Ex vivo culture of intact human patient derived pancreatic tumour tissue

AbstractThe poor prognosis of pancreatic ductal adenocarcinoma (PDAC) is attributed to the highly fibrotic stroma and complex multi-cellular microenvironment that is difficult to fully recapitulate in pre-clinical models. To fast-track translation of therapies and to inform personalised medicine, we aimed to develop a whole-tissue ex vivo explant model that maintains viability, 3D multicellular architecture, and microenvironmental cues of human pancreatic tumours. Patient-derived surgically-resected PDAC tissue was cut into 1–2 mm explants and cultured on gelatin sponges for 12 days. Immunohistochemistry revealed that human PDAC explants were viable for 12 days and maintained their original tumour, stromal and extracellular matrix architecture. As proof-of-principle, human PDAC explants were treated with Abraxane and we observed different levels of response between patients. PDAC explants were also transfected with polymeric nanoparticles + Cy5-siRNA and we observed abundant cytoplasmic distribution of Cy5-siRNA throughout the PDAC explants. Overall, our novel model retains the 3D architecture of human PDAC and has advantages over standard organoids: presence of functional multi-cellular stroma and fibrosis, and no tissue manipulation, digestion, or artificial propagation of organoids. This provides unprecedented opportunity to study PDAC biology including tumour-stromal interactions and rapidly assess therapeutic response to drive personalised treatment.

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Bringing the pancreas patient back to the bench: Ex vivo culture of intact human patient derived pancreatic tumour tissue

10.1101/2020.07.30.223925 ◽

2020 ◽

Author(s):

John Kokkinos ◽

George Sharbeen ◽

Koroush S. Haghighi ◽

Rosa Mistica C. Ignacio ◽

Chantal Kopecky ◽

...

Keyword(s):

Ex Vivo ◽

Personalised Medicine ◽

Fold Increase ◽

Ductal Adenocarcinoma ◽

Human Patient ◽

Pancreatic Tumours ◽

Tissue Explants ◽

Original Tumour ◽

Tissue Manipulation ◽

Pdac Tissue

AbstractThe poor prognosis of pancreatic ductal adenocarcinoma (PDAC) is attributed to the highly fibrotic stroma and complex multi-cellular microenvironment that is difficult to fully recapitulate in pre-clinical human models. To fast-track translation of therapies and to inform personalised medicine, we aimed to develop a whole-tissue ex vivo explant model that maintains viability, 3D multicellular architecture, and microenvironmental cues present in human pancreatic tumours. Patient-derived surgically-resected PDAC tissue was cut into 2 mm explants, cultured on gelatin sponges, and grown for 12 days. Immunohistochemistry revealed that human PDAC tissue explants were viable for 12 days and maintained their original tumour, stromal and extracellular matrix architecture. As proof-of-principle, human PDAC tissue explants responded to Abraxane® treatment with a 3.7-fold increase in cell-death (p=0.0007). PDAC explants were also transfected with polymeric nanoparticles+Cy5-siRNA and we observed abundant cytoplasmic distribution of nanoparticle+Cy5-siRNA throughout the PDAC explant tissue. Our novel model retains the 3D architecture of human pancreatic tumours and has several advantages over standard organoids: presence of functional multi-cellular stroma and fibrosis and no tissue manipulation, digestion, or artificial propagation of organoids. This provides an unprecedented opportunity to study PDAC biology, tumour-stromal interactions and rapidly assess therapeutic response that could drive personalised treatment for PDAC.

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Modelling Pancreatic Neuroendocrine Cancer: From Bench Side to Clinic

Cancers ◽

10.3390/cancers12113170 ◽

2020 ◽

Vol 12 (11) ◽

pp. 3170

Author(s):

Alexander Ney ◽

Gabriele Canciani ◽

J. Justin Hsuan ◽

Stephen P. Pereira

Keyword(s):

