Delivery of molecules into biological cells

Abstract

The present invention relates to the delivery of molecules into biological cells, and particularly, although not exclusively, to methods and associated apparatus for delivering exogenous molecules into cells involving the application of a weight or pressure to the cells. The invention also encompasses the use of the apparatus or 5 method in diagnosing or prognosing disease in biological cells, such as cancer and metastasis.

Description

[0001] Delivery of molecules into biological cells

[0002] The present invention relates to the delivery of molecules into biological cells, and particularly, although not exclusively, to methods and associated apparatus for delivering exogenous molecules into cells involving the application of a weight or pressure to the cells. The invention also encompasses the use of the apparatus or method in diagnosing or prognosing disease in biological cells, such as cancer and metastasis.

[0003] A diverse range of methodologies exist which enable the delivery of foreign genetic material, gene products, and chemical agents into mammalian cells. The selection of the appropriate delivery method depends on the mammalian cell type, the molecular characteristics of the foreign materials and the overall experimental objectives of the study. The delivery of genetic material (DNA and RIMA) into cells, which is termed transfection, is achieved using chemical, physical, and biological methods. Chemical methods utilise cationic polymers (Schenborn et al., 2000), cationic amino acids (Midoux et al., 2002; Eliyahu et al., 2005), cationic lipids (Jarnagin et al., 1992; Mukalel et al., 2019) and calcium phosphate (Kwon et al., 2013; Kumar et al., 2019) to form complexes with the negatively charged nucleic acids. Lipid nanoparticle-based messenger RNA (mRNA) delivery methods proved highly effective during the Covid-19 pandemic (Hou et al., 2021; Vogel et al., 2021). The major advantage of the chemical-based transfection methods is their ease of use. However, the major disadvantages are the variable transfection efficiency, which can range from very high to very low depending on the cell type, and the potential for cytotoxicity. Additionally, polyethylene glycol (PEG)-based lipid nanoparticle transfection methods can lead to hypersensitivity reactions and immunogenicity (Ibrahim et al., 2022; Guerrini et al., 2022; Tenchov et al., 2023). Finally, chemical methods vary in cost; for instance, calcium phosphate-based methods are inexpensive, whereas lipid-based formulations are costly.

[0004] Common physical-mediated transfection methods include electroporation, microinjection, biolistic, magnetic and laser-assisted delivery (Fus-Kujawa et al., 2021). Electroporation is widely used for both research and commercial applications. The entry of the genetic material into the cell is facilitated via the generation of membrane pores created by the application of high voltage pulses (Xie et al., 1990; Sukharev et al 1992). The major drawbacks of electroporation are the high cell death rates (Batista Napotnik et al., 2021), the need for extensive optimisation, and the usage of expensive electroporation buffers and consumables. Like other physical transfection methods, electroporation requires costly equipment and time-consuming sample preparation.

[0005] Biological transfection methods, which involve the use of viruses (called viral transduction), are highly efficient. However, the major disadvantages include potential damage to the host genome due to insertional mutagenesis, immunogenicity, limitations on DNA the cargo size, health and safety concerns on laboratory personnel, and the need for dedicated containment level 2 facilities (Kim et al., 2010).

[0006] Moreover, methods that are effective for delivering nucleic acids may not always be directly applicable for delivering other biomolecules into cells, such as proteins, or large molecules (e.g. antibodies) or small molecules (e.g. drugs). While some delivery methods are more versatile, differences in their molecular properties and cellular uptake mechanisms often necessitate specific optimisations for each type of molecule.

[0007] There is, therefore, a need for an improved and safer means of delivering exogenous molecules into cells.

[0008] The inventors have devised a novel way to deliver, into mammalian cells, a wide variety of molecules, including nucleic acids, proteins, antibodies, and peptides into cells, through weight-induced compression and consecutive relaxation of the cells, in a process, which may be referred to herein as "mechanofection".

[0009] Thus, according to a first aspect of the invention, there is provided a method of introducing at least one exogenous molecule into at least one biological cell, the method comprising:

[0010] (i) contacting the at least one biological cell with the at least one exogenous molecule; and

[0011] (ii) exerting force on the at least one biological cell, to thereby introduce the at least one exogenous molecule into the at least one biological cell.

[0012] The inventors have devised an apparatus which reliably and reproducibly enables the delivery of exogenous molecules into cells using the method of the first aspect.

[0013] Thus, in a second aspect of the invention, there is provided an apparatus for introducing at least one exogenous molecule into at least one biological cell, the apparatus comprising: (i) means for contacting the at least one biological cell with the at least one exogenous molecule; and

[0014] (ii) means for exerting force on the at least one biological cell, to thereby introduce the at least one exogenous molecule into the at least one biological cell.

[0015] Advantageously, the inventors believe that their innovative method and apparatus using the exertion of force on the or each biological cell is much safer and more physiologically natural than currently used transfection or transformation methods, and allows the introduction of exogenous molecules in such a way that does not cause immune reactions and without compromising the viability of the treated biological cells.

[0016] The method may be performed in vivo, in vitro, or ex vivo.

[0017] The inventors have exemplified their method using of three different cell lines, namely MCF10A (non-tumorigenic), MDA-MB-231 (highly metastatic and triple-negative) and HCC38 (non-metastatic basal-like) cell lines.

[0018] Thus, the at least one biological cell may comprise at least one mammalian cell. In one embodiment, therefore, the at least one biological cell may be selected from a group of cell types consisting of: epithelial cell, muscle cell, nerve cell, blood cell, connective tissue cell, cancer cell, and stem cell.

[0019] In one embodiment, the at least one biological cell may comprise a cell line comprising a plurality of cells. The at least one biological cell may comprise the same cell type or a plurality of two or more different cell types.

[0020] In one embodiment, the at least one cell may comprise a plurality of cells. The at least one biological cell may comprise at least 50, 100, 250, 500 or 1000 cells. The at least one biological cell may comprise at least 1500, 2000, 2500, 3000 or 3500 cells. The at least one biological cell may comprise at least 4000, 4500, 5000, 7500 or 10,000 cells.

[0021] As discussed in the Examples, the inventors maintained the cells as a monolayer.

[0022] In one embodiment, therefore, the at least one biological cell may comprise a plurality of biological cells. In another embodiment, the at least one biological cell may comprise a monolayer comprising a plurality of biological cells. In another embodiment, the at least one biological cell may comprise a plurality of layers of biological cells. Typically, however, the at least one biological cell comprises a monolayer of cells.

[0023] As discussed in the Examples, the inventors seeded cells onto a surface, e.g., a coverslip. It will be appreciated, therefore, that the diameter of the or each layer of biological cells may be the same as the diameter as the surface to which the biological cells are adhered.

[0024] In one embodiment, the diameter of the or each layer of biological cells may be between 0.001mm and 1000mm, between 0.01 and 500mm, or between 1.1mm and 100mm. In another embodiment, the diameter of the or each layer of biological cells may be between 2mm and 90mm, between 3mm and 80mm, between 4mm and 70mm, between 5mm and 60mm, or between 6mm and 50mm. In another embodiment, the diameter of the or each layer of biological cells may be between 7mm and 40mm, between 8mm and 30mm, between 9mm and 20mm, between 9.5mm and 15mm, or between 10mm and 12.5mm. Typically, however, the diameter of the or each layer of biological cells is about 11mm.

[0025] In one embodiment, the plurality of cells may comprise a cell density of between 2 and 2,000,000 cells per cm2. In another embodiment, the plurality of cells may comprise between 10 and 1,000,000 cells per cm2, between 100 and 100,000 cells per cm2, between 1,000 and 500,000 cells per cm2, between 2,000 and 250,000 cells per cm2, between 3,000 and 200,000 cells per cm2, between 4,000 and 150,000 cells per cm2, or between 5,000 and 100,000 cells per cm2. In another embodiment, the plurality of cells may comprise a cell density of between 6,000 and 90,000 cells per cm2, between 7,000 and 80,000 cells per cm2, between 8,000 and 70,000 cells per cm2, between 9,000 and 60,000 cells per cm2, between 10,000 and 50,000 cells per cm2, between 11,000 and 40,000 cells per cm2, or between 12,000 and 30,500 cells per cm2. In another embodiment, the plurality of cells may comprise a cell density of between 13,000 and 30,000 cells per cm2, between 14,000 and 28,000 cells per cm2, between 15,000 and 27,000 cells per cm2, between 16,000 and 26,000 cells per cm2, between 17,000 and 25,000 cells per cm2, between 18,000 and 24,000 cells per cm2, or between 18,000 and 23,000 cells per cm2. In one embodiment, the plurality of cells may comprise a cell density of between 19,000 and 21,000 cells per cm2. Typically, however, the plurality of cells comprises a cell density of around 20,000 cells per cm2. The inventors envisage that the plurality of cells may be in a three-dimensional formation. It will be appreciated that liver cell density is about 100 million cells per cm3, bone marrow cell density is about 1 billion cells per cm3, and skin tissue cell density is about 1 billion cells per cm3.

[0026] Accordingly, in one embodiment, the plurality of cells may comprise a cell density of between 2 and 1,909,000,000 cells per cm3, between 100 and 1,800,000,000 cells per cm3, between 1,000 and 1,700,000,000 cells per cm3, between 10,000 and 1,600,000,000 cells per cm3, or between 100,000 and 1,500,000,000 cells per cm3. In another embodiment, the plurality of cells may comprise a cell density of between 1,000,000 and 1,400,000,000 cells per cm3, between 10,000,000 and 1,300,000,000 cells per cm3, between 100,000,000 and 1,200,000,000 cells per cm3, between 250,000,000 and 1,100,000,000 cells per cm3, or between 500,000,000 and 1,000,500,000 cells per cm3. In another embodiment, the plurality of cells may comprise a cell density of between 750,000,000 and 1,000,250,000 cells per cm3, or between 900,000,000 and 1,000,000,000 cells per cm3. Typically, however, the plurality of cells comprises a cell density of around 1,000,000,000 cells per cm3.

[0027] In one embodiment, the at least one biological cell may comprise a healthy cell. In yet another embodiment, the at least one biological cell may comprise an unhealthy cell, such as a cancer cell. In one embodiment, the cancer cell may comprise a breast cancer cell.

[0028] In one embodiment, the at least one cell may be an adherent cell, i.e., one which grows while adhering to a surface, for example a surface of a culture vessel. Typically, the at least one cell comprises an adherent cell.

[0029] Thus, in one embodiment, the method further comprises a step of adhering the at least one biological cell to a surface before the step of contacting the at least one biological cell with the at least one exogenous molecule and / or the step of exerting force on the at least one biological cell.

[0030] It will be appreciated that the step of adhering the at least one biological cell to the surface by incubation may be referred to as the "adherence step", as shown in Figure IB.

[0031] As discussed in the Examples, the force exerted on the cells is defined by the physical mass of the weights, making the method highly reproducible and the apparatus straightforward to calibrate. Advantageously, since the load is applied directly to the cells, the mechanical input is uniform and can be quantified precisely. In contrast, should the method and / or apparatus utilise a flexible surface to which the cells are adhered (e.g., microporous membranes), several sources of variability would be introduced. For example, a membrane itself can deform non-uniformly, leading to different force distributions between the centre and the periphery. Additionally, the mechanical properties of a membrane (e.g., tension, elasticity, thickness, pore density) can vary between batches, making it harder to ensure consistent loading conditions. As a result, such membrane-based methods would be less direct, less predictable, and often more complex to interpret. Accordingly, the inventors' selection of a solid, non-flexible surface enables a simpler, more accurate, and more consistent method for applying defined compressive stimuli to cells compared with membranebased systems.

