Phenotype screens of murine pancreatic cancer identify a Tgf-α-Ccl2-paxillin axis driving human-like neural invasion
Xiaobo Wang, Rouzanna Istvanffy, Linhan Ye, Steffen Teller, Melanie Laschinger, Kalliope N. Diakopoulos, Kıvanç Görgülü, Qiaolin Li, Lei Ren, Carsten Jäger, Katja Steiger, Alexander Muckenhuber, Baiba Vilne, Kaan Çifcibaşı, Carmen Mota Reyes, Ümmügülsüm Yurteri, Maximilian Kießler, Ibrahim Halil Gürçınar, Maya Sugden, Saliha Elif Yıldızhan, Osman Uğur Sezerman, Sümeyye Çilingir, Güldal Süyen, Maximilian Reichert, Roland M. Schmid, Stefanie Bärthel, Rupert Oellinger, Achim Krüger, Roland Rad, Dieter Saur, Hana Algül, Helmut Friess, Marina Lesina, Güralp Onur Ceyhan, Ihsan Ekin Demir
Xiaobo Wang, Rouzanna Istvanffy, Linhan Ye, Steffen Teller, Melanie Laschinger, Kalliope N. Diakopoulos, Kıvanç Görgülü, Qiaolin Li, Lei Ren, Carsten Jäger, Katja Steiger, Alexander Muckenhuber, Baiba Vilne, Kaan Çifcibaşı, Carmen Mota Reyes, Ümmügülsüm Yurteri, Maximilian Kießler, Ibrahim Halil Gürçınar, Maya Sugden, Saliha Elif Yıldızhan, Osman Uğur Sezerman, Sümeyye Çilingir, Güldal Süyen, Maximilian Reichert, Roland M. Schmid, Stefanie Bärthel, Rupert Oellinger, Achim Krüger, Roland Rad, Dieter Saur, Hana Algül, Helmut Friess, Marina Lesina, Güralp Onur Ceyhan, Ihsan Ekin Demir
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Research Article Oncology

Phenotype screens of murine pancreatic cancer identify a Tgf-α-Ccl2-paxillin axis driving human-like neural invasion

Abstract

Solid cancers like pancreatic ductal adenocarcinoma (PDAC), a type of pancreatic cancer, frequently exploit nerves for rapid dissemination. This neural invasion (NI) is an independent prognostic factor in PDAC, but insufficiently modeled in genetically engineered mouse models (GEMM) of PDAC. Here, we systematically screened for human-like NI in Europe’s largest repository of GEMM of PDAC, comprising 295 different genotypes. This phenotype screen uncovered 2 GEMMs of PDAC with human-like NI, which are both characterized by pancreas-specific overexpression of transforming growth factor α (TGF-α) and conditional depletion of p53. Mechanistically, cancer-cell-derived TGF-α upregulated CCL2 secretion from sensory neurons, which induced hyperphosphorylation of the cytoskeletal protein paxillin via CCR4 on cancer cells. This activated the cancer migration machinery and filopodia formation toward neurons. Disrupting CCR4 or paxillin activity limited NI and dampened tumor size and tumor innervation. In human PDAC, phospho-paxillin and TGF-α–expression constituted strong prognostic factors. Therefore, we believe that the TGF-α-CCL2-CCR4-p-paxillin axis is a clinically actionable target for constraining NI and tumor progression in PDAC.

Authors

Xiaobo Wang, Rouzanna Istvanffy, Linhan Ye, Steffen Teller, Melanie Laschinger, Kalliope N. Diakopoulos, Kıvanç Görgülü, Qiaolin Li, Lei Ren, Carsten Jäger, Katja Steiger, Alexander Muckenhuber, Baiba Vilne, Kaan Çifcibaşı, Carmen Mota Reyes, Ümmügülsüm Yurteri, Maximilian Kießler, Ibrahim Halil Gürçınar, Maya Sugden, Saliha Elif Yıldızhan, Osman Uğur Sezerman, Sümeyye Çilingir, Güldal Süyen, Maximilian Reichert, Roland M. Schmid, Stefanie Bärthel, Rupert Oellinger, Achim Krüger, Roland Rad, Dieter Saur, Hana Algül, Helmut Friess, Marina Lesina, Güralp Onur Ceyhan, Ihsan Ekin Demir

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Figure 3

The migration behavior of cancer cells triggered by neurons is regulated by the CCL2/CCR4 axis.

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The migration behavior of cancer cells triggered by neurons is regulated...
(A) Schematic representation of the 3D migration assay. Arrows indicate the direction of migrating cells. (B) Representative images of migrating SU.86.86 pancreatic cancer cells in the MF (toward DRGs) and BF (opposite to DRGs) analyzed by confocal microscopy and labeled filopodia (pink lines). (C) Diagrams showing the number and length of filopodia in the cancer cells in BF and MF, quantified with the FiloQuant software. (D) Representative images of SU.86.86 cancer cells from the migration assay, stained with phalloidin (red) and phospho-paxillinY118 (green), and counterstained with DAPI (blue). (E) Diagram showing the number of p-paxillin-positive points per ×10 magnification. (F) Representative Western blots of SU.86.86 and T3M4 cancer cells treated with recombinant CCL2 (100 ng/mL). (G) Graphs with relative content of proteins identified by Western blot measured with the ImageJ software (n=3 biological replicates). (H) Western blots of SU.86.86 and T3M4 cancer cells treated with CCR4 inhibitor C021 (140 nM). (I) Graphs with relative content of proteins identified by Western blot measured with the ImageJ software. (J) Scheme of experiment: cancer cells were pretreated with rCCL2 or C021 for 15 minutes and placed into the 3D migration assay with DRG neurons. As control, cells pretreated with vehicle were used. (K) Graphs indicating FMI, velocity, and migrated distance of cancer cells in the 3D migration assay. All results in graphs are shown as a mean ± SEM. For statistical analyses we used unpaired t test (B and E), 1-way ANOVA (G, H, and K), Dunnett’s multiple comparisons test (G, I, and K), and for the distribution, Shapiro-Wilk normality test (all panels). The P value ˂ 0.05 was considered to have significance. Scale bars: 20 μm.