Pancreatic ductal adenocarcinoma (PDAC) is characterized by a fibroblast-rich desmoplastic stroma. Cancer-associated fibroblasts (CAFs) have been shown to display a high degree of interconvertible ...states including quiescent, inflammatory, and myofibroblastic phenotypes; however, the mechanisms by which this plasticity is achieved are poorly understood. Here, we aim to elucidate the role of CAF plasticity and its impact on PDAC biology.
To investigate the role of mesenchymal plasticity in PDAC progression, we generated a PDAC mouse model in which CAF plasticity is modulated by genetic depletion of the transcription factor Prrx1. Primary pancreatic fibroblasts from this mouse model were further characterized by functional in vitro assays. To characterize the impact of CAFs on tumor differentiation and response to chemotherapy, various coculture experiments were performed. In vivo, tumors were characterized by morphology, extracellular matrix composition, and tumor dissemination and metastasis.
Our in vivo findings showed that Prrx1-deficient CAFs remain constitutively activated. Importantly, this CAF phenotype determines tumor differentiation and disrupts systemic tumor dissemination. Mechanistically, coculture experiments of tumor organoids and CAFs showed that CAFs shape the epithelial-to-mesenchymal phenotype and confer gemcitabine resistance of PDAC cells induced by CAF-derived hepatocyte growth factor. Furthermore, gene expression analysis showed that patients with pancreatic cancer with high stromal expression of Prrx1 display the squamous, most aggressive, subtype of PDAC.
Here, we define that the Prrx1 transcription factor is critical for tuning CAF activation, allowing a dynamic switch between a dormant and an activated state. This work shows that Prrx1-mediated CAF plasticity has significant impact on PDAC biology and therapeutic resistance.
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The regulation of metastatic organotropism in pancreatic ductal a denocarcinoma (PDAC) remains poorly understood. We demonstrate, using multiple mouse models, that liver and lung metastatic ...organotropism is dependent upon p120catenin (p120ctn)-mediated epithelial identity. Mono-allelic p120ctn loss accelerates KrasG12D-driven pancreatic cancer formation and liver metastasis. Importantly, one p120ctn allele is sufficient for E-CADHERIN-mediated cell adhesion. By contrast, cells with bi-allelic p120ctn loss demonstrate marked lung organotropism; however, rescue with p120ctn isoform 1A restores liver metastasis. In a p120ctn-independent PDAC model, mosaic loss of E-CADHERIN expression reveals selective pressure for E-CADHERIN-positive liver metastasis and E-CADHERIN-negative lung metastasis. Furthermore, human PDAC and liver metastases support the premise that liver metastases exhibit predominantly epithelial characteristics. RNA-seq demonstrates differential induction of pathways associated with metastasis and epithelial-to-mesenchymal transition in p120ctn-deficient versus p120ctn-wild-type cells. Taken together, P120CTN and E-CADHERIN mediated epithelial plasticity is an addition to the conceptual framework underlying metastatic organotropism in pancreatic cancer.
•Independent mouse models reveal that liver metastasis requires P120CTN/E-CADHERIN•P120CTN/E-CADHERIN interaction is not required for lung metastasis in these models•p120ctn isoform 1A restoration in p120ctn null cells reinstates liver tropism•Human liver metastases exhibit more epithelial properties relative to primary PDAC
The functional basis of metastatic organotropism sheds light on the properties required for successful colonization of distant organs. Reichert et al. demonstrate that epithelial plasticity is a determinant of metastatic organotropism in pancreatic cancer with differing properties required for liver and lung colonization.
Abstract
Background: Pancreatic ductal adenocarcinoma (PDAC) is characterized by a fibroblast-rich desmoplastic stroma which plays a critical role in the progression and therapeutic resistance of ...PDAC. The stroma is composed of extracellular matrix proteins, mainly deposited by the cancer-associated-fibroblasts (CAFs) and various types of immune cells. Cancer-associated fibroblasts display a high degree of interconvertible states including quiescent, inflammatory and myofibroblastic phenotypes. However, the mechanisms by which this plasticity is achieved are poorly understood. Here, we demonstrate that CAF plasticity promotes PDAC cell aggressiveness through multiple mechanism, particularly promoting Epithelial-to-Mesenchymal Transition and immune cell infiltration.