[0032] In one embodiment, therefore, the surface may be solid, hard, rigid, and / or non- flexible.

[0033] The surface may be selected from a list of surfaces consisting of: glass, polystyrene, plastic, extracellular matrix coated, fibrin, silicon, ecoflex, hydrogel, spun silk fibroin, thermoplastic elastomer, polymeric film, polyurethane, polydimethylsiloxane, polyacrylamide, polyethylene terephthalate, polycarbonate, polylactic acid, poly(lactic- co-glycolic acid), polycaprolactone, chitosan, poly(N-isopropylacrylamide), graphene, graphene oxide, hydroxyapatite, carbon nanotube, polyethylene glycol, and alginate. Typically, however, the surface comprises glass, for example, a glass coverslip or glass slide.

[0034] In one embodiment, the at least one biological cell may be incubated to allow the or each cell to substantially adhere to the surface. In one embodiment, the at least one biological cell may be adhered to the surface by incubation at a temperature of between 20°C and 50°C, or between 30°C and 35°C. In one embodiment, the at least one biological cell may be adhered to the surface by incubation in an atmosphere of between 0.1% and 10% CO2, or between 1% and 7% CO2. In one embodiment, the at least one biological cell may be adhered to the surface by incubation for between 1 minute and 10 hours, or between 1 hour and 5 hours.

[0035] In one embodiment, the at least one biological cell may be adhered to the surface by incubation in a humidified cell incubator. Typically, therefore, the at least one biological cell is adhered to the surface by incubation in a humidified cell incubator at about 37°C and in an atmosphere of about 5% CO2 for at least 2 hours. In one embodiment, subsequent to the adherence step, the at least one biological cell comprises a free apical cell surface.

[0036] It will be appreciated that the term "free apical cell surface", as used herein, can refer to the cell surface area that is not adhered to the surface.

[0037] Typically, therefore, the free apical cell surface of the at least one biological cell is not contacted with, or adhered to, a surface or membrane.

[0038] Once adherence of the cells to the surface was established, the inventors subsequently contacted the cells with complete media and performed a further incubation under the same conditions, but for a longer period of time. It will be appreciated that this incubation with complete media may be referred to as the "pre-treatment incubation step", as shown in Figure IB.

[0039] In one embodiment, therefore, the at least one biological cell may be contacted and incubated with complete media. Typically, the free apical cell surface of the at least one biological cell may be contacted and incubated with complete media. Typically, the at least one biological cell is contacted and incubated with complete media subsequent to the adherence step.

[0040] It will be appreciated that the term complete media can mean a culture medium that is enriched to contain all of the growth requirements of the at least one biological cell. It will also be appreciated, therefore, that the complete media can be a culture medium enriched with a variety of different components, which will be dictated by the type of cell and the growth requirements of that cell. It will also be appreciated that the term "complete media" and the term "media" may be used interchangeably herein.

[0041] As can be seen in the Examples and Materials and Methods, the inventors cultured cells of the non-malignant breast epithelial cell line MCF10A in DMEM / F-12 media, MDA-MB-231 cells in DMEM high glucose (4.5 g / L glucose) media, and HCC38 cells in RPMI 1640 media. The DMEM / F-12 media was supplemented with 100 ng / ml cholera toxin, 20 ng / ml epidermal growth factor, 2 mM L-glutamine, 500 ng / ml hydrocortisone, 0.01 mg / ml insulin, 1% Penicillin-Streptomycin, and 5% Horse Serum. The DMEM high glucose (4.5 g / L glucose) media and RPMI 1640 media were supplemented with 10% FBS, 2 mM L-Glutamine, and 1% Penicillin-Streptomycin. In one embodiment, therefore, the complete media may comprise DMEM / F-12 media, DMEM high glucose media, and / or RPMI 1640 media.

[0042] In one embodiment, the DMEM high glucose media may comprise at least 1 g / L, 2 g / L, 3 g / L, or 4 g / L glucose. Typically, however, the DMEM high glucose media comprises at least 4.5 g / L glucose.

[0043] In one embodiment, the complete media may comprise cholera toxin, epidermal growth factor, L-Glutamine, hydrocortisone, insulin, Penicillin-Streptomycin, Horse Serum, and / or FBS.

[0044] The at least one biological cell may be incubated with complete media at a temperature of between 20°C and 50°C, or between 30°C and 35°C. In one embodiment, the at least one biological cell may be incubated with complete media in an atmosphere of between 0.1% and 10% CO2, or between 1% and 7% CO2. In one embodiment, the at least one biological cell may be incubated with the complete media for between 5 and 100 hours, between 10 and 50 hours, between 15 and 35 hours, or between 20 and 30 hours. Typically, however, the at least one biological cell is incubated with complete media at about 37°C in an atmosphere of about 5% CO2 for about 24 hours.

[0045] Typically, the at least one biological cell is removed from the complete media prior to the step of exerting the force on the at least one cell (i.e., the first incubation period).

[0046] In one embodiment, subsequent to incubating the at least one biological cell with complete media (i.e., the pre-treatment incubation step), the method may further comprise a step of inverting the surface on which the at least one biological cell is adhered. It will be appreciated that inverting the surface on which the at least one biological cell is adhered means that the at least one biological cell is facing downwards, as shown in Figure IB. Typically, the surface on which the at least one cell is adhered is inverted before the step of exerting force on the at least one cell.

[0047] In one embodiment, the method may comprise a first incubation period comprising (i) contacting the at least one biological cell with the at least one exogenous molecule; and (ii) exerting force on the at least one biological cell, as described in the first aspect, and as depicted in Figure IB. In one embodiment, the surface on which the at least one cell is adhered may be contacted with a medium comprising the at least one exogenous molecule, such that the at least one cell is contacted with the at least one molecule. In one embodiment, the medium may comprise a solid, semi-solid, semi-liquid, or liquid. In one embodiment, the medium comprising the at least one exogenous molecule is serum- free. In another embodiment, the medium comprising the at least one molecule comprises PBS (Phosphate Buffered Saline). Typically, the medium comprising the at least one molecule comprises DPBS (Dulbeccos's Phosphate Buffered Saline).

[0048] In one embodiment, the at least one exogenous molecule may comprise a biological molecule. The biological molecule may be selected from a group of biological molecules consisting of: protein, nucleic acid, nucleotide, carbohydrate, lipid, DNA, RIMA, antibody, antibody fragment, peptide, enzyme, hormone, growth factor, neurotransmitter, cytokine, antibiotic, oligonucleotide, synthetic or bioengineered pharmaceutical drug, chemotherapy agent, nutraceutical, and toxin.

[0049] In one embodiment, the at least one exogenous molecule may not be complexed with a carrier (e.g., lipid-based carrier complex). In one embodiment, the at least one exogenous molecule may not form part of a carrier complex.

[0050] As described in the Examples, the inventors have exemplified the delivery of exogenous GFP-encoding bare DNA and mRNA into cells, resulting in the production of the GFP protein by the cells, as shown in Figures 2B and 3A (DNA), and Figures 5C and Figure 8E (RNA). As such, the inventors have demonstrated that the method and apparatus of the invention can be used to successfully introduce bare DNA and RNA encoding a protein into a cell, resulting in the production of a functional protein by the cell. The skilled person would readily appreciate that the GFP is an exemplar acting as a representative of any gene, because the data presented herein proves that the method of the invention is able to introduce, in vitro and ex vivo, bare DNA and mRNA encoding a protein into a cell. As such, the GFP provides robust evidence of the proof of concept that the method of the invention can be used to introduce any proteinencoding bare DNA or RNA molecule into a cell.

[0051] In one embodiment, therefore, the at least one molecule may comprise a proteinencoding DNA or RNA. Typically, the protein-encoding DNA or RNA is bare or naked. It will be appreciated that the terms "bare" or "naked", as used herein can refer to a DNA or RIMA molecule which is not complexed with a carrier (e.g., lipid-based carrier complexes).

[0052] In another embodiment, the at least one exogenous molecule may be selected from a group of molecules consisting of: small molecule, nanoparticle, liposome, exosome, quantum dot, polymer, dendrimer, ion, nanomaterial, and antioxidant.

[0053] In one embodiment, the molecular weight of the exogenous molecule may be at least 10 Da, 20 Da, 30 Da, 40 Da, or 50 Da. In another embodiment, the molecular weight of the exogenous molecule may be at least 60 Da, 70 Da, 80 Da 90 Da, or 95 Da. In another embodiment, the molecular weight of the exogenous molecule may be at least 96 Da, 97 Da, 98 Da, or 99 Da. Typically, however, the molecular weight of the exogenous molecule is at least 100 Da.

[0054] In one embodiment, the molecular weight of the exogenous molecule may be between 10 Da and 10 MDa, between 20 Da and 9 MDa, between 30 Da and 8 MDa, between 40 Da and 7 MDa, or between 50 Da and 6 MDa. In another embodiment, the molecular weight of the exogenous molecule may be between 60 Da and 5 MDa, between 70 Da and 4 MDa, between 80 Da and 3 MDa, between 90 Da and 2 MDa, or between 95 Da and 1.9 MDa. In another embodiment, the molecular weight of the exogenous molecule may be between 96 Da and 1.8 MDa, between 97 Da and 1.7 MDa, between 98 Da and 1.6 MDa, or between 99 Da and 1.5 MDa. Typically, however, the molecular weight of the exogenous molecule is between about 100 Da and about 1.45 MDa.

[0055] The force that is exerted on the at least one biological cell may be a push force or a pull force acting on the or each cell, or a result of its interaction with another object. Typically, exerting the force on the at least one biological cell results in compression of the or each cell, to allow the or each exogenous molecule to pass through the cell's outer cell membrane.

[0056] In an embodiment, the force that is exerted on the at least one biological cell may be a contact force applied to the at least one biological cell. Thus, the force may be selected from a group of contact forces consisting of: applied force, spring force, air resistance force, normal force, tension force and frictional force. In another embodiment, the force that is exerted on the at least one biological cell may be a non-contact force which is applied to the at least one biological cell from a distance. Therefore, the force may be selected from a group of non-contact forces consisting of: magnetic force, electrical force and gravitational force.

[0057] Typically, however, the force is a mechanical force. It will be appreciated that the term mechanical force can mean the direct contact between the at least one biological cell and another object. It will also be appreciated that exerting mechanical force or applying pressure on the at least one biological cell results in compression of the cell.

[0058] In one embodiment, the at least one biological cell may be contacted with the at least one molecule (i.e., step (i) of the first aspect) before the force is exerted on the at least one biological cell (i.e., step (ii) of the first aspect). However, in another embodiment, the at least one biological cell may be contacted with the at least one molecule (i.e., step (i) of the first aspect) after the force is exerted on the at least one biological cell (i.e., step (ii) of the first aspect). In yet another embodiment, the at least one biological cell may be contacted with the at least one molecule (i.e., step (i) of the first aspect) at the same time as the force is exerted on the at least one biological cell (i.e., step (ii) of the first aspect). It will be appreciated that each of the above three embodiments results in the introduction of the at least one exogenous molecule into the at least one biological cell.

