Marcus Binner

3D extracellular matrix interactions modulate tumour cell growth, invasion and angiogenesis in engineered tumour microenvironments.

UNLABELLED Interactions between tumour cells and extracellular matrix proteins of the tumour microenvironment play crucial roles in cancer progression. So far, however, there are only a few experimental platforms available that allow us to study these interactions systematically in a mechanically defined three-dimensional (3D) context. Here, we have studied the effect of integrin binding motifs found within common extracellular matrix (ECM) proteins on 3D breast (MCF-7) and prostate (PC-3, LNCaP) cancer cell cultures, and co-cultures with endothelial and mesenchymal stromal cells. For this purpose, matrix metalloproteinase-degradable biohybrid poly(ethylene) glycol-heparin hydrogels were decorated with the peptide motifs RGD, GFOGER (collagen I), or IKVAV (laminin-111). Over 14days, cancer spheroids of 100-200μm formed. While the morphology of poorly invasive MCF-7 and LNCaP cells was not modulated by any of the peptide motifs, the aggressive PC-3 cells exhibited an invasive morphology when cultured in hydrogels comprising IKVAV and GFOGER motifs compared to RGD motifs or nonfunctionalised controls. PC-3 (but not MCF-7 and LNCaP) cell growth and endothelial cell infiltration were also significantly enhanced in IKVAV and GFOGER presenting gels. Taken together, we have established a 3D culture model that allows for dissecting the effect of biochemical cues on processes relevant to early cancer progression. These findings provide a basis for more mechanistic studies that may further advance our understanding of how ECM modulates cancer cell invasion and how to ultimately interfere with this process. STATEMENT OF SIGNIFICANCE Threedimensional in vitro cancer models have generated great interest over the past decade. However, most models are not suitable to systematically study the effects of environmental cues on cancer development and progression. To overcome this limitation, we have developed an innovative hydrogel platform to study the interactions between breast and prostate cancer cells and extracellular matrix ligands relevant to the tumour microenvironment. Our results show that hydrogels with laminin- and collagen-derived adhesive peptides induce a malignant phenotype in a cell-line specific manner. Thus, we have identified a method to control the incorporation of biochemical cues within a three dimensional culture model and anticipate that it will help us in better understanding the effects of the tumour microenvironment on cancer progression.

High Conductivity in Molecularly p‐Doped Diketopyrrolopyrrole‐Based Polymer: The Impact of a High Dopant Strength and Good Structural Order

[3]-Radialene-based dopant CN6-CP studied herein, with its reduction potential of +0.8 versus Fc/Fc+ and the lowest unoccupied molecular orbital level of -5.87 eV, is the strongest molecular p-dopant reported in the open literature, so far. The efficient p-doping of the donor-acceptor dithienyl-diketopyrrolopyrrole-based copolymer having the highest unoccupied molecular orbital level of -5.49 eV is achieved. The doped films exhibit electrical conductivities up to 70 S cm(-1) .

A three-dimensional ex vivo tri-culture model mimics cell-cell interactions between acute myeloid leukemia and the vascular niche

Ex vivo studies of human disease, such as acute myeloid leukemia, are generally limited to the analysis of two-dimensional cultures which often misinterpret the effectiveness of chemotherapeutics and other treatments. Here we show that matrix metalloproteinase-sensitive hydrogels prepared from poly(ethylene glycol) and heparin functionalized with adhesion ligands and pro-angiogenic factors can be instrumental to produce robust three-dimensional culture models, allowing for the analysis of acute myeloid leukemia development and response to treatment. We evaluated the growth of four leukemia cell lines, KG1a, MOLM13, MV4-11 and OCI-AML3, as well as samples from patients with acute myeloid leukemia. Furthermore, endothelial cells and mesenchymal stromal cells were co-seeded to mimic the vascular niche for acute myeloid leukemia cells. Greater drug resistance to daunorubicin and cytarabine was demonstrated in three-dimensional cultures and in vascular co-cultures when compared with two-dimensional suspension cultures, opening the way for drug combination studies. Application of the C-X-C chemokine receptor type 4 (CXCR4) inhibitor, AMD3100, induced mobilization of the acute myeloid leukemia cells from the vascular networks. These findings indicate that the three-dimensional tri-culture model provides a specialized platform for the investigation of cell-cell interactions, addressing a key challenge of current testing models. This ex vivo system allows for personalized analysis of the responses of patients’ cells, providing new insights into the development of acute myeloid leukemia and therapies for this disease.