Methods: To manipulate fibroblast plasticity in PDAC, we generated genetically engineered mouse models (GEMMs) in which CAF plasticity is modulated by genetical depletion of the transcription factor Prrx1 in fibroblasts by using orthotopic implantation models (Sm22-CreERT, Prrx1fl/fl, Rosa26mTmG) as well as dual recombinase-driven GEMMs (Pdx-Flp, FSF-KrasG12D/w t, p53fr/wtt, Sm22-CreERT, Prrx1fl/fl). To characterize the impact of CAFs on tumor differentiation, immune cell infiltration and response to chemotherapy various in vivo and in vitro co-culture experiments were performed.
Results: Our in vivo results demonstrate that restraining CAF plasticity by Prrx1-depletion leads to more differentiated tumors, disrupts systemic tumor dissemination, including circulating tumor cells as well as metastases. Interestingly in tumors with Prrx1-deficient stroma, infiltration of macrophages and lymphocytes was increased. Specifically, we observed more B-cells as well as cytotoxic T-cells. Gene expression profiling of primary murine fibroblast samples revealed that Prrx1-deficient CAFs express myofibroblastic gene signatures characterized by ECM secretion phenotype. Indeed, on a functional level Prrx1-deficient CAFs secret more collagen and are highly migratory. Additionally, co-culture experiments of tumor cells and CAFs revealed that Prrx1-driven CAF-derived hepatocyte growth factor confers to a more invasive PDAC cell phenotype and resistant to therapy-induced apoptosis by inducing EMT in vitro. Importantly, in line with our in vitro and in vivo findings, compartment specific-gene expression analysis of human data revealed that pancreatic cancer patients with high stromal expression of Prrx1 display the squamous, most aggressive, subtype of PDAC.
Conclusions: Here, we define that the Prrx1 transcription factor is critical for CAF plasticity, allowing a dynamic switch between different states. This work demonstrates that Prrx1-mediated CAF plasticity has significant impact on PDAC biology and therapeutic resistance.
Citation Format: Karin Feldmann, Carlo Maurer, Katja Peschke, Steffen Teller, Kathleen Schuck, Katja Steiger, Thomas Engleitner, Rupert Öllinger, Aristeidis Papargyriou, Rim Sabrina Jahan Sarker, Wilko Weichert, Anil K. Rustgi, Roland M. Schmid, Roland Rad, Günter Schneider, Dieter Saur, Maximilian Reichert. Fibroblast plasticity driven by Prrx1 interferes the tumor cells - tumor microenvironment crosstalk towards a more aggressive pancreatic ductal adenocarcinoma abstract. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PR003.
Pancreatic ductal adenocarcinoma remains a major challenge in cancer medicine. Given the increase in incidence and mortality, interdisciplinary research is necessary to translate basic knowledge into ...therapeutic strategies improving the outcome of patients. On the 4th and 5th of February 2021, three German pancreatic cancer research centers, the Clinical Research Unit 5002 from Göttingen, the Collaborative Research Center 1321 from Munich, and Clinical Research Unit 325 from Marburg organized the 1st Virtual Göttingen-Munich-Marburg Pancreatic Cancer Meeting in order to foster scientific exchange. This report summarizes current research and proceedings presented during that meeting.
One of the major challenges in using pancreatic cancer patient-derived organoids (PDOs) in precision oncology is the time from biopsy to functional characterization. This is particularly true for ...endoscopic ultrasound-guided fine-needle aspiration biopsies, typically resulting in specimens with limited tumor cell yield. Here, we tested conditioned media of individual PDOs for cell-free DNA to detect driver mutations already early on during the expansion process to accelerate the genetic characterization of PDOs as well as subsequent functional testing. Importantly, genetic alterations detected in the PDO supernatant, collected as early as 72 hours after biopsy, recapitulate the mutational profile of the primary tumor, indicating suitability of this approach to subject PDOs to drug testing in a reduced time frame. In addition, we demonstrated that this workflow was practicable, even in patients for whom the amount of tumor material was not sufficient for molecular characterization by established means. Together, our findings demonstrate that generating PDOs from very limited biopsy material permits molecular profiling and drug testing. With our approach, this can be achieved in a rapid and feasible fashion with broad implications in clinical practice.