[0059] In one embodiment, the force is exerted on the at least one biological cell by a weight (W) and / or a microweight (MW). It will be appreciated that the terms weight and microweight can mean objects which are known to weigh a definite amount. It will also be appreciated that a microweight is a type of weight.

[0060] In one embodiment, the force may be exerted on the at least one biological cell by positioning the cell between two solid (e.g., horizontal) planar surfaces and positioning a weight (W) and / or a microweight (MW) adjacent one of the surfaces (e.g., on top of the uppermost surface) in order to thereby compress the cell.

[0061] In one embodiment, the weight may be contacted with the at least one biological cell, typically the free apical cell surface of the at least one biological cell, and / or the surface to which the at least one biological cell is adhered. In one embodiment, the weight may be contacted with the at least one cell and / or the surface to which the at least one cell is adhered at the same time as the at least one cell is contacted with the at least one molecule. In one embodiment, the weight may weigh between 0.01 g and 50 g. In another embodiment, the weight may weigh between 0.5 g and 40 g, between 1 g and 30 g, or between 2 g and 25 g. Typically, however, the weight weighs between 5 g and 20 g-

[0062] It will be appreciated that contacting a weight with the at least one biological cell results in the exertion of pressure on the at least on the cell, where Pressure = Force / Area. In one embodiment, therefore, a pressure is exerted on the at least one biological cell. In one embodiment, the pressure exerted on the at least one biological cell may be between 0.0001 Pascal and 10,000 Pascal. In another embodiment, the pressure exerted on the at least one biological cell may be between 0.001 Pascal and 8,000 Pascal, between 0.01 Pascal and 7,000 Pascal, between 0.1 Pascal and 6,000 Pascal, or between 1 Pascal and 5,000 Pascal. The pressure exerted on the at least one biological cell may be between 10 Pascal and 4,000 Pascal, or between 100 Pascal and 3,000 Pascal. In another embodiment, the pressure exerted on the at least one biological cell may be between 200 Pascal and 3750 Pascal, between 300 Pascal and 3500 Pascal, between 400 Pascal and 3250 Pascal, or between 500 Pascal and 3000 Pascal.

[0063] In one embodiment, the pressure exerted on the at least one biological cell by a 5 gram weight may be around 516 Pascal. In one embodiment, the pressure exerted on the at least one biological cell by a 10 gram weight may be around 1031 Pascal. In another embodiment, the pressure exerted on the at least one biological cell by a 20 gram weight may be around 2064 Pascal.

[0064] In one embodiment, the force (or pressure) may be exerted on the at least one biological cell for between 1 second and 10 minutes, between 2 seconds and 9 minutes, between 3 seconds and 8 minutes, or between 4 seconds and 7 minutes. The force (or pressure) may be exerted on the at least one biological cell for between 5 seconds and 6 minutes, between 6 seconds and 5 minutes, between 7 seconds and 4 minutes, between 8 seconds and 3 minutes, or between 9 seconds and 2 minutes. Typically, however, the force (or pressure) is exerted on the at least one biological cell for about 10 seconds. Any of the above weights and periods of contacting between the weight and the biological cell may be combined in any combination to create the desired force (or pressure) on the at least one biological cell. As described above, it will be appreciated that contacting the at least one biological cell with the at least one exogenous molecule whilst exerting force on the at least on the cell (either before, after, or simultaneously) may be referred to as a "first incubation period".

[0065] Accordingly, in one embodiment, the method may comprise a first incubation period comprising (i) contacting the at least one biological cell with the at least one molecule, and (ii) exerting force on the at least one biological cell. Typically, however, the first incubation period comprises (i) contacting the at least one biological cell with the at least one molecule, and (ii) exerting force on the at least one biological cell by exerting a pressure of between 500 Pascal and 2100 Pascal on the at least one cell for about 10 seconds.

[0066] In one embodiment, subsequent to the first incubation period, the method may comprise a step of inverting the surface on which the at least one biological cell is adhered. It will be appreciated that inverting the surface on which the at least one biological cell is adhered subsequent to the first incubation period means that the at least one biological cell is facing upwards.

[0067] In one embodiment, the method may comprise a second incubation period comprising contacting and incubating the at least one biological cell with media, but does not comprise exerting force on the at least one cell. In one embodiment, subsequent to the first incubation period, the at least one biological cell may be subjected to a second incubation period in which it is contacted and incubated with media.

[0068] In one embodiment, the media may comprise DMEM / F-12 media, DMEM high glucose media, and / or RPMI 1640 media.

[0069] In one embodiment, the DMEM high glucose media may comprise at least 1 g / L, 2 g / L, 3 g / L, or 4 g / L glucose. Typically, however, the DMEM high glucose media comprises at least 4.5 g / L glucose.

[0070] In one embodiment, the media may comprise cholera toxin, epidermal growth factor, L-Glutamine, hydrocortisone, insulin, Penicillin-Streptomycin, Horse Serum, and / or FBS.

[0071] In one embodiment, the second incubation period may comprise incubating the at least one biological cell with media for between 1 second and 10 minutes, between 2 seconds and 9 minutes, between 3 seconds and 8 minutes, or between 4 seconds and 7 minutes. The at least one biological cell may be incubated with media for between 5 seconds and 6 minutes, between 6 seconds and 5 minutes, between 7 seconds and 4 minutes, between 8 seconds and 3 minutes, or between 9 seconds and 2 minutes.

[0072] Typically, however, the second incubation period comprises incubating the at least one cell with media for about 10 seconds.

[0073] It will be appreciated that subjecting the at least one biological cell to a second incubation period, i.e., contacting and incubating the at least one cell with media in the absence of the application of force, results in relaxation of the at least one cell following force-induced compression of the at least one cell in the first incubation period.

[0074] It will also be appreciated that subjecting the at least one biological cell to a first and second incubation period, as defined above, may be referred to as a treatment.

[0075] In one embodiment, the method of the first aspect may comprise one or more treatments. Typically, the method of the first aspect comprises two treatments.

[0076] Accordingly, it will be appreciated that the method may comprise cycles of compression (i.e., exerting a force on cells, such as defined by the first incubation period) and consecutive relaxation (i.e., removing the force from cells, such as defined by the second incubation period) of cells.

[0077] In one embodiment, therefore, the method may comprise an additional first incubation period.

[0078] The features and parameters of the additional first incubation period may be defined as in the first incubation period, as described above.

[0079] In one embodiment, the method may comprise an additional second incubation period.

[0080] The features and parameters of the additional second incubation period may be defined as in the second incubation period, as described above.

[0081] In one embodiment, subsequent to one or more treatments, (i.e., one or more rounds of first and second incubation periods), the at least one cell may be contacted and incubated with media. This step may be referred to as a "post-treatment incubation step", as shown in Figure IB.

[0082] In one embodiment, the media may comprise DMEM / F-12 media, DMEM high glucose media, and / or RPMI 1640 media.

[0083] In one embodiment, the DMEM high glucose media may comprise at least 1 g / L, 2 g / L, 3 g / L, or 4 g / L glucose. Typically, however, the DMEM high glucose media comprises at least 4.5 g / L glucose.

[0084] In one embodiment, the media may comprise cholera toxin, epidermal growth factor, L-Glutamine, hydrocortisone, insulin, Penicillin-Streptomycin, Horse Serum, and / or FBS.

[0085] In one embodiment, subsequent to one or more treatments, the post-treatment incubation step may comprise contacting and incubating the at least one biological cell with media at between 20°C and 50°C, or between 30°C and 35°C. In one embodiment, the at least one biological cell may be incubated with complete media in an atmosphere of between 0.1% and 10% CO2, or between 1% and 7% CO2. In one embodiment, the at least one biological cell may be incubated with the complete media for between 5 and 100 hours, between 10 and 50 hours, between 15 and 35 hours, or between 20 and 30 hours. Typically, however, the at least one biological cell is incubated with media at about 37°C in an atmosphere of about 5% CO2 for about 24 hours.

[0086] As described herein, the inventors have exemplified an apparatus used to reliably and reproducibly deliver exogenous molecules into cells through weight-induced compression and consecutive relaxation of the cells.

[0087] In one embodiment, the apparatus of the second aspect may be used to perform the method of the first aspect. The apparatus may be used in a method of introducing at least one exogenous molecule into the at least one biological cell in vivo, in vitro, or ex vivo.

[0088] In one embodiment, the means for contacting the at least one biological cell with the at least one molecule may comprise a surface onto which the at least one cell may be adhered. The surface may be defined as in relation to the first aspect. The means for contacting the at least one biological cell with the at least one exogenous molecule may comprise a surface onto which the at least one molecule may be dispersed. The surface onto which the at least one molecule may be dispersed may comprise a film, such as a parafilm. In one embodiment, the parafilm may be positioned on a plate, such as a glass plate. In another embodiment, the parafilm may be hydrophobic.

[0089] In one embodiment, the means for contacting the at least one biological cell with the at least one molecule may comprise lowering the surface onto which the at least one cell may be adhered such that the at least one cell contacts the at least one molecule which may be dispersed on a surface.

[0090] In one embodiment, the means for exerting force on the at least one biological cell may comprise a weight which may be placed adjacent to the surface onto which the at least one cell may be adhered.

[0091] As described in the Examples, the inventors have discovered that there are various distinguishing features between non-malignant, malignant, and non-tumorigenic cell lines in vitro that may be identified using the microweight approaches described herein. These distinguishing features, alone or combined, may be referred to as a biomechanical signature.

[0092] Advantageously, the distinguishing features that the inventors have discovered reveal a novel biomechanical signature of malignancy that does not rely on traditional molecular markers. Thus, the inventors have discovered a novel biomechanical signature that reliably differentiates tumour cells from non-malignant and non- tumorigenic counterparts, which may provide a powerful indicator of malignant transformation. Advantageously, such a metric could significantly enhance diagnostic accuracy and prognostic assessment by capturing mechanical behaviours intrinsically linked to tumour aggressiveness.

[0093] As discussed in the Examples, these distinguishing factors include:

[0094] (i) uptake of external substances into cells, since the inventors have found that cancer cells more readily take up exogenous material than healthy, non-tumorigenic cells;

[0095] (ii) MW-induced organelle status and / or MW-induced cell viability, since delivery of cytotoxic material into the cell has been found to result in nuclear shrinking and decreased cell viability; (iii) Responsiveness Factor (RF) ratio, which the inventors have found to be high for metastatic cells, and very low for non-metastatic cells;

[0096] (iv) MW-induced nuclear expansion, since the inventors have found that the nucleus of metastatic cells expands more easily, while the nucleus of a healthy, non-tumorigenic cell does not; and / or

[0097] (v) MW-induced cell viability relative to cell density.

[0098] Firstly, the inventors have identified that exogenous molecules can be introduced into cancer cells (i.e., mechanofected) more efficiently than healthy cells, likely due to the nature and elasticity of their cell membrane, and can thereby distinguish between a cancer cell and a healthy cell based on cell stiffness, e.g., the more malleable the cell, the more molecules will be introduced into the cell, and / or the more cancer cells a tissue biopsy contains, the more cells will be mechanofected and the more molecules will be introduced into the cells of the biopsy. Furthermore, since it is known that increased malleability is common in metastatic cells, the inventors envisage that they can use their methods to determine the likelihood of a cell to metastasize, whereby the more molecules that are introduced into a cell by mechanofection, the more likely the cell is to metastasize.