Enhanced targeting of invasive glioblastoma cells by peptide-functionalized gold nanorods in hydrogel-based 3D cultures

Cancer stem cells (CSCs) are responsible for drug resistance, tumor recurrence, and metastasis in several cancer types, making their eradication a primary objective in cancer therapy. Glioblastoma Multiforme (GBM) tumors are usually composed of a highly infiltrating CSC subpopulation, which has Nestin as a putative marker. Since the majority of these infiltrating cells are able to elude conventional therapies, we have developed gold nanorods (AuNRs) functionalized with an engineered peptide capable of specific recognition and selective eradication of Nestin positive infiltrating GBM-CSCs. These AuNRs generate heat when irradiated by a near-infrared laser, and cause localized cell damage. Nanoparticle internalization assays performed with GBM-CSCs or Nestin negative cells cultured as two-dimensional (2D) monolayers or embedded in three-dimensional (3D) biodegradable-hydrogels of tunable mechanical properties, revealed that the AuNRs were mainly internalized by GBM-CSCs, and not by Nestin negative cells. The AuNRs were taken up via energy-dependent and caveolae-mediated endocytic mechanisms, and were localized inside endosomes. Photothermal treatments resulted in the selective elimination of GBM-CSCs through cell apoptosis, while Nestin negative cells remained viable. Results also indicated that GBM-CSCs embedded in hydrogels were more resistant to AuNR photothermal treatments than when cultured as 2D monolayers. In summary, the combination of our engineered AuNRs with our tunable hydrogel system has shown the potential to provide an in vitro platform for the evaluation and screening of AuNR-based cancer therapeutics, leading to a substantial advancement in the application of AuNRs for targeted GBM-CSC therapy. STATEMENT OF SIGNIFICANCE There is an urgent need for reliable and efficient therapies for the treatment of Glioblastoma Multiforme (GBM), which is currently an untreatable brain tumor form with a very poor patient survival rate. GBM tumors are mostly comprised of cancer stem cells (CSCs), which are responsible for tumor reoccurrence and therapy resistance. We have developed gold nanorods functionalized with an engineered peptide capable of selective recognition and eradication of GBM-CSCs via heat generation by nanorods upon NIR irradiation. An in vitro evaluation of nanorod therapeutic activities was performed in 3D synthetic-biodegradable hydrogel models with distinct biomechanical cues, and compared to 2D cultures. Results indicated that cells cultured in 3D were more resistant to photothermolysis than in 2D systems.

Defined Geldrop Cultures Maintain Neural Precursor Cells

Distinct micro-environmental properties have been reported to be essential for maintenance of neural precursor cells (NPCs) within the adult brain. Due to high complexity and technical limitations, the natural niche can barely be studied systematically in vivo. By reconstituting selected environmental properties (adhesiveness, proteolytic degradability, and elasticity) in geldrop cultures, we show that NPCs can be maintained stably at high density over an extended period of time (up to 8 days). In both conventional systems, neurospheres and monolayer cultures, they would expand and (in the case of neurospheres) differentiate rapidly. Further, we report a critical dualism between matrix adhesiveness and degradability. Only if both features are functional NPCs stay proliferative. Lastly, Rho-associated protein kinase was identified as part of a pivotal intracellular signaling cascade controlling cell morphology in response to environmental cues inside geldrop cultures. Our findings demonstrate that simple manipulations of the microenvironment in vitro result in an important preservation of stemness features in the cultured precursor cells.