Abstract Chronic graft-versus-host disease (cGVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (HSCT) and a leading cause of non-relapse mortality (NRM). Currently, ...biology-based markers are lacking both for diagnosis and for monitoring the activity of cGVHD. Seventy patients who received HSCT were enrolled in a pilot study, including 21 without cGVHD and 49 with active or resolved cGVHD. Evaluations were comprised of clinical parameters including cGVHD severity and infections. Peripheral blood cells were analyzed by multi-parameter flow cytometry. The CD19+ B cell compartment was further subdivided by staining for surface IgD, CD21 and CD27. No significant differences in absolute B, T, and natural killer (NK) cell numbers were observed between the groups with and without cGVHD. However, elevated numbers (>15% of B lymphocytes) of immature/transitional CD19+ /CD21- B cells were associated with the occurrence of severe infections ( P = .003). Most significantly, all patients with active cGVHD and elevated numbers of CD19+ /CD21- B lymphocytes experienced severe infections ( P = .00016). The numbers of both non-class-switched and class-switched memory B cells were significantly lower in patients with active cGVHD when compared to patients who never experienced cGVHD ( P = .002 and P = .001). Perturbation of circulating B lymphocyte compartments may serve as a novel biomarker for monitoring cGVHD activity and its impact on the immune system. A prospective study on unselected patients assessed serially for B cell reconstitution after HSCT is warranted.
Introduction: Chronic graft-versus-host-disease (cGvHD) is a major complication of allogeneic hematopoietic stem cell transplantation (HSCT) and a leading cause of non-relapse mortality due to ...profound immunodeficiency. The approach currently used to establish the diagnosis of cGvHD depends almost exclusively on the clinical history, physical examination, and histopathologic confirmation. Biology-based markers for confirmation of diagnosis or monitoring progression of cGvHD are urgently warranted. The pathogenesis of cGvHD is poorly understood. Mounting evidence implies a role of B-cells in this autoimmune-like disorder. In this study we analyzed whether changes in B-cell subpopulations in the peripheral blood (PB) of HSCT recipients could serve as a biomarker for immune-modulatory consequences of cGvHD.
Methods: Within six months 70 consecutive patients (45 men, 25 women; median age 47.5 years) with complete donor cell engraftment a median of 45.5 (range, 5–149) months after allogeneic HSCT were enrolled including 21 never having cGvHD and 49 with cGvHD (35 active and/or progressive, 14 resolved at study entry). The latter received standard anti-infective prophylaxis and immunosuppressive therapy with a median duration of 36 (range, 3–104) months. A third of patients with active/progressive cGvHD had more than 2 organs involved. Evaluations in the study consisted of clinical parameters including cGvHD severity and treatment, serum immunoglobulin (Ig) levels, and infections. PB was analyzed by multiparameter flow cytometry to define B-lymphocytes (CD19+), which were further subdivided by staining for surface Ig (IgD+/M+) and the B-cell memory marker CD27+ and more immature B-lymphocytes (CD19+/CD21−).
Results: No significant differences in absolute B, T, and NK-cell counts between the groups with and without cGvHD were seen. However, the relative number of both class-switched and non-class-switched memory B-cells was significantly lower (below 3%) in patients with active cGvHD compared to patients never having cGvHD (up to 7%) (p<0.001, c2 =22.0 for class-switched and p=0.002, c2 =9.2 for non-class-switched). Absolute cell counts confirmed these findings (10.3 vs. 27.2 cells/uL, p=0.02 for class-switched and 12.1 vs. 22.1 cells/ul, p=0.047 for non-class-switched). Significantly more patients with active cGvHD had elevated numbers of immature CD19+/CD21− B-cells (>15% cut-off) than the groups with resolved or never having cGvHD (37% vs 21% vs 9%, p=0.024). In patients with >15% immature CD19+/CD21− B-cells significantly more severe infections (according to NCI-CTC Infection module grade III–IV) were seen compared to patients with low CD19+/CD21− B-cell counts (p=0.002, c2 =9.5). In the group of active cGvHD 12/13 (92%) patients with >15% CD19+/CD21− B-cells compared to 4/22 (18%) patients with ≤ 15% CD19+/CD21− B-cells experienced severe infections (p<0.001, c2 =18.1). First results in an ongoing prospective study showed an increase of memory B-cell numbers in patients with decreasing cGvHD activity.
Discussion: Monitoring of class-switched and non-class-switched memory B-cells may allow measuring of disease activity of chronic GvHD. In combination with assessment of immature CD19+/CD21− B-cells new insights into immune-modulatory consequences of cGvHD focusing on B-cell defects and activation status are possible.
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