Ex Vivo ◽

Neuroendocrine Differentiation ◽

Genetically Engineered ◽

Treatment Modalities ◽

Ductal Adenocarcinoma ◽

Disease Models ◽

Tissue Remodelling ◽

In Vivo Models ◽

Original Tumour

Pancreatic neuroendocrine tumours (pNETs) are a heterogeneous group of epithelial tumours with neuroendocrine differentiation. Although rare (incidence of <1 in 100,000), they are the second most common group of pancreatic neoplasms after pancreatic ductal adenocarcinoma (PDAC). pNET incidence is however on the rise and patient outcomes, although variable, have been linked with 5-year survival rates as low as 40%. Improvement of diagnostic and treatment modalities strongly relies on disease models that reconstruct the disease ex vivo. A key constraint in pNET research, however, is the absence of human pNET models that accurately capture the original tumour phenotype. In attempts to more closely mimic the disease in its native environment, three-dimensional culture models as well as in vivo models, such as genetically engineered mouse models (GEMMs), have been developed. Despite adding significant contributions to our understanding of more complex biological processes associated with the development and progression of pNETs, factors such as ethical considerations and low rates of clinical translatability limit their use. Furthermore, a role for the site-specific extracellular matrix (ECM) in disease development and progression has become clear. Advances in tissue engineering have enabled the use of tissue constructs that are designed to establish disease ex vivo within a close to native ECM that can recapitulate tumour-associated tissue remodelling. Yet, such advanced models for studying pNETs remain underdeveloped. This review summarises the most clinically relevant disease models of pNETs currently used, as well as future directions for improved modelling of the disease.

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Phagocytosis of Liposome Particles by Rat Splenic Immature Monocytes Makes Them Transiently and Highly Immunosuppressive In Ex Vivo Culture Conditions

Journal of Pharmacology and Experimental Therapeutics ◽

10.1124/jpet.110.172510 ◽

2011 ◽

Vol 337 (1) ◽

pp. 42-49 ◽

Cited By ~ 8

Author(s):

Daisuke Takahashi ◽

Hiroshi Azuma ◽

Hiromi Sakai ◽

Keitaro Sou ◽

Daiko Wakita ◽

...

Keyword(s):

Ex Vivo ◽

Culture Conditions ◽

Ex Vivo Culture

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Mimicking of Blood Flow Results in a Distinct Functional Phenotype in Human Non-Adherent Classical Monocytes

Biology ◽

10.3390/biology10080748 ◽

2021 ◽

Vol 10 (8) ◽

pp. 748

Author(s):

Elisa Wirthgen ◽

Melanie Hornschuh ◽

Ida Maria Wrobel ◽

Christian Manteuffel ◽

Jan Däbritz

Keyword(s):

Blood Flow ◽

Shear Flow ◽

Ex Vivo ◽

Culture Conditions ◽

Inflammatory Signaling ◽

Gm Csf ◽

Beneficial Effects ◽

Migratory Capacity ◽

Ex Vivo Culture ◽

Static Conditions

Ex vivo culture conditions during the manufacturing process impact the therapeutic effect of cell-based products. Mimicking blood flow during ex vivo culture of monocytes has beneficial effects by preserving their migratory ability. However, the effects of shear flow on the inflammatory response have not been studied so far. Hence, the present study investigates the effects of shear flow on both blood-derived naïve and activated monocytes. The activation of monocytes was experimentally induced by granulocyte-macrophage colony-stimulating factor (GM-CSF), which acts as a pro-survival and growth factor on monocytes with a potential role in inflammation. Monocytes were cultured under dynamic (=shear flow) or static conditions while preventing monocytes' adherence by using cell-repellent surfaces to avoid adhesion-induced differentiation. After cultivation (40 h), cell size, viability, and cytokine secretion were evaluated, and the cells were further applied to functional tests on their migratory capacity, adherence, and metabolic activity. Our results demonstrate that the application of shear flow resulted in a decreased pro-inflammatory signaling concurrent with increased secretion of the anti-inflammatory cytokine IL-10 and increased migratory capacity. These features may improve the efficacy of monocyte-based therapeutic products as both the unwanted inflammatory signaling in blood circulation and the loss of migratory ability will be prevented.

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Stem cell integrins: Implications for ex-vivo culture and cellular therapies

Stem Cell Research ◽

10.1016/j.scr.2010.09.005 ◽

2011 ◽

Vol 6 (1) ◽

pp. 1-12 ◽

Cited By ~ 110

Author(s):

Andrew B.J. Prowse ◽

Fenny Chong ◽

Peter P. Gray ◽

Trent P. Munro

Keyword(s):

Stem Cell ◽

Ex Vivo ◽

Cellular Therapies ◽

Ex Vivo Culture

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A GATA6-centred gene regulatory network involving HNFs and ΔNp63 controls plasticity and immune escape in pancreatic cancer

Gut ◽

10.1136/gutjnl-2020-321397 ◽

2021 ◽

pp. gutjnl-2020-321397

Author(s):

Bernhard Kloesch ◽

Vivien Ionasz ◽

Sumit Paliwal ◽

Natascha Hruschka ◽

Jaime Martinez de Villarreal ◽

...

