Recapitulating the Angiogenic Switch in a Hydrogel-Based 3D In Vitro Tumor-Stroma Model

Abstract

To ensure nutrient and oxygen supply, tumors beyond a size of 1–2 mm3 need a connection to the vascular system. Thus, tumor cells modify physiological tissue homeostasis by secreting inflammatory and angiogenic cytokines. This leads to the activation of the tumor microenvironment and the turning of the angiogenic switch, resulting in tumor vascularization and growth. To inhibit tumor growth by developing efficient anti-angiogenic therapies, an in depth understanding of the molecular mechanism initiating angiogenesis is essential. Yet so far, predominantly 2D cell cultures or animal models have been used to clarify the interactions within the tumor stroma, resulting in poor transferability of the data obtained to the in vivo situation. Consequently, there is an abundant need for complex, humanized, 3D models in vitro. We established a dextran-hydrogel-based 3D organotypic in vitro model containing microtumor spheroids, macrophages, neutrophils, fibroblasts and endothelial cells, allowing for the analysis of tumor–stroma interactions in a controlled and modifiable environment. During the cultivation period of 21 days, the microtumor spheroids in the model grew in size and endothelial cells formed elongated tubular structures resembling capillary vessels, that appeared to extend towards the tumor spheroids. The tubular structures exhibited complex bifurcations and expanded without adding external angiogenic factors such as VEGF to the culture. To allow high-throughput screening of therapeutic candidates, the 3D cell culture model was successfully miniaturized to a 96-well format, while still maintaining the same level of tumor spheroid growth and vascular sprouting. The quantification of VEGF in the conditioned medium of these cultures showed a continuous increase during the cultivation period, suggesting the contribution of endogenous VEGF to the induction of the angiogenic switch and vascular sprouting. Thus, this model is highly suitable as a testing platform for novel anticancer therapeutics targeting the tumor as well as the vascular compartment.

Neutrophils Culture in Collagen Gel System

“…Although the 3D culture of neutrophil-like cell line or primary Neu co-culture with other cells have been used for improving the viability of Neu ( 15 , 16 ), however, the detailed phenomena of Neu in 3D culturing system have never evaluated. The hydrogel is a biodegradable and biocompatible porous material with the ability to maintain high water content, used in a wide range of medical applications ( 15 , 37 , 38 ), including the drug release carriers, corneal contact lenses, in bone and soft tissue regeneration, in reconstruction and in the treatment of burns ( 39 ). They have good permeability, allowing a variety of nutrients, gases, and metabolites to pass through them freely and resembling the extracellular environment of the body’s tissues ( 17 ).…”

Challenges and exploration for immunotherapies targeting cold colorectal cancer

“…An insufficient number of pericyclic cells or loose vascular basement membrane connections in tumor tissue that cover endothelial cells increases the permeability of tumor blood vessels, thereby promoting tumor metastasis[ 26 , 27 ]. As tumor cells continue to grow, the increased supply of nutrients and oxygen enhance the expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), which promote the production of tumor cell blood vessels and create a hypoxic and acidic environment[ 28 - 30 ]. Hypoxia recruits immunosuppressive cells to the tumor immune microenvironment (TME) and can inhibit the function of immune effector T cells[ 31 ].…”

The mechanistic immunosuppressive role of the tumour vasculature and potential nanoparticle-mediated therapeutic strategies

“…Angiogenesis is the process of new blood vessel formation from existing vasculature and is abundant in growing tumours ( 1 ). As tumours increase in size, the accompanying increased demand for nutrients and oxygen upregulates the excess production of pro-angiogenic factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) from tumour cells, termed the ‘angiogenic switch’ ( 2 ). However, the new vasculature often displays increased permeability, tortuosity, and immaturity, thus facilitating metastasis but not supplying the metabolic demands of the tumour ( 3 , 4 ).…”