Decellularised Human Pancreata and Liver for the study of Pancreatic Ductal Adenocarcinoma: Development, Metastasis and Chemoresistance

Background and Aims: Over 80% of patients with pancreatic ductal adenocarcinoma (PDAC) are diagnosed with concurrent metastases. Over the last 50 years, conventional treatment approaches have had little impact on the course of this disease. Therefore, the development of new treatment strategies to control PDAC is needed. We propose the use of 3D extracellular matrix (ECM) scaffolds that could redefine in vitro models of PDAC and preclinical testing of novel therapies. Methods: Decellularised human pancreata and livers were characterised for the elimination of cellular material and preservation of ECM proteins and micro-architecture using histology, immunohistochemistry (IHC) and quantification kits. Both primary (PANC-1 and MIA PaCa-2) and metastatic pancreatic tumour cells (PK-1) were seeded onto 5 mm3 scaffolds, as well as 2D cultures. Histological analyses were used to confirm cell attachment and migration/invasion. Further, changes in protein expression (IHC) and gene expression (qPCR and RNAseq) were evaluated at day 14 post reseeding. Treatments with doxorubicin and Gemcitabine were performed; viability (AlamarBlue), protein expression (IHC) and gene expression (RNAseq) analyses were performed to test therapy-resistance in the 3D systems. Results: All primary PDAC cell lines were able to migrate and invade the pancreas scaffolds whereas several of these cells were only able to attach superficially onto the liver scaffolds. PK1 cells were able to exclusively migrate and invade the liver scaffolds and only attached superficially onto the pancreatic scaffolds. These differences were supported by significant deregulations in gene and protein expression (i.e. MMP9, WNT1, β-CATENIN) between pancreas scaffolds, liver scaffolds and 2D culture. Interestingly, both primary and metastatic cells were found significantly more resistant to all chemotherapy treatments in the 3D models when compared to 2D cultures, even though confocal microscopy confirmed the uptake of drugs into the cells. Conclusion: Our results suggest that primary and metastatic pancreatic cancer cells manifest a conserved invasive behaviour depending on the 3D ECM structure of origin. Moreover, there is an evident alteration in cell response to different cancer-therapies in the presence of a natural ECM niche. These observations provide a proof of concept for the development of an effective bio-engineered model for drug discovery, therapy screening and biomarker discovery

Modified Cantilever Arrays Improve Sensitivity and Reproducibility of Nanomechanical Sensing in Living Cells

Mechanical signaling involved in molecular interactions lies at the heart of materials science and biological systems, but the mechanisms involved are poorly understood. Here we use nanomechanical sensors and intact human cells to provide unique insights into the signaling pathways of connectivity networks, which deliver the ability to probe cells to produce biologically relevant, quantifiable and reproducible signals. We quantify the mechanical signals from malignant cancer cells, with 10 cells per ml in 1000-fold excess of non-neoplastic human epithelial cells. Moreover, we demonstrate that a direct link between cells and molecules creates a continuous connectivity which acts like a percolating network to propagate mechanical forces over both short and long length-scales. The findings provide mechanistic insights into how cancer cells interact with one another and with their microenvironments, enabling them to invade the surrounding tissues. Further, with this system it is possible to understand how cancer clusters are able to co-ordinate their migration through narrow blood capillaries

Whole Human liver decellularisation-recellularisation for future liver transplantation and extracorporeal device application

Background and Aims: An estimated 29 million people in the European Union (EU) suffer from a chronic liver condition, with liver transplantation still remaining the only treatment for end-stage hepatic disease. Currently, there are approximately 6700 people awaiting liver transplantation in the EU. Considering that 15- 25% of donated organs are discarded, whole human liver regeneration represents a novel approach to overcome current organ shortages. One possible approach is the use of native extracellular matrix (ECM) as a suitable environment for cells to restore tissue function. Therefore, the aim of this project was to demonstrate, for the first time, the recellularisation of a decellularised whole human liver for future transplantation and extracorporeal device applications. Method: A human liver explant, diagnosed with Crigler–Najjar syndrome, was decellularised using a well- established method, previously characterized for cellular material elimination and preservation of ECM proteins and micro-architecture. Temperature, pH, oxygen and pressure sensors were incorporated into the Harvard Apparatus’ ORCA system, as well as compressed air, O 2 and CO 2 reservoirs. Whole human liver scaffolds (840g) was recellularised by IVC infusion with 2x10 9 HepG2. The liver was maintained in 6 L of complete media with a flow-rate of 400ml/min. The media was changed by replacing 3L of existing media with fresh complete media after 48 hours. The experiment was stopped after 72 hours and the liver was fixed in 4% formaldehyde. The liver was sectioned into 21 parts to investigate repopulation by H&E stating. Albumin secretion was measured using an ELISA kit at 0, 24 and 72 hours. Results: Histological analysis using H&E staining showed that cells have infiltrated all liver segments, excluding segment one. HepG2 cells were seen microscopically to have been migrating from the central vein towards the portal triad, including penetrating into the parenchymal space. Oxygen consumption during the course of three days decreased from 20% to 10%. Additionally, pH was reduced by 0.4. Finally, albumin present in the media increased from 0 ng/ml on day 0, to 200 ng/ml on day 1, to 1500 ng/ml on day 3. Conclusion: This is the first report describing the recellularisation of whole human liver ECM scaffolds with a human hepatocyte cell line. This is a key advance in the development of a bioengineered human liver for future liver transplantation and extracorporeal device applications

