Tissue engineering the cancer microenvironment-challenges and opportunities

Abstract

Mechanosensing is increasingly recognised as important for tumour progression. Tumours become stiff and the forces that normally balance in the healthy organism break down and become imbalanced, leading to increases in migration, invasion and metastatic dissemination. Here, we review recent advances in our understanding of how extracellular matrix properties, such as stiffness, viscoelasticity and architecture control cell behaviour. In addition, we discuss how the tumour microenvironment can be modelled in vitro, capturing these mechanical aspects, to better understand and develop therapies against tumour spread. We argue that by gaining a better understanding of the microenvironment and the mechanical forces that govern tumour dynamics, we can make advances in combatting cancer dormancy, recurrence and metastasis.

Keywords: Adhesion; Cancer microenvironment; Cell migration; Cytoskeleton; Extracellular matrix; Hydrogels; Mechanosensing; Motility.

Fig. 1

During embryogenesis, forces balance as cells proliferate, differentiate and sort into specific tissues and organs. Angiogenesis allows oxygenation of the growing embryo and migration, both collectively and individually, drives sorting and homing of cells and tissues. Embryonic tissue shows plasticity in cell fate, but as development progresses, cells become more committed, and stem cells form in specific niches, where they continue to maintain tissues and organism in the adult. Programmed cell death is also important for pruning out cells during sculpting, such as in the formation of digits. The differentiated epithelium (shown right) is an example of a tissue that maintains stem cells in a niche, progenitor cells and differentiated cells in a continuous state of equilibrium in the adult. There is much less cell motility in adult tissues than embryonic, and growth is generally balanced by death and pruning. Unlike the well-organised embryo, tumours behave in more unpredictable and chaotic ways. However, in common with embryos, they show increased angiogenesis and cell migration. The blood vessels in tumours are generally leaky and tortuous, resulting from and causing further force imbalances. Tumours also have stem-like cells and have altered capacity for proliferation, often hyperproliferating or suppressing programmed cell death to become crowded and deprived of nutrients. If the stem-like cells escape from the primary tumour, they may land in lymph nodes or travel through the bloodstream, where they can seed new tumours (metastases) at distant sites. Most escaping tumour cells are thought to die due to the hostile conditions and the body’s surveillance system, but if even a few survive, they can start new tumours. New tumour formation can start immediately or after years of dormancy, a poorly understood state where the cells lie in the host tissue, but the tumour is not detectable. Dormancy may be quiescence and fails to grow, or may be a balance of growth and death that keeps the small cluster undetectable. However, these small micrometastases re-awaken and can result in full metastasis. Metastases can also shed cells into the bloodstream that return to the primary tumour and increase its aggressiveness and diversity

Fig. 2

Integrin activation and importance for balanced growth. Integrins lie at the roots of cellular mechanosensing, as they are considered to be the main membrane receptors mediating cell-ECM interactions. They are heterodimers of α- and β-subunits forming an elongated extracellular ligand binding domain and a short cytoplasmic tail. In the absence of stimuli, integrin subunits have an inactive bent conformation. Integrin subunit elongation and activation can occur either through ECM protein ligand binding on the extracellular site (‘outside-in’) or by intracellular signalling events mediated mainly by focal complex or actin cytoskeleton associated protein such as talin (‘inside-out’). Integrin activity can enhance remodelling of the surrounding microenvironment which can also promote more integrin activation indicating a positive loop. Tension and mechanical force arising either from ECM or cytoskeletal dynamics can also extend, activate and cluster integrin subunits. Non-transformed cells require a degree of ECM adhesion and integrin signalling to sustain their proliferation and growth. Malignant transformation, however, maintains cell proliferation even in the absence of ECM adhesion. At the same time though, transformed cells display integrin enrichment and imbalanced cell-ECM dynamics. Tumours frequently display an increase in ECM stiffness, which can be further enhanced by inflammation and fibrosis. This can drive increased cytoskeletal activation as well as signalling downstream of integrin activation

Fig. 3

Cells generate force against stiff ECM, leading to clutch engagement. When cells experience soft or viscous matrix, where adhesions do not generate enough tension to stretch mechanosensitive proteins and trigger a response, the molecular clutch remains unengaged. In this situation, actin polymerisation at the leading edge is uncoupled from adhesion, and retrograde flow of newly generated filaments occurs in the direction away from the plasma membrane. Adhesions remain small, and the cell is not able to use actin-based protrusion to move against the substratum. However, upon a threshold of ECM stiffness, mechanosensitive cytoskeletal linkers, such as vinculin and talin, engage and form a molecular clutch. The clutch catches the ECM-derived force and transmits it to the cytoskeletal cortex. As adhesions increase in size due to integrin clustering and the cytoskeleton couples to the rigid matrix, actin polymerisation results in membrane protrusion and promotes motility. During tumorigenesis, high ECM stiffness, enrichment and hyperactivation, the mechanosensing machinery can promote invasion, migration and metastatic dissemination

Fig. 4

Nuclear forces are balanced by the cytoskeleton. The nucleus is connected to the cytoskeleton via transmembrane proteins, including nesprins and SUN proteins. These assemblies are called the LINC, linker of the nucleoskeleton and cytoskeleton complex. The LINC complex connects to the cytoskeleton, including actin filaments, microtubules and intermediate filaments through the nuclear envelope to chromatin. The LINC complex is usually composed by the SUN protein subunits connected to lamins intranuclearly and the nesprin proteins on the cytoplasm. This complex is thought to relay cytoskeletal changes to alterations in chromatin organisation and affect gene expression. Additionally, increased force can lead to stretching of nuclear pores and increased exchange of proteins between the nucleus and the cytoplasm. When cells invade through pores of the ECM or intravasate into a blood vessel and travel through the bloodstream; the associated squeezing and shear forces affect chromatin organisation and stability of the genome

Fig. 5

Hydrogels recapitulate mechanical aspects of the microenvironment. a Sketch of a hydrogel, showing cells embedded in the 3D environment. b Details of an example hydrogel, showing crosslinker, which can be varied to control pore size and stiffness; polymer, which can also be varied to change mechanical and chemical properties; protein, which can represent an endogenous tissue or tumour matrix protein such as fibronectin; growth factor, which can be included in the hydrogel and presented either upon stimulus or constitutively. c Micrograph showing spheroid of mouse pancreatic cancer cells growing in a hydrogel. Sketches courtesy of Sara Trujillo-Munoz, University of Glasgow