Three-Dimensional In Vitro Hydro- and Cryogel-Based Cell-Culture Models for the Study of Breast-Cancer Metastasis to Bone

Bone is the most common site for breast-cancer invasion and metastasis, and it causes severe morbidity and mortality. A greater understanding of the mechanisms leading to bone-specific metastasis could improve therapeutic strategies and thus improve patient survival. While three-dimensional in vitro culture models provide valuable tools to investigate distinct heterocellular and environmental interactions, sophisticated organ-specific metastasis models are lacking. Previous models used to investigate breast-to-bone metastasis have relied on 2.5D or singular-scaffold methods, constraining the in situ mimicry of in vitro models. Glycosaminoglycan-based gels have demonstrated outstanding potential for tumor-engineering applications. Here, we developed advanced biphasic in vitro microenvironments that mimic breast-tumor tissue (MCF-7 and MDA-MB-231 in a hydrogel) spatially separated with a mineralized bone construct (human primary osteoblasts in a cryogel). These models allow distinct advantages over former models due to the ability to observe and manipulate cellular migration towards a bone construct. The gels allow for the binding of adhesion-mediating peptides and controlled release of signaling molecules. Moreover, mechanical and architectural properties can be tuned to manipulate cell function. These results demonstrate the utility of these biomimetic microenvironment models to investigate heterotypic cell–cell and cell–matrix communications in cancer migration to bone.

Hydrogel-based in vitro models of tumor angiogenesis

In situ forming hydrogels prepared from multi-armed poly(ethylene glycol) (PEG), glycosaminoglycans (GAG) and various peptides enable the development of advanced three dimensional (3D) culture models. Herein, we report methods for the PEG-GAG gel-based 3D co-cultivation of human umbilical vein endothelial cells, mesenchymal stromal cells, and different cancer cell lines. The resulting constructs allow for the exploration of interactions between solid tumors with 3D vascular networks in vitro to study the mechanistic aspects of cancer development and to perform drug testing.

Cell-instructive starPEG-heparin-collagen composite matrices

Polymer hydrogels can be readily modulated with regard to their physical properties and functionalized to recapitulate molecular cues of the extracellular matrix (ECM). However, they remain structurally different from the hierarchical supramolecular assemblies of natural ECM. Accordingly, we herein report a set of hydrogel composite materials made from starPEG-peptide conjugates, maleimide-functionalized heparin and collagen type I that combine semisynthetic and ECM-derived components. Collagen fibrillogenesis was controlled by temperature and collagen concentration to form collagen microstructures which were then homogeneously distributed within the 3D composite matrix during hydrogel formation. The collagen-laden hydrogel materials showed a heterogeneous local variation of the stiffness and adhesion ligand density. Composite gels functionalized with growth factors and cell adhesive peptides (RGDSP) supported the growth of embedded human umbilical cord vein endothelial cells (HUVECs) and induced the alignment of embedded bone marrow-derived human mesenchymal stem cells (MSCs) to the collagen microstructures in vitro. The introduced composite hydrogel material is concluded to faithfully mimic cell-instructive features of the ECM. STATEMENT OF SIGNIFICANCE Cell-instructive materials play an important role in the generation of both regenerative therapies and advanced tissue and disease models. For that purpose, biofunctional polymer hydrogels recapitulating molecular cues of the extracellular matrix (ECM) were successfully applied in various different studies. However, hydrogels generally lack the hierarchical supramolecular structure of natural ECM. We have therefore developed a hydrogel composite material made from starPEG-peptide conjugates, maleimide-functionalized heparin and collagen type I fibrils. The collagen-laden scaffolds showed a heterogeneous local variation in the stiffness of the material. The composite gels were successfully tested in culture experiments with human umbilical cord vein endothelial cells and bone marrow-derived human mesenchymal stem cells. It was concluded that the composite scaffold was able to faithfully mimic important cell-instructive features of the ECM.