Flexible microdevices for characterization of bionanomechanics in cancer

There have been extensive efforts to study the role of gene mutations in cancer and an accumulation of mutations has been proposed as being necessary for cancer development. However, the results from gene-based drug treatments of cancers have been variable [1] and less satisfactory than desired [2], being much less than antibiotic effects on infectious diseases for example. Recently, evidence has accumulated that cancer is not only a disease of genetic mutations, but that the micro- and nano-environments of cells may be essential factors in triggering tumour growth [3]. Tumorigenic growth patterns can be induced by inappropriate mechanical forces [4-6]. For example, the dysfunctional collagen crosslinking in the cell extracellular matrix (ECM) of old individuals may be the trigger for cancer in old age [7-9]. Furthermore, the earlier onset of breast cancer, compared to cancers of other organs [10], is explained by accelerated aging of healthy breast tissue [11]. For cancer to spread, invade or metastasise, a cancer cell must exert physical forces [12]. Thus, we propose that a disrupted environment (a mutation-independent element) is necessary for the local condition to promote cancer development. However, there is no coherent quantitative data on the nature and level of mechanical forces that influence the interactions between the physical micro- and nano-environment and cancer cells [12].

Research work plan
We will develop a microfluidic platform to perform force measurements on three dimensional spheroid clusters [15]. Clusters of cells, often called ‘spheroids’, will be formed suspended in culture medium. We will place clusters of cells in arrays and cages of elastomeric and conductive polymer pillars to study spheroids in a 3D environment. Our collaborator Prof. Vieu recently demonstrated the measurement of interior forces in multi-cellular 3-D spheroid tumour models using high aspect ratio pillars made of PDMS [16]. During culturing, clusters increase in size by cell proliferation and gradually exert greater force on the pillars and on themselves.

In this work we will extend these experiments to endometrial cancer cells and add the ability to artificially impose mechanical forces on the spheroids. Cells being compressed will transfer force to adjacent pillars, allowing the magnitude to be quantified from the pillar deflection. For force sensing we will directly compare elastomeric and conductive polymer pillar arrays, thus adding functionality and producing more detailed and accurate force measurements. Our existing pillar arrays, developed for multi-point force measurement with nematodes [17], have recently been miniaturized. Elastomeric pillars 30 μm high and with diameters as small as 7 μm, were used for force sensing on fungal hyphae, demonstrating nano-Newton resolution [18]. We will use soft-lithography and replica moulding developed for this work to fabricate the elastomeric pillar arrays and direct writing [19] on prefabricated electrode arrays to produce the conductive polymer pillar arrays. The spheroids will be collected at selected time points and submitted to immunohistochemistry, which will identify differences between cells on the inside of the spheroid from those on the outside, noting for example changes to focal adhesion complexes and the cytoskeleton.

Building on the force measurement devices described above, we will extend the work further by developing a microfluidic platform capable of applying controlled forces to multi-cellular models. Current force application techniques designed to study single and 2-D cell behaviour under mechanical stress do not integrate force feedback or allow for active force modulation [20, 21]. The ability to dynamically alter forces to which cells are exposed [22] will provide more relevant information. Active force modulation with direct sensor feedback will allow us to test for a variety of compressive force levels to determine the effect on multi-cellular cancer models. PDMS membrane-based Quake-type [23] and side valve devices [24] capable of applying compressive force will be developed. A microfluidic cell handling platform will be developed that will allow for precise control of force level, duration and dynamics in a high throughput fashion. Integrated fluid handling will allow us to study gene and protein expression of the cancer cells during mechanobiologically relevant force profiles on-chip.

This PhD project has the following specific objectives:
1) Design and fabricate a microfluidic platform for cell-spheroid force application and measurement.
2) Characterize the bionanomechanics of tumour growth and effect of force stimuli.
3) Explore gene and protein expression during mechanobiologically relevant force profiles on-chip.