[0099] Secondly, the inventors have identified that their methods enable the study of the function of internal cellular proteins, as they have shown that the delivery of nesprin-2 antibodies into the cells by mechanofection results in nuclear shrinking, and can thereby distinguish between a cancer cell and a healthy cell based on the study of an organelle, in this case, the nucleus. In addition, the inventors have identified that the entry by mechanofection of a toxic peptide into cells affects cell viability, and can thereby distinguish between a cancer cell and a healthy cell based on cell viability.

[0100] Thirdly, the inventors have discovered that the Responsiveness Factor (RF) ratio is high for metastatic cells, and very low for non-metastatic cells, and thus this RF ratio can be used to differentiate between metastatic and non-metastatic cells. RF is defined as the ratio of the perinuclear F-actin ring response (%) at 10 g (MW-10) to the response at 0.5 g (MW-0.5) (RF = MW-10 I MW-0.5), providing a measure of how the cell lines adapt from high to low mechanical load.

[0101] Fourthly, the inventors have found that the nucleus of metastatic cells expands more easily, while the nucleus of a healthy, non-tumorigenic cell does not. Highly metastatic and triple-negative cells harbour softer nuclei than non-tumorigenic cells; MW treatment enlarges the nuclei of these metastatic cells, while the nuclei of non- tumorigenic cells are resilient to MW-induced nuclear shape changes.

[0102] Fifthly, the inventors have identified that MW-induced cell viability, relative to cell density, can differentiate between differentiate between metastatic, non-metastatic cells, and non-tumorigenic cells, since MW treatment affected cell viability of each these types of cell line tested.

[0103] It will be appreciated that (i) uptake of external substances into cells; (ii) MW-induced organelle status and / or MW-induced cell viability; (iii) MW-induced nuclear expansion; and (iv) MW-induced cell viability relative to cell density are factors which may be used to distinguish a healthy, non-tumorigenic cell from a cancer cell.

[0104] It will also be appreciated that (i) Responsiveness Factor (RF) ratio and (ii) MW- induced cell viability relative to cell density are factors which may be used to distinguish a metastatic cancer cell from a non-metastatic cancer cell.

[0105] As such, based on these observations, the inventors also envisage that the method of the first aspect and / or apparatus of the second aspect can be used for diagnostic or prognostic purposes, as well as for determining the likelihood of a cell to metastasize.

[0106] Accordingly, in a third aspect of the invention, there is provided a method of diagnosing or prognosing cancer in a subject, the method comprising, either: (A)

[0107] (i) contacting at least one biological test cell obtained from a test subject with at least one exogenous molecule;

[0108] (ii) contacting at least one biological non-cancerous control cell with at least one exogenous molecule;

[0109] (iii) exerting force on the at least one biological test cell and the at least one biological control cell, to thereby introduce the at least one exogenous molecule into the at least one biological test cell and the at least one biological control cell;

[0110] (iv) (a) detecting the amount of the at least one molecule which has been introduced into the at least one biological test cell and the at least one biological control cell, (b) determining the status of at least one organelle in the at least one biological test cell and the at least one biological control cell, and / or (c) determining the cell viability of the at least one biological test cell and the at least one biological control cell; and (v) (a) comparing the amount of the at least one molecule which has been introduced into the at least one biological test cell to the amount of the at least one molecule which has been introduced into a control cell, (b) comparing the status of the at least one organelle in the at least one biological test cell to the status of the at least one organelle in the at least one biological control cell, and / or (c) comparing the cell viability of the at least one biological test cell to the cell viability of the at least one biological control cell, to thereby diagnose or prognose cancer in the subject; and / or

[0111] (B)

[0112] (i) (a) determining the size and / or shape of the nuclei of at least one biological test cell and at least one biological control cell and / or (b) determining the cell viability of at least one biological test cell and at least one biological control cell;

[0113] (ii) exerting force on the at least one biological test cell and the at least one biological control cell;

[0114] (iii) subsequent to exerting force on the cells, (a) determining the size of the nuclei of the at least one biological test cell and the at least one biological control cell, and / or (b) determining the cell viability of the at least one biological test cell and the at least one biological control cell; and

[0115] (iv) (a) comparing the sizes and / or shapes of the nuclei of the at least one biological test cell and the at least one biological control cell prior to and subsequent to exerting force on the cells; and / or (b) comparing the cell viabilities of the at least one biological test cell and the at least one biological control cell prior to and subsequent to exerting force on the cells, to thereby diagnose or prognose cancer in the subject.

[0116] Steps (i) and (ii) of method part (A) may be as defined with respect to the method of the first aspect.

[0117] In a fourth aspect of the invention, there is provided a method of determining the likelihood of a cell to metastasize, the method comprising, either:

[0118] (A)

[0119] (i) contacting at least one biological test cell obtained from a test subject with at least one exogenous molecule;

[0120] (ii) contacting at least one biological non-cancerous control cell with at least one exogenous molecule;

[0121] (iii) exerting force on the at least one biological test cell and the at least one biological control cell, to thereby introduce the at least one exogenous molecule into the at least one biological test cell and the at least one biological control cell;

[0122] (iv) detecting the amount of the at least one molecule which has been introduced into the at least one biological test cell and the at least one biological control cell; and

[0123] (v) comparing the amount of the at least one molecule which has been introduced into the at least one biological test cell to the amount of the at least one molecule which has been introduced into a control cell, to thereby determine the likelihood of a cell to metastasize; and / or

[0124] (B)

[0125] (i) (a) determining the Responsiveness Factor (RF) ratio of at least one biological test cell and at least one biological control cell and / or (b) determining the cell viability of at least one biological test cell and at least one biological control cell;

[0126] (ii) exerting force on the at least one biological test cell and the at least one biological control cell;

[0127] (iii) subsequent to exerting force on the cells, (a) determining the Responsiveness Factor (RF) ratio of the at least one biological test cell and the at least one biological control cell, and / or (b) determining the cell viability of the at least one biological test cell and the at least one biological control cell; and

[0128] (iv) (a) comparing the Responsiveness Factor (RF) ratios of the at least one biological test cell and the at least one biological control cell prior to and subsequent to exerting force on the cells; and / or (b) comparing the cell viabilities of the at least one biological test cell and the at least one biological control cell prior to and subsequent to exerting force on the cells, to thereby determine the likelihood of a cell to metastasize.

[0129] Steps (i) and (ii) may be as defined with respect to the method of the first aspect.

[0130] The methods of the third or fourth aspect may be performed in vivo, in vitro, or ex vivo.

[0131] The inventors have exemplified the delivery of exogenous molecules into cells, which can be demonstrated by a signal in the cell. For example, the inventors have shown that successful delivery of GFP-encoding bare DNA and mRNA into cells may be demonstrated by a green signal in cells which were processed for immunofluorescence microscopy, indicating GFP protein expression. In one embodiment, therefore, the at least one molecule may result in a signal in the cell into which the at least one molecule has been introduced.

[0132] In one embodiment, the at least one molecule may be selected from a list of molecules consisting of: fluorescent protein, fluorescent protein-encoding DNA, fluorescent-encoding mRNA, fluorescent dye and conjugate, luciferase protein, luciferase-encoding DNA, luciferase-encoding mRNA, luminescent protein, chromogenic molecule, siRNA, chemical probe, nanoparticle (e.g., nanoparticle with optical properties), fluorescently-tagged antibody, labelled peptide, chromobody, labelled protein, quantum dot, and near-infrared dye. Typically, the at least one molecule is GFP protein, GFP-encoding DNA, or GFP-encoding mRNA.

[0133] In one embodiment, analysing the amount of the at least one molecule which has been introduced into the at least one mammalian cell may comprise detecting the at least one molecule in the at least one cell.

[0134] In one embodiment, detecting the at least one molecule in the at least one cell may comprise a method of detection selected from the list of methods of detection consisting of: fluorescence microscopy, immunofluorescence, bioluminescence assay, flow cytometry, quantitative PCR, western blotting, northern blotting, fluorometric assay, radiolabelling, electron microscopy, mass spectrometry, positron emission tomography, magnetic resonance imaging, computer tomography, and single-photon emission computed tomography. Typically, detecting the at least one molecule in the at least one cell comprises immunofluorescence.

[0135] In one embodiment, the control may comprise at least one healthy or normal cell. In one embodiment, the healthy or normal cell may be from a cell line. In another embodiment, the control cell may be obtained from a healthy subject. In one embodiment, the control may comprise the same number of healthy or normal cells as the at least one biological cell obtained from the subject.

[0136] In one embodiment, comparing the amount of the at least one molecule which has been introduced into the at least one mammalian cell to a control may comprise identifying whether the same amount or a greater amount of the at least one molecule has been introduced into the at least one mammalian cell compared to the control. Typically, if a greater amount of the at least one molecule has been introduced into the at least one biological test cell than the at least one biological control cell, a diagnosis or prognosis of cancer in the subject is made. Conversely, if a lower amount of the at least one molecule is introduced into the test cell compared to the control cell, then cancer may not be diagnosed or prognosed.

[0137] In one embodiment, the amount of the at least one molecule that has been introduced into the at least one biological test cell may be determined as the same as or greater than the amount introduced into the at least one biological control cell using statistical analysis.

[0138] In one embodiment, the organelle may be selected from a group of organelles consisting of: nucleus, mitochondrion, centrosome, cilium, cell membrane, smooth endoplasmic reticulum, rough endoplasmic reticulum, golgi apparatus, peroxisome, lysosome, transport vesicle, ribosome, and cytoskeleton. Typically, however, the organelle is a nucleus.

[0139] In one embodiment, determining the status of the at least one organelle may comprise determining the size, shape, and / or function of the at least one organelle. Furthermore, a cancer cell often has more than two centrosomes whereas a healthy cell has only two.

[0140] In one embodiment, determining the size, shape, and / or function of the at least one organelle may comprise immunofluorescence examination, electron microscopy, confocal microscopy, live cell imaging, Western blotting, mitochondrial membrane potential assay, electron paramagnetic resonance, metabolomics, proteomics, gene expression profiling (e.g. RNA-seq), or flow cytometry of the at least one biological test cell and the at least one biological control cell. Typically, however, determining the size, shape, and / or function of the at least one organelle comprises immunofluorescence examination of the at least one biological test cell and the at least one biological control cell.

[0141] In another embodiment, determining the size, shape, and / or function of the at least one organelle may comprise statistical analysis of the results of the immunofluorescence examination of the at least one biological test cell and the at least one biological control cell.

[0142] In one embodiment, the method comprises identifying whether the status of the at least one organelle in the at least one biological test cell is different to the status of the at least one organelle in the at least one biological control cell. Typically, if the status of the at least one organelle in the at least one biological test cell is different to the status of the at least one organelle in the at least one biological control cell, a diagnosis or prognosis of cancer in the subject is made.

[0143] In one embodiment, the status of the at least one organelle in the at least one biological test cell may be identified as different to the status of the at least one organelle in the at least one biological control cell using statistical analysis.