Amyloid persistence in decellularized liver: Biochemical and histopathological characterization

Systemic amyloidoses are a group of debilitating and often fatal diseases in which fibrillar protein aggregates are deposited in the extracellular spaces of a range of tissues. The molecular basis of amyloid formation and tissue localization is still unclear. Although it is likely that the extracellular matrix (ECM) plays an important role in amyloid deposition, this interaction is largely unexplored, mostly because current analytical approaches may alter the delicate and complicated three-dimensional architecture of both ECM and amyloid. We describe here a decellularization procedure for the amyloidotic mouse liver which allows high-resolution visualization of the interactions between amyloid and the constitutive fibers of the extracellular matrix. The primary structure of the fibrillar proteins remains intact and the amyloid fibrils retain their amyloid enhancing factor activity

Tissue-Specific Human Extracellular Matrix Scaffolds Promote Pancreatic Tumour Progression and Chemotherapy Resistance.

Over 80% of patients with pancreatic ductal adenocarcinoma (PDAC) are diagnosed at a late stage and are locally advanced or with concurrent metastases. The aggressive phenotype and relative chemo- and radiotherapeutic resistance of PDAC is thought to be mediated largely by its prominent stroma, which is supported by an extracellular matrix (ECM). Therefore, we investigated the impact of tissue-matched human ECM in driving PDAC and the role of the ECM in promoting chemotherapy resistance. Decellularized human pancreata and livers were recellularized with PANC-1 and MIA PaCa-2 (PDAC cell lines), as well as PK-1 cells (liver-derived metastatic PDAC cell line). PANC-1 cells migrated into the pancreatic scaffolds, MIA PaCa-2 cells were able to migrate into both scaffolds, whereas PK-1 cells were able to migrate into the liver scaffolds only. These differences were supported by significant deregulations in gene and protein expression between the pancreas scaffolds, liver scaffolds, and 2D culture. Moreover, these cell lines were significantly more resistant to gemcitabine and doxorubicin chemotherapy treatments in the 3D models compared to 2D cultures, even after confirmed uptake by confocal microscopy. These results suggest that tissue-specific ECM provides the preserved native cues for primary and metastatic PDAC cells necessary for a more reliable in vitro cell culture

Tissue-Specific Human Extracellular Matrix Scaffolds Promote Pancreatic Tumour Progression and Chemotherapy Resistance

Over 80% of patients with pancreatic ductal adenocarcinoma (PDAC) are diagnosed at a late stage and are locally advanced or with concurrent metastases. The aggressive phenotype and relative chemo- and radiotherapeutic resistance of PDAC is thought to be mediated largely by its prominent stroma, which is supported by an extracellular matrix (ECM). Therefore, we investigated the impact of tissue-matched human ECM in driving PDAC and the role of the ECM in promoting chemotherapy resistance. Decellularized human pancreata and livers were recellularized with PANC-1 and MIA PaCa-2 (PDAC cell lines), as well as PK-1 cells (liver-derived metastatic PDAC cell line). PANC-1 cells migrated into the pancreatic scaffolds, MIA PaCa-2 cells were able to migrate into both scaffolds, whereas PK-1 cells were able to migrate into the liver scaffolds only. These differences were supported by significant deregulations in gene and protein expression between the pancreas scaffolds, liver scaffolds, and 2D culture. Moreover, these cell lines were significantly more resistant to gemcitabine and doxorubicin chemotherapy treatments in the 3D models compared to 2D cultures, even after confirmed uptake by confocal microscopy. These results suggest that tissue-specific ECM provides the preserved native cues for primary and metastatic PDAC cells necessary for a more reliable in vitro cell culture

Tissue CD14+CD8+ T cells reprogrammed by myeloid cells and modulated by LPS

The liver is bathed in bacterial products, including lipopolysaccharide transported from the intestinal portal vasculature, but maintains a state of tolerance that is exploited by persistent pathogens and tumours1-4. The cellular basis mediating this tolerance, yet allowing a switch to immunity or immunopathology, needs to be better understood for successful immunotherapy of liver diseases. Here we show that a variable proportion of CD8+ T cells compartmentalized in the human liver co-stain for CD14 and other prototypic myeloid membrane proteins and are enriched in close proximity to CD14high myeloid cells in hepatic zone 2. CD14+CD8+ T cells preferentially accumulate within the donor pool in liver allografts, among hepatic virus-specific and tumour-infiltrating responses, and in cirrhotic ascites. CD14+CD8+ T cells exhibit increased turnover, activation and constitutive immunomodulatory features with high homeostatic IL-10 and IL-2 production ex vivo, and enhanced antiviral/anti-tumour effector function after TCR engagement. This CD14+CD8+ T cell profile can be recapitulated by the acquisition of membrane proteins-including the lipopolysaccharide receptor complex-from mononuclear phagocytes, resulting in augmented tumour killing by TCR-redirected T cells in vitro. CD14+CD8+ T cells express integrins and chemokine receptors that favour interactions with the local stroma, which can promote their induction through CXCL12. Lipopolysaccharide can also increase the frequency of CD14+CD8+ T cells in vitro and in vivo, and skew their function towards the production of chemotactic and regenerative cytokines. Thus, bacterial products in the gut-liver axis and tissue stromal factors can tune liver immunity by driving myeloid instruction of CD8+ T cells with immunomodulatory ability