymal (M) cells, or back to E cells, by passing through a series of intermediate E/M states, a phenomenon termed E-M plasticity (EMP). The induction of MET has clinical potential as it can block the initial EMT stages that favor tumor cell dissemination, while its inhibition can curb metastatic outgrowth at distant sites. In pancreatic ductal adenocarcinoma (PDAC), cellular models with which to study EMP or MET induction are scarce. Here, we have generated single cell-derived clonal cultures of the quasimesenchymal PDAC-derived cell line, PANC-1, and found that these differ strongly with respect to cell morphology and EMT marker expression, allowing for their tentative classification as E, E/M or M. Interestingly, the different EMT phenotypes were found to segregate with differences in tumorigenic potential in vitro, as measured by colony forming and invasive activities, and in circadian clock function. Moreover, the individual clones the phenotypes of which remained stable upon prolonged culture also responded differently to treatment with transforming growth factor (TGF)β1 in regard to regulation of growth and individual TGFβ target genes, and to culture conditions that favour ductal-to-endocrine transdifferentiation as a more direct measure for cellular plasticity. Of note, stimulation with TGFβ1 induced a shift in parental PANC-1 cultures towards a more extreme M and invasive phenotype, while exposing the cells to a combination of the proinflammatory cytokines IFNγ, IL1β and TNFα (IIT) elicited a shift towards a more E and less invasive phenotype resembling a MET-like process. Finally, we show that the actions of TGFβ1 and IIT both converge on regulating the ratio of the small GTPase RAC1 and its splice isoform, RAC1b. Our data provide strong evidence for dynamic EMT-MET transitions and qualify this cell line as a useful model with which to study EMP.
肿瘤内异质性（ITH）是恶性肿瘤的一个内在特征，最终会使一部分耐药癌细胞发生克隆进化，导致治疗失败或复发。ITH、细胞可塑性和肿瘤进展是由上皮-间质转化（EMT）及其逆向过程间质-上皮转化（MET）驱动的。在这些发育过程中，上皮（E）细胞通过一系列中间E/M状态依次转化为侵袭性间质（M）细胞，或再变回E细胞，这种现象称为E-M可塑性（EMP）。MET的诱导具有临床潜力，因为它可以阻断有利于肿瘤细胞播散的初始EMT阶段，而抑制MET则可以抑制远处转移灶的生长。在胰腺导管腺癌（PDAC）中，用于研究EMP或MET诱导的细胞模型很少。在这里，我们建立了源自准间质PDAC细胞系PANC-1的单细胞克隆培养物，发现这些培养物在细胞形态和EMT标志物表达方面有很大差异，从而可初步将它们分类为E、E/M或M。有趣的是，通过集落形成和侵袭活性以及昼夜节律功能测量发现，不同的EMT表型在体外与致瘤潜力的差异相关。此外，其表型在长期培养后保持稳定的单个克隆，在生长调节和单个TGFβ靶基因方面，以及在有利于导管向内分泌转分化的培养条件下（作为细胞可塑性的更直接指标），对转化生长因子（TGF）β1治疗的反应也不同。值得注意的是，用TGFβ1刺激会使亲代PANC-1培养物向更极端的M和侵袭性表型转变，而将细胞暴露于促炎细胞因子IFNγ、IL1β和TNFα（IIT）的组合中则会引发向更E和侵袭性更小的表型转变，类似于MET样过程。最后，我们表明TGFβ1和IIT的作用都集中在调节小GTP酶RAC1及其剪接异构体RAC1b的比例上。我们的数据为动态EMT-MET转变提供了有力证据，并证明该细胞系是研究EMP的有用模型。