[0144] In one embodiment, determining the cell viability of the at least one biological test cell and the at least one biological control cell may comprise examination of the metabolic activity of and / or performing a live / dead cell counting assay on the at least one biological test cell and the at least one biological control cell. Suitable cell viability methods may comprise trypan blue exclusion assay, flow cytometry, microscopy or colony formation assay-cell growth studies. Typically, however, determining the cell viability of the at least one biological test cell and the at least one biological control cell comprises performing a MTT assay on the at least one biological test cell and the at least one biological control cell.

[0145] Typically, the cell viability of the at least one biological test cell and the at least one biological control cell is determined subsequent to introducing a cytotoxic molecule into the at least one biological test cell and the at least one biological control cell.

[0146] In one embodiment, the method comprises identifying whether the cell viability of the at least one biological test cell is equal to or less than the cell viability of the at least one biological control cell. Typically, if the cell viability of the at least one biological test cell is less than the cell viability of the at least one biological control cell, a diagnosis or prognosis of cancer in the subject is made.

[0147] In one embodiment, the cell viability of the at least one biological test cell may be identified as equal to or less than the cell viability of the at least one biological control cell using statistical analysis.

[0148] In one embodiment, with regards to the third aspect, if more of the at least one molecule has been introduced into the at least one biological cell than the control, a diagnosis of cancer in the subject is made. In one embodiment, with regards to the fourth aspect, if more of the at least one molecule has been introduced into the at least one biological cell than the control, the greater the likelihood that the at least one biological cell will metastasize.

[0149] In one embodiment, comparing the amount of the at least one molecule which has been introduced into the at least one biological test cell to the amount of the at least one molecule which has been introduced into a control cell may comprise comparing the amounts of the at least one molecule that have been introduced into the at least one biological test cell and the control cell to a mechanofection standard.

[0150] In one embodiment, the mechanofection standard may comprise a set of molecules and corresponding mechanofection values for said molecules. In one embodiment, the mechanofection values may comprise a number and / or percentage, and / or a range of numbers and / or percentages, which represents a known amount and / or amounts of the at least one molecule which is introduced into a control (i.e. healthy) cell.

[0151] In one embodiment, comparing the amounts of the at least one molecule that have been introduced into the at least one biological test cell and the control cell to a mechanofection standard may comprise deriving a mechanofection score for the at least one biological test cell.

[0152] In one embodiment, the mechanofection score may be determined by the difference between the amount of the at least one molecule that has been introduced into the at least one biological test cell and the mechanofection standard for the at least one molecule.

[0153] It will be appreciated that the amount of the at least one molecule that has been introduced into the at least one control cell should be within the number and / or percentage, and / or range of numbers and / or percentages, of the mechanofection standard if such a standard for the at least one molecule exists.

[0154] In one embodiment, the difference between the amount of the at least one molecule that has been introduced into the at least one biological test cell and the mechanofection standard for the at least one molecule may be determined by statistical analysis.

[0155] In one embodiment, if the difference between the amount of the at least one molecule that has been introduced into the at least one biological test cell and the mechanofection standard for the at least one molecule is statistically significant, there is a high likelihood that the cell will metastasize. In another embodiment, the greater the statistically significant difference between the amount of the at least one molecule that has been introduced into the at least one biological test cell and the mechanofection standard for the at least one molecule, the higher the mechanofection score and / or the greater the likelihood that the cell will metastasize.

[0156] In one embodiment, determining the size and / or shape of the nuclei may comprise immunofluorescence examination, electron microscopy, confocal microscopy, live cell imaging, Western blotting, mitochondrial membrane potential assay, electron paramagnetic resonance, metabolomics, proteomics, gene expression profiling (e.g. RNA-seq), or flow cytometry of the at least one biological test cell and the at least one biological control cell. Typically, however, determining the size and / or shape of the nuclei comprises immunofluorescence examination of the at least one biological test cell and the at least one biological control cell.

[0157] In another embodiment, determining the size and / or shape of the nuclei may comprise statistical analysis of the results of the immunofluorescence examination of the at least one biological test cell and the at least one biological control cell.

[0158] In one embodiment, the method comprises identifying whether the size and / or shape of the nucleus in the at least one biological test cell is different to the size and / or shape of the at least one organelle in the at least one biological control cell. Typically, if the size and / or shape of the nucleus of the at least one biological test cell is different to the size and / or shape of the nucleus of the nucleus of the at least one biological control cell, a diagnosis or prognosis of cancer in the subject is made.

[0159] In one embodiment, the size and / or shape of the nucleus of the at least one biological test cell may be identified as different to the size and / or shape of the nucleus of the at least one biological control cell using statistical analysis.

[0160] The inventors determined cell viability relative to cell density using an MTT assay. It will be appreciated, however, that other methods of determining cell viability relative to cell density may be used, such as LDH (lactate dehydrogenase enzyme) presence in media.

[0161] In one embodiment, determining the cell viability of the at least one biological test cell and / or the at least one biological control cell may comprise determining the cell viability of the at least one biological test cell and / or the at least one biological control cell relative to cell density.

[0162] In one embodiment, determining the cell viability of the at least one biological test cell and the at least one biological control cell may comprise examination of the metabolic activity of and / or performing a live / dead cell counting assay on the at least one biological test cell and the at least one biological control cell. Suitable cell viability methods may comprise trypan blue exclusion assay, flow cytometry, microscopy, colony formation assay-cell growth studies, or detecting LDH (lactate dehydrogenase enzyme) presence in media in which cells are grown. Typically, however, determining the cell viability of the at least one biological test cell and the at least one biological control cell comprises performing a MTT assay on the at least one biological test cell and the at least one biological control cell.

[0163] Typically, the cell viability of the at least one biological test cell and the at least one biological control cell is determined prior to and subsequent to exerting force on the at least one biological test cell and the at least one biological control cell.

[0164] In one embodiment, the method comprises identifying whether the cell viability of the at least one biological test cell is equal to or less than the cell viability of the at least one biological control cell. Typically, if the cell viability of the at least one biological test cell is less than the cell viability of the at least one biological control cell, a diagnosis or prognosis of cancer in the subject is made. Typically, if the cell viability of the at least one biological test cell is less than the cell viability of the at least one biological control cell, the greater the likelihood of the biological test cell to metastasize.

[0165] In one embodiment, the cell viability of the at least one biological test cell may be identified as equal to or less than the cell viability of the at least one biological control cell using statistical analysis.

[0166] It will be appreciated that the Responsiveness Factor (RF) ratio is defined as the ratio of the perinuclear F-actin ring response (%) at a first MW treatment to the response at a second MW treatment (e.g., 10 g (MW-10) to the response at 0.5 g (MW-0.5) (RF = MW-10 / MW-0.5)).

[0167] It will also be appreciated that the perinuclear F-actin ring response (5) is defined as the degree of the presence of F-actin rings around the nucleus, thus providing a measure of how the cell lines adapt from low to high mechanical load. The F-actin cytoskeleton may be visualised using FITC-conjugated phalloidin, while the nuclei are counterstained with DAPI.

[0168] In one embodiment, determining the Responsiveness Factor (RF) ratio of the at least one biological test cell and the at least one biological control cell may comprise determining the presence and / or amount of F-actin rings around the nuclei of the cells. Suitable methods for determining the presence and / or amount of F-actin rings around the nuclei of the cells may comprise staining, typically with FITC-conjugated phalloidin and / or DAPI, most typically with FITC-conjugated phalloidin and DAPI.

[0169] Typically, the Responsiveness Factor (RF) ratio of the at least one biological test cell and the at least one biological control cell is determined prior to and subsequent to exerting force on the at least one biological test cell and the at least one biological control cell.

[0170] In one embodiment, the method comprises identifying whether the Responsiveness Factor (RF) ratio of the at least one biological test cell is equal to or greater than the cell viability of the at least one biological control cell. Typically, if the Responsiveness Factor (RF) ratio of the at least one biological test cell is greater than the cell viability of the at least one biological control cell, the greater the likelihood of the biological test cell to metastasize. Typically, if the Responsiveness Factor (RF) ratio of the at least one biological test cell is high, the greater the likelihood of the biological test cell to metastasize. Typically, if the Responsiveness Factor (RF) ratio of the at least one biological test cell is low, the lesser the likelihood of the biological test cell to metastasize.

[0171] In one embodiment, a high RF ratio may comprise a RF ratio of >5.0, >10.0, or >12.0. Typically, a high RF ratio comprises a RF ratio of >12.4. In one embodiment, an intermediate RF ratio may comprise a RF ratio of between >1.0 and <20.0, between >1.5 and <15.0, or between >1.8 and <13. Typically, an intermediate RF ratio comprises a RF ratio of between >1.9 and <12.4. In one embodiment, a low RF ratio may comprise a RF ratio of <10.0, <5.0, or <2.0. Typically, a low RF ratio comprises a RF ratio of <1.9.

[0172] In one embodiment, a high RF ratio may indicate that a cell is cancerous metastatic.

[0173] In one embodiment, an intermediate RF ratio may indicate that a cell is cancerous non-metastatic. In one embodiment, a low RF ratio may indicate that a cell is normal, or non-cancerous.

[0174] In one embodiment, the Responsiveness Factor (RF) ratio of the at least one biological test cell may be identified as equal to, greater than, or less than the Responsiveness Factor (RF) ratio of the at least one biological control cell using statistical analysis.

[0175] As discussed in Example 25, the MCF10A cell line (non-tumorigenic) showed high sensitivity at low weights, peaking around 0.5-1 g, however, the response diminished at higher weights. In contrast, MD-AMB-231 cell line (highly metastatic tumorigenic) showed minimal response at very low weights, but exhibited a strong, sustained increase at higher weights. Thus, MCF10A behaviour suggests a switch-like response, compared to the gradual adaptation seen in MDA-MB-231. These results highlight differences in mechanofection, with cancer cells (MDA-MB-231) showing resilience and altered force adaptation. Further, HCC38 (non-metastatic basal-like) cells displayed a very strong response at MW-1, but this response was not sustained. Fluctuating responses were noted at higher weights, but never reaching the high levels seen in MDA-MB-231 cells. In addition, HCC38 cells exhibit more "burst-like" sensitivity rather than a smooth, dose-response.

[0176] In one embodiment, therefore, exerting force on the at least one biological test cell and the at least one biological control cell may comprise exerting force for one or more periods. The one or more periods may comprise exerting varying amounts of force on the cells. The force may be defined as under the first aspect.

[0177] The inventors envisage that the diagnostic and prognostic methods of the third and fourth aspects may be used as standalone approaches, or in combination with a molecular biomarker-based approach. Such a biomarker-based approach may utilise detection molecules (e.g., antibodies) to detect cancer biomarker presence or absence in a sample obtained from a subject, and / or organisation (e.g., nuclear / cell architecture). The combination of these approaches may increase specificity and sensitivity of the diagnostic and prognostic methods disclosed herein.

[0178] In one embodiment, therefore, the methods of the third or fourth aspect may be combined with a biomarker-based and / or cell and / or nuclear organisation-based diagnostic or prognostic assay. As discussed in Example 13, the inventors have shown that mechanical stimulation sensitises metastatic cancer cells, which are doxorubicin-resistant, to doxorubicin chemotherapy treatment. As such, the methods of the invention provide advantageous synergistic effects (i.e., synthetic lethality) if mechanical stimulation and chemotherapy is combined. Furthermore, the inventors envisage that mechanical stimulation may advantageously enable the usage of lower cytotoxic doses of anticancer drugs, since more of the external drug can enter a cancer cell if mechanically stimulated. The inventors have also found that the cytotoxic effects of mechanical stimulation (i.e., compressive forces) are pronounced in dense cell cultures, as shown in Figure 9.