Keyword(s):

Pancreatic Cancer ◽

Immune Escape ◽

Lung Metastases ◽

Personalised Medicine ◽

Molecular Taxonomy ◽

Classification Systems ◽

Ductal Adenocarcinoma ◽

Tissue Samples ◽

Basal Phenotype ◽

Phenotype Switch

ObjectiveMolecular taxonomy of tumours is the foundation of personalised medicine and is becoming of paramount importance for therapeutic purposes. Four transcriptomics-based classification systems of pancreatic ductal adenocarcinoma (PDAC) exist, which consistently identified a subtype of highly aggressive PDACs with basal-like features, including ΔNp63 expression and loss of the epithelial master regulator GATA6. We investigated the precise molecular events driving PDAC progression and the emergence of the basal programme.DesignWe combined the analysis of patient-derived transcriptomics datasets and tissue samples with mechanistic experiments using a novel dual-recombinase mouse model for Gata6 deletion at late stages of KRasG12D-driven pancreatic tumorigenesis (Gata6LateKO).ResultsThis comprehensive human-to-mouse approach showed that GATA6 loss is necessary, but not sufficient, for the expression of ΔNp63 and the basal programme in patients and in mice. The concomitant loss of HNF1A and HNF4A, likely through epigenetic silencing, is required for the full phenotype switch. Moreover, Gata6 deletion in mice dramatically increased the metastatic rate, with a propensity for lung metastases. Through RNA-Seq analysis of primary cells isolated from mouse tumours, we show that Gata6 inhibits tumour cell plasticity and immune evasion, consistent with patient-derived data, suggesting that GATA6 works as a barrier for acquiring the fully developed basal and metastatic phenotype.ConclusionsOur work provides both a mechanistic molecular link between the basal phenotype and metastasis and a valuable preclinical tool to investigate the most aggressive subtype of PDAC. These data, therefore, are important for understanding the pathobiological features underlying the heterogeneity of pancreatic cancer in both mice and human.

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Ex vivo culture of Fancc-/- stem/progenitor cells predisposes cells to undergo apoptosis, and surviving stem/progenitor cells display cytogenetic abnormalities and an increased risk of malignancy

Blood ◽

10.1182/blood-2004-06-2483 ◽

2005 ◽

Vol 105 (9) ◽

pp. 3465-3471 ◽

Cited By ~ 48

Author(s):

Xiaxin Li ◽

Michelle M. Le Beau ◽

Samantha Ciccone ◽

Feng-Chun Yang ◽

Brian Freie ◽

...

Keyword(s):

Progenitor Cells ◽

Genome Stability ◽

Bone Marrow Cells ◽

Ex Vivo ◽

Acquired Resistance ◽

Intrinsic Defects ◽

Cytogenetic Abnormalities ◽

Increased Risk ◽

Ex Vivo Culture ◽

The Impact

AbstractCurrent strategies for genetic therapy using Moloney retroviruses require ex vivo manipulation of hematopoietic cells to facilitate stable integration of the transgene. While many studies have evaluated the impact of ex vivo culture on normal murine and human stem/progenitor cells, the cellular consequences of ex vivo manipulation of stem cells with intrinsic defects in genome stability are incompletely understood. Here we show that ex vivo culture of Fancc-/- bone marrow cells results in a time-dependent increase in apoptosis of primitive Fancc-/- progenitor cells in conditions that promote the proliferation of wild-type stem/progenitor cells. Further, recipients reconstituted with the surviving Fancc-/- cells have a high incidence of cytogenetic abnormalities and myeloid malignancies that are associated with an acquired resistance to tumor necrosis factor α (TNF-α). Collectively, these data indicate that the intrinsic defects in the genomic stability of Fancc-/- stem/progenitor cells provide a selective pressure for cells that are resistant to apoptosis and have a propensity for the evolution to clonal hematopoiesis and malignancy. These studies could have implications for the design of genetic therapies for treatment of Fanconi anemia and potentially other genetic diseases with intrinsic defects in genome stability.

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