[0179] Thus, in a fifth aspect of the invention, there is provided a method of:

[0180] (i) sensitising, to an anti-cancer drug, a subject that is suffering from cancer that is insensitive to the anti-cancer drug; or

[0181] (ii) enhancing the sensitivity, to an anti-cancer drug, a subject that is suffering from cancer that is sensitive to the anti-cancer drug, the method comprising exerting force on an area of the subject comprising a cancerous tumour.

[0182] In one embodiment, the method of the fifth aspect further comprises administering, to the subject, the anti-cancer drug.

[0183] Typically, the anti-cancer drug comprises doxorubicin. Typically, the subject is insensitive to doxorubicin.

[0184] In one embodiment, the at least one biological cell may be obtained from a resected tissue or biopsy sample from the subject. In one embodiment, where the at least one biological cell is obtained from a resected tissue or biopsy sample from the subject, the methods of the third or fourth aspect may be used to inform cancer treatment.

[0185] In one embodiment, it may be determined that the subject has been sensitised to the anti-cancer drug, or that the sensitivity of the subject to the anti-cancer drug has been enhanced, by determining the response of the cancerous tumour to the treatment. In one embodiment, the response of the cancerous tumour to the treatment may be determined by:

[0186] 1) a radiologic response, whereby tumour size is measured with CT / MRI, and a shrinkage or disappearance of the tumour, or increased rate of shrinkage of the tumour, indicates sensitisation to the drug or enhanced sensitivity to the drug, or whereby a PET scan is used to measure metabolic activity, whereby decreased metabolic activity in the area in which the tumour is disposed indicates sensitisation to the drug or enhanced sensitivity to the drug;

[0187] 2) tumour biomarkers, whereby a decrease in the level of one or more tumour biomarker in a sample obtained from the subject indicates sensitisation to the drug or enhanced sensitivity to the drug; and / or

[0188] 3) a pathologic response, whereby a biopsy of the area in which the tumour is disposed is taken; if fewer viable tumour cells or no tumour cells are present, this indicates sensitisation to the drug or enhanced sensitivity to the drug.

[0189] Furthermore, the inventors envisage therapeutic and prognostic approaches built on mammography, notably without the X-ray imaging modality. Instead, the procedure would be performed with Adaptive kPa-controlled Cyclic Compression (ACC), which may comprise stepwise pressure plateaus, followed by brief relaxation intervals, followed by final target compression, with imaging at plateaus. Imaging would be based on the mechanofection concept.

[0190] The inventors envisage that therapy for tumours that are positive (i.e., at the histology and imaging level) may comprise chemotherapy combined with Adaptive kPa-controlled Cyclic Compression (ACC), based on the inventors' findings disclosed herein that in vitro mechanical stimulation can overcome drug resistance, restore chemosensitivity, and enable cytotoxic effects at a lower drug concentration.

[0191] In one embodiment, the method of the fifth aspect may comprise administering, to the subject, the anti-cancer drug prior to exerting force on the area of the subject comprising the cancerous tumour. In one embodiment, the method of the fifth aspect may comprise administering, to the subject, the anti-cancer drug subsequent to exerting force on the area of the subject comprising the cancerous tumour. In one embodiment, the method of the fifth aspect may comprise administering, to the subject, the anti-cancer drug concurrent to exerting force on the area of the subject comprising the cancerous tumour.

[0192] Thus, in a sixth aspect of the invention, there is provided a method of treating a subject suffering from cancer, the method comprising:

[0193] (i) exerting force on an area of the subject comprising a cancerous tumour; and

[0194] (ii) administering, to the subject, an anti-cancer drug. Typically, the cancer is breast cancer. Typically, the area of the subject comprising a cancerous tumour comprises breast tissue. Typically, the force is exerted by a mammography device. Typically, the force is exerted on breast tissue of the subject. Typically, the force is exerted using Adaptive kPa-controlled Cyclic Compression (ACC). It will be appreciated that Adaptive kPa-controlled cyclic compression (ACC) is a technique that allows for the control of mechanical stress in a range of kPa values.

[0195] In one embodiment, force may be exerted on the area of the subject comprising a cancerous tumour for one or more periods. Typically, the one or more periods may be separated by one or more intervals, during which no force is exerted on the area of the subject comprising a cancerous tumour.

[0196] In one embodiment, the force exerted on the area of the subject comprising a cancerous tumour may vary between the one or more periods. In one embodiment, the force exerted on the area of the subject comprising a cancerous tumour may increase with each subsequent period. Typically, the force exerted on the area of the subject comprising a cancerous tumour increases with each subsequent period to thereby reach a final, target force. In one embodiment, the force exerted on the area of the subject comprising a cancerous tumour may decrease with each subsequent period.

[0197] Advantageously, in applying pressure locally to a tumour region, regional enhancement of anti-cancer drug uptake is enabled, without increasing systemic exposure.

[0198] In one embodiment, exerting force on the area of the subject comprising the cancerous tumour causes an increase in the uptake of the anti-cancer drug into the cancerous tumour. In one embodiment, the anti-cancer drug may be administered to the subject at a decreased dosage when force is exerted on the area of the subject comprising the cancerous tumour. In one embodiment, the method may comprise increasing cellular uptake of the anti-cancer drug in the area of the subject comprising the cancerous tumour, and / or decreasing cellular uptake of the anti-cancer drug in an area of the subject not comprising the cancerous tumour.

[0199] In one embodiment, the method of the sixth aspect may comprise administering, to the subject, the anti-cancer drug prior to exerting force on the area of the subject comprising the cancerous tumour. In one embodiment, the method of the sixth aspect may comprise administering, to the subject, the anti-cancer drug subsequent to exerting force on the area of the subject comprising the cancerous tumour. In one embodiment, the method of the sixth aspect may comprise administering, to the subject, the anti-cancer drug concurrent to exerting force on the area of the subject comprising the cancerous tumour.

[0200] Advantageously, the method of the sixth aspect enables effective treatment at reduced drug doses, thus minimising off-target toxicity to healthy tissue. In one embodiment, therefore, the method of the sixth aspect may reduce or eliminate off- target toxicity to healthy tissue.

[0201] The inventors also envisage, based on the data discussed in Example 13, that their mechanofection methodology can overcome drug resistance. In one embodiment, therefore, the cancer may be drug-resistant. In one embodiment, the method may comprise administering, to the subject, an anti-cancer drug which the cancer is resistant to. In one embodiment, the method may comprise sensitising the cancer to the anti-cancer drug.

[0202] In one embodiment, the method of the sixth aspect may be combined with one or more existing cancer treatment.

[0203] The inventors also envisage that uptake levels of a molecule, such as imaging dye, measured under compression (i.e., following exerting force) may be used to adjust drug dosing or treatment schedules in subsequent drug administrations. In one embodiment, therefore, the method may comprise administering, to the subject, an imaging substance, such as an imaging dye, and exerting force on the area of the subject comprising the cancerous tumour. In one embodiment, the method may comprise detecting the amount of the imagining substance which has been introduced into the area of the subject comprising the cancerous tumour, before and / or after administering, to the subject, the anti-cancer drug.

[0204] In one embodiment, subsequent to detecting the amount of the imagining substance which has been introduced into the area of the subject comprising the cancerous tumour, the method may comprise comparing the amount of the imagine substance that is introduced into the area of the subject comprising the cancerous tumour before and after administering, to the subject, the anti-cancer drug. In one embodiment, where the amount of the imaging substance that is introduced into the area of the subject comprising the cancerous tumour after administering, to the subject, the anticancer drug is less than the amount of the imaging substance that is introduced into the area of the subject comprising the cancerous tumour before administering, to the subject, the anti-cancer drug, the method may comprise reducing the dose of the anticancer drug or reducing the frequency of the administration of the anti-cancer drug. In one embodiment, where the amount of the imaging substance that is introduced into the area of the subject comprising the cancerous tumour after administering, to the subject, the anti-cancer drug is greater than the amount of the imaging substance that is introduced into the area of the subject comprising the cancerous tumour before administering, to the subject, the anti-cancer drug, the method may comprise increasing the dose of the anti-cancer drug or increasing the frequency of the administration of the anti-cancer drug.

[0205] It will be appreciated that, where the amount of the imaging substance that is introduced into the area of the subject comprising the cancerous tumour after administering, to the subject, the anti-cancer drug is less than the amount of the imaging substance that is introduced into the area of the subject comprising the cancerous tumour before administering, to the subject, the anti-cancer drug, this indicates that the cancerous tumour comprises fewer cancerous cells, since uptake of the imaging substance is reduced after treatment with force. It will also be appreciated that, where the amount of the imaging substance that is introduced into the area of the subject comprising the cancerous tumour after administering, to the subject, the anti-cancer drug is greater than the amount of the imaging substance that is introduced into the area of the subject comprising the cancerous tumour before administering, to the subject, the anti-cancer drug, this indicates that the cancerous tumour comprises more cancerous cells, since uptake of the imaging substance is increased.

[0206] All of the features described herein (including any accompanying claims, abstracts, and drawings), and / or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some features and / or steps are mutually exclusive.

[0207] For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying Figures, in which:-

[0208] Figure 1 is a schematic summarising one embodiment of microweight-induced delivery of a molecule into a biological cell using a process referred to herein as "mechanofection". On day 1, 20,000 cells are seeded and adhered onto a glass coverslip (adherence step) and allowed to grow for 24 hours in a cell incubator (37°C, 5% CO2) (pre-treatment incubation step). The next day (day 2), the coverslip is flipped over (cells face downwards) (remove cells from media and invert surface) and placed either alone (MW-0) or with a microweight (MW-10; 10 gram) on top of a 25 pl droplet on a parafilm sheet (first incubation period). The droplet is either serum-free medium or a DPBS (Dulbecco's Phosphate-Buffered Saline) solution containing a biological molecule (e.g. nucleic acid, or protein) or a chemical agent (e.g. TRITC- phalloidin). After 10 seconds, the coverslip is inverted (cells facing upwards) and placed back into the 24-well tissue culture plate and allowed to grow for another 24 hours (post treatment incubation step). On day 3, the cells are processed for further analysis. The insets are higher magnifications of the dashed boxed areas.

[0209] Figure 2 is an analysis of MDA-MB-231 cells which have been incubated for 10 seconds (sec) with a GFP (green fluorescent protein)-encoding plasmid, which was diluted either in media (panels A-D) or in DPBS (E) in the absence (MW-0) and presence of microweights (MW). The panels (A, B) present a fluorescence microscopy analysis of cells stained with phalloidin (denotes F-actin) and DAPI (nuclear stain). This analysis reveals the expression of GFP (green channel) exclusively in cells that have been treated with a 10 gram microweight (MW-10). Scale bar 20 pm. (C) Statistical analysis of GFP-expressing cells without (-) and with (+) 5 and 10 gram microweight (MW-5, MW-10) incubations (10 sec). Either single (lx) or repeated rounds (2x) of MW incubation were performed. (D) Statistical analysis of GFP- expressing cells without (-) and with (+) 20 gram microweight (MW-20) incubations (10 sec). Either single (lx) or repeated rounds (2x) of MW incubation were performed. (E) Statistical analysis of GFP-expressing cells without (-) and with (+) 10 gram microweight (MW-10) incubations (10 sec). The transfections were performed in DPBS. Single (lx) or repeated rounds (2x) of MW incubation are indicated. (C-E) The data represent the mean ±SEM (Standard Error of the Mean), with n = 3. Statistical significance, indicated by (*) was determined using a one-way ANOVA with a Dunnett's post-hoc test, comparing the data to the treatment where no MWs were used. Non-significant differences are denoted as "ns". Statistical significance levels are denoted as follows: ** p < 0.01 and **** p < 0.0001.

[0210] Figure 3 is an analysis of MCF10A cells, which have been incubated for 10 seconds (sec) with a DPBS solution containing 2 pg of GFP (green fluorescent protein) - encoding plasmid, in the absence (MW-0) and presence of microweights (MW). Panel (A) presents a fluorescence microscopy analysis of cells stained with phalloidin (denotes F-actin) and DAPI (nuclear stain). This analysis reveals the expression of GFP (green channel) in cells that have been treated with a 10 gram microweight (MW-10) for 10 seconds. Scale bar 20 |jm. (B) Statistical analysis of GFP-expressing cells without (MW-0) and with either 1 (MW-1) and 10 gram microweights (MW-10) incubations (10 sec). The data represent the mean ±SEM (Standard Error of the Mean), with n = 3 and are the outcome of a single (lx) MW incubation. Statistical significance, indicated by (*, p < 0.05) was determined using a one-way ANOVA with a Dunnett's post-hoc test, comparing the data to the treatment where no MWs were used. Non-significant differences are denoted as "ns".

[0211] Figure 4 is a statistical analysis of a MW-10 single mechanofection (lx 10 seconds) of GFP-encoding plasmids using DPBS as a mechanofection buffer and either MCF10A or MDA-MB-231 adherent cells. The error bars represent the Standard Error of the Mean (SEM). Statistical significance (*** p < 0.001) was assessed using a Student's unpaired t-test.

[0212] Figure 5 is an analysis of MDA-MB-231 cells which have been incubated for 10 seconds (sec) with 10 pg GFP (green fluorescent protein) mRNA 5-Methyoxyuridine, which was diluted in DPBS in the absence (MW-0) and presence of 10 gram microweights (MW-10), relative to conventional Lipofectamine-mediated GFP mRNA transfection. The panels (A) present a fluorescence microscopy analysis of cells stained with anti-GFP (red channel) and DAPI (nuclear stain). The green channel indicates GFP autofluorescence and therefore the expression of GFP protein within cells. In panel (B), the absence of GFP-expressing cells is evident when no microweights (MW-0) are used. In panel (C) the expression of GFP (green channel; autofluorescence) is evident when the cells are treated with a 10 gram microweight (MW-10) and then subjected to anti-GFP (red channel) immunofluorescence. Scale bars for panels (A-C) is 40 pm. (D) Statistical analysis of GFP-expressing cells without (MW-0) and with 10 gram microweights (MW-10) single incubations (lx 10 sec), relative to conventional lipofectamine-mediated GFP mRNA transfection. The data represent the mean ±SEM (Standard Error of the Mean). Statistical significance, indicated by (*) was determined using a one-way ANOVA with a Dunnett's post-hoc test, comparing the data to the treatment where no MWs (MW-0) were used.

[0213] Statistical significance levels are denoted as follows: * p < 0.05 and **** p < 0.0001.

[0214] Figure 6 is an analysis of MDA-MB-231 cells, which have been incubated for 10 seconds (sec) with a serum-free medium solution containing 3 pg of anti-Nesprin-2 polyclonal antibody (pAbKl), in the absence (mechanofection negative; MW-0) and presence of microweights (MW-10; mechanofection positive). Panel (A) is a conventional immunofluorescence examination using pAbKl (red channel) and DAPI (stains nuclei; blue channel) staining, on permeabilised cells. This positive control indicates the functionality of the pAbKl antibody reagent, which detects the nuclear envelope Nesprin-2 protein. Inset (red-channel only) is higher magnification of boxed area (dashed lines). (B) Immunofluorescence examination of cells without MW-induced mechanofection (MW-0) in the presence of exogenous pAbKl antibody. Inset (red- channel only) is higher magnification of boxed area (dashed lines). (C) Immunofluorescence examination of cells after MW-10-induced mechanofection in the presence of exogenous pAbKl antibody. Note the prominent pAbKl staining at the nucleus (arrowheads). Inset (red-channel only) is higher magnification of boxed area (dashed lines). Scale bar for panels (A-C) is 20 pm. (D) Statistical analysis of pAbKl positive cells without MWs (MW-0) and with MW-10 for single (lx) and double (2x) 10-second incubation steps. The data represent the mean ±SEM (Standard Error of the Mean). Statistical significance, indicated by (*) was determined using a one-way ANOVA with a Dunnett's post-hoc test, comparing the data to the treatment were no MWs (MW-0) were used. Non-significant differences are denoted as "ns". Statistical significance levels are denoted as follows: * p < 0.05 and **** p < 0.0001. (E) Statistical analysis of the impact on nuclear area of a double (2x) 10-second incubation with MW-10 relative to a control (MW-0) in the presence of pAbKl antibodies. The error bars represent the Standard Error of the Mean (SEM). Statistical significance (** p < 0.01) was assessed using a Student's unpaired t-test.

[0215] Figure 7 is an analysis of MDA-MB-231 cells, which have been incubated for 10 seconds (sec) with a serum-free medium solution containing 0.4 mg / ml TRITC- conjugated phalloidin (TRITC-phall), in the absence (mechanofection negative; MW-0) and presence of microweights (MW-10; mechanofection positive). Panels (A, A') are a conventional fluorescence microscopy examination using TRITC-phalloidin (red channel) and DAPI (stains nuclei; blue channel) staining on permeabilised cells. This positive control demonstrates that the TRITC-phalloidin conjugate is biologically active and binds specifically to filamentous actin (F-actin) structures. In panels (B, B') MDA- MB-231 cells without MW-induced mechanofection (MW-0) of TRITC-phalloidin were processed for conventional immunofluorescence analysis using DAPI staining (to visualise the nuclei; blue channel). Note the lack of TRITC-phalloidin (red channel) staining. In panel (C) MDA-MB-231 cells were subjected to MW-10 induced mechanofection of TRITC-phalloidin and then processed for immunofluorescence analysis using DAPI counterstain to reveal the nuclei (blue channel). Note that in contrast to panels (A, A') that some cells (denoted by asterisks) are either not positive or weakly stained for TRITC-phalloidin conjugate labelling. Scale bar for panel (C) is 20 pm and is identical for other micrographs (A-C')- (D) Statistical analysis of TRITC- phalloidin positive cells without MWs (MW-0) and with MW-10 for single (lx) and double (2x) 10-second incubation steps in the presence of exogenous TRITC-phalloidin solution. The data represent the mean ±SEM (Standard Error of the Mean). Statistical significance, indicated by (*) was determined using a one-way ANOVA with a Dunnett's post-hoc test, comparing the data to the treatment where no MWs (MW-0) were used. Non-significant differences are denoted as "ns". Statistical significance levels are denoted as follows: ** p < 0.01. (E) Statistical analysis of the impact on cell viability of a single (lx) and double (2x) 10-second incubation with MW-10 relative to a control (MW-0) in the presence and absence of TRITC-phalloidin solution using an MTT metabolic activity assay. The MTT assay was performed 24 hours after MW- treatment. The error bars represent the Standard Error of the Mean (SEM). Statistical significance (* p < 0.05) was assessed between groups (MW-0 versus MW-10) using a Student's unpaired t-test. Non-significant differences are denoted as "ns". (F) Statistical analysis of the impact on cell viability of a single 10-second incubation with MW-10 relative to a control (MW-0) in the presence and absence of TRITC-phalloidin solution using an MTT metabolic activity assay 48 hours after MW-treatment. The data represent the mean ±SEM (Standard Error of the Mean). Statistical significance, indicated by (*) was determined using a one-way ANOVA with a Dunnett's post-hoc test, comparing the data to the treatment where no MWs (MW-0) were used. Nonsignificant differences are denoted as "ns". Statistical significance levels are denoted as follows: *** p < 0.001 and **** p < 0.0001.

[0216] Figure 8 is an analysis of HCC38 cells, which have been incubated for 10 seconds (sec) with 10 pg GFP (green fluorescent protein) mRNA 5-Methyoxyuridine, which was diluted in DPBS in the absence (MW-0) and presence of 10 gram microweights (MW- 10), relative to conventional lipofectamine-mediated GFP mRNA transfection. The panels (A) present a fluorescence microscopy analysis of cells transfected with GFP mRNA using Lipofectamine. The cells were stained with anti-GFP (red channel) and DAPI (nuclear stain). The green channel indicates GFP autofluorescence and therefore the expression of GFP protein within cells. In panel (B), it is shown that a single 10- second (lx 10 sec) treatment involving the weight of the glass coverslip alone, to which the cells are attached, is sufficient to induce GFP protein expression, without the usage of microweights (MW-0). In panel (C), the results of a single 10-second (lx lOsec) mechanofection treatment involving 1 gram microweights (MW-1) is shown. Expression of GFP protein is indicated by autofluorescence (green channel) and positive anti-GFP antibody immunostaining (red channel). In panel (D), the results of a single 10-second (lx lOsec) mechanofection treatment involving 10 gram microweights (MW-10) is shown. Expression of GFP protein is indicated by autofluorescence (green channel) and positive anti-GFP antibody immunostaining (red channel). In panel (E), the results of a double 10-second (2x lOsec) mechanofection treatment involving 10 gram microweights (MW-10) is shown. Expression of GFP protein is indicated by autofluorescence (green channel) and positive anti-GFP antibody immunostaining (red channel). Scale bars for panels (A-E) is 40 pm. (F) Statistical analysis of GFP-expressing cells without (MW-0) and with microweights (MW-1 and MW-10) single (lx 10 sec) and double incubations (2x 10 sec), relative to conventional lipofectamine-mediated GFP mRNA transfection. The data represent the mean ±SEM (Standard Error of the Mean). Statistical significance, indicated by (*) was determined using a one-way ANOVA with a Dunnett's post-hoc test, comparing the MW-1 and MW-10 data to the treatment were no MWs (MW-0) were used. Statistical significance levels are denoted as follows: * p < 0.05. The statistical significance (**** p < 0.0001) between the Lipofectamine and MW-0 data was assessed using a Student's unpaired t-test.

[0217] Figure 9 is an analysis of the cell viability effects of doxorubicin and MW-treatment in doxorubicin-resistant MDA-MB-231 cells. Cell viability was determined by an MTT assay, which was performed 24 hours (i.e., day 2) after the MW-treatment (day 1). (A) Cell viability of a single (lx) MW-10 treatment lasting 10 seconds in the presence of 20 pg / mL doxorubicin. (B) Cell viability results after a double (2x) MW-10 treatment lasting 10 seconds each, in the presence of 20 pg / mL doxorubicin. (C) Cell viability results after a single (lx) MW-10 treatment lasting 10 seconds in the presence of 10 pg / mL doxorubicin. (D) Cell viability results after a single (lx) MW-10 treatment lasting 10 seconds in the presence of 2.5, 5.0, and 10 pg / mL doxorubicin. Original seeding cell density for experiments A-D is 20,000 cells (day 0). (E-F) Cell viability results after a single (lx) MW-1 (panel E) or MW-10 (F) treatment lasting 10 seconds in the presence of 10 pg / mL doxorubicin. Seeding cell density 100,000 (day 0; high confluency). The error bars in all graphs represent the Standard Error of the Mean (SEM). Statistical significance, indicated by (*) was determined using one-way ANOVA with a Dunnett's post-hoc test (A-C, E-F). Statistical significance for panel (D) was assessed using a Student's unpaired t-test. Statistical significance levels are denoted as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 and ****** p < 0.000001. Non-significant differences are denoted as "ns".

[0218] Figure 10 is an analysis of MDA-MB-231 cells incubated with microweights (MW) in the presence and absence of latrunculin (1 pg / mL), one day after seeding (day 1). (A, B) MW-treatment induces perinuclear F-actin rings. Immunofluorescence examination of F-actin architecture in MW-0 (control) and MW-20 treated cells (10-minute MW- incubation). The F-actin cytoskeleton is visualised using FITC-conjugated phalloidin (green channel), while the nuclei are counterstained with DAPI (blue channel). Panels A', B' in grayscale aid the detection of perinuclear F-actin (denoted with arrowheads). (C) Statistical analysis of perinuclear F-actin presence (%) in cells exposed to 0.5-, 1-, 2-, 5-, 10-, 20-, and 50-grams of MWs (10-minute incubation) relative to controls (MW-0) or cells. (D) Cell viability results for the various MW-treatments, as determined by MTT assay, which was performed 24 hours (i.e., day 2) after the MW- treatment (day 1). (E, F) Immunofluorescence analysis of latrunculin B (latB) treated cells without (MW-0) and with microweight (MW-20) treatment (10 minutes) using TRITC-conjugated phalloidin (red channel) and DAPI (blue channel). Latrunculin B is an inhibitor of actin polymerisation. Note the drastic reduction of phalloidin staining in both MW-0 and MW-20 treated cells. In contrast to MW-20 treated cells, MW-0 exposed cells exhibit several small nuclei (arrowheads). Note the absence of perinuclear F-actin in MW-20 and latrunculin B co-treated cells (panel F). Scale bar for panels A-F is 20 pm. (G) Statistical analysis of the effects of latrunculin B on perinuclear F-actin presence without (MW-0) and with microweights (MW-20). Note that the formation of perinuclear F-actin is abolished upon latrunculin B treatment. (H) Statistical analysis of the impact of MW-20 and latrunculin B treatment on nuclear area (left graph). The right graph displays a statistical analysis of the normalised nuclear area data. The error bars represent the Standard Error of the Mean (SEM). Statistical significance (* p < 0.05, **** p < 0.0001) was assessed using a Student's unpaired t-test. (I) Statistical analysis of the impact on cell viability of a single (lx) 10-minute incubation with a MW-10 relative to a control (MW-0) in the presence and absence of latrunculin B solution using an MTT metabolic activity assay. The error bars in all graphs represent the Standard Error of the Mean (SEM). Statistical significance, indicated by (*) was determined using one-way ANOVA with a Dunnett's post-hoc test. Statistical significance levels are denoted as follows: * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. Non-significant differences are denoted as "ns". Seeding density for examined conditions was 20,000 cells (day 0).

[0219] Figure 11 is a nuclear morphometric analysis of MDA-MB-231 cells incubated with microweights (MW), one day post-seeding (day 1). A single MW-10 treatment for 10 minutes on MDA-MB-231 cells leads to nuclear enlargement. (A) Immunofluorescence examination of MDA-MB-231 cells (20,000 seeded cells, day 0) treated with 10-gram (MW-10) and without microweights (MW-0) for 10 minutes using TRITC-conjugated phalloidin (red channel) and DAPI (blue). Grayscale images shown aid the observation of perinuclear F-actin structures (arrowheads) and nuclear shape differences. Note the presence of prominent F-actin rings for the MW-10 treatment. Scale bar is 20 pm. (B) Statistical evaluation of the effect of MW-0 (green bar) and MW-10 (blue bar) on nuclear area. The error bars represent the Standard Error of the Mean (SEM). Statistical significance (**** p < 0.0001) was assessed using a Student's unpaired t- test.

[0220] Figure 12 is a statistical analysis of perinuclear F-actin ring formation (%) in MDA- MB-231 cells incubated with MW-20 for 10 minutes, assessed either immediately after treatment or following a 20-minute recovery period (MW-removal). The error bars represent the Standard Error of the Mean (SEM). Statistical significance, indicated by (*) was determined using one-way ANOVA with a Dunnett's post-hoc test. Statistical significance levels are: **** p < 0.0001. Non-significant differences are denoted as "ns".

[0221] Figure 13 is an analysis of MDA-MB-231 cells treated with a single (lx) MW-10, for 5, 10, 30, 60, 120, 600, and 1200 seconds (sec); or two consecutive (2x), or ten (lOx) consecutive (lOx) MW-10 10 second treatments, one day after cell seeding (day 1). (A-H') Immunofluorescence examination of F-actin architecture in MW-0 (control) and MW-10 treated cells (20,000 seeded cells at day 0). The F-actin cytoskeleton is visualised using TRITC-conjugated phalloidin (red channel), while the nuclei are counterstained with DAPI (blue channel). Grayscale images (A'-H') indicated aid the detection of perinuclear F-actin (denoted by asterisks). (I) Statistical analysis of perinuclear F-actin ring formation (%) following a MW-10 treatment at the indicated time points. (J) Cell viability results for the MW-10 treatment, at the indicated time points, as determined by MTT assay. (K-L) Cell viability results for the one (lx), two (2x) (panel K), and ten (lOx) (panel L) consecutive MW-10 treatments (10 second duration), as determined by MTT assay (day 2). The error bars in all graphs represent the Standard Error of the Mean (SEM). Statistical significance, indicated by (*) was determined using one-way ANOVA with a Dunnett's post-hoc test. Statistical significance levels are denoted as follows: * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. Non-significant differences are denoted as "ns".

[0222] Figure 14 is an analysis of a variable number of MDA-MB-231 cells (seeding number at day 0: 1,000; 5,000; 10,000; 20,000; 50,000; 100,000; and 250,000 cells) subjected to a single MW-10 treatment for 10-seconds, one day after seeding (day 1). (A-G') Immunofluorescence examination of F-actin (TRITC-phalloidin, red channel) and nuclear architecture (DAPI, blue channel) in MW-0 (control) and MW-10 treated cells. Magnification is identical in all panels. Insets in A and A' are higher magnifications of dashed boxes, highlighting effects on nuclear size. (H) Graph depicting the (%) of perinuclear F-actin rings for the indicated MDA-MB-231 cell seeding densities (day 0) subjected to a single (lx) MW-10 treatment that lasted 10 seconds. (I) Statistical evaluation of the effects of a single MW-10 treatment (10 second duration) on nuclear area for the indicated cell densities. (J) Cell viability (MTT assay) effects of a single MW-10 treatment for 10 seconds, for the indicated cell seeding densities, one day post seeding (day 1). Error bars in all graphs represent the Standard Error of the Mean (SEM). Statistical significance, indicated by (*) was determined using one-way ANOVA with a Dunnett's post-hoc test. Statistical significance levels are denoted as follows: * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. Non-significant differences are denoted as "ns".

[0223] Figure 15 is an analysis of perinuclear F-actin ring formation (%) in MCF10A cells upon MW-treatment, relative to the indicated microweight values. Statistical analysis of the impact of single (lx) 0.5-, 1-, 2-, 5-, 10-, 20-, and 50-gram microweight treatment lasting 10 minutes on the perinuclear F-actin cytoskeleton (%) in MCF10A cells. The data represent the mean ±SEM (Standard Error of the Mean), and were collected one day post seeding. Statistical significance, indicated by (*) was determined using one-way ANOVA with a Dunnett's post-hoc test, comparing the data to the condition where no exogenous MWs (MW-0) were used (green bar). Statistical significance levels are denoted as follows: ** p < 0.01, *** p < 0.001 and **** p < 0.0001. Non-significant differences are denoted as "ns". Seeding density at day 0 is 20,000.

[0224] Figure 16 is an analysis of cell viability for MCF10A cells following MW-treatment, as determined by an MTT assay. Statistical analysis of the impact of single 0.5-, 1-, 2-, 5-, 10-, 20-, and 50-gram microweight treatment lasting 10 minutes on MCF10A cell viability (%) one day post seeding. The data represent the mean ±SEM (Standard Error of the Mean), and were collected one day post seeding. Statistical significance, indicated by (*) was determined using one-way ANOVA with a Dunnett's post-hoc test, comparing the data to the condition where no exogenous MWs (MW-0) were used (green bar). Statistical significance levels are denoted as follows: ** p < 0.01, *** p < 0.001 and **** p < 0.0001. Non-significant differences are denoted as "ns". Seeding density at day 0 is 20,000 cells.

[0225] Figure 17 is an analysis of MW-induced perinuclear F-actin ring formation at the periphery and the central area of MCF10A cell colonies. (A-B) Immunofluorescence examination of MCF10A cells (20,000 seeded cells at day 0) treated with 20-gram (MW-20) and without microweights (MW-0) for 10 minutes using TRITC-conjugated phalloidin (red channel) one day post seeding (day 1). Perinuclear F-actin structures are observable upon MW-20 treatment (panel B) but are absent in MW-0 treated cells (panel A). Perinuclear F-actin structures are indicated by blue and orange asterisks in peripheral (zone-1) and central (zone-2) colony cells. Scale bar 20 pm. (C) Statistical analysis of perinuclear F-actin rings (%) in MW-20 treated MCF10A cells within zone-1 and zone-2. The error bars represent the Standard Error of the Mean (SEM). Statistical significance (*** p < 0.001) was assessed using a Student's unpaired t-test.

[0226] Figure 18 is a nuclear morphometric analysis of MCF10A cells subjected to a single (lx) MW-10 treatment (duration 10 minutes) relative to cell density. Statistical evaluation of the effect of MW-0 (green bar; 5,000 cells seeded at day 0) and MW-10 (blue bars) treatments on the nuclear area of variable MCF10A cell densities (1,000; 5,000; 10,000; 20,000; 50,000 seeded cells at day 0). The error bars represent the Standard Error of the Mean (SEM). Data were collected one day post seeding. Statistical significance, indicated by (* p < 0.05) was determined using one-way ANOVA with a Dunnett's post-hoc test, comparing the data to the condition where no exogenous MWs (MW-0; 5,000 cells) were used (green bar). Non-significant differences are denoted as "ns".

[0227] Figure 19 is an analysis of cell viability of MCF10A cells subjected to a single MW-10 treatment (duration: 10 seconds) relative to cell density. MCF10A cell density affects the impact of MW-10 treatment on cell viability effects. Statistical analysis of the impact on cell viability following a single (lx) 10-second treatment with a MW-10 (blue bars) relative to the control (MW-0; green bar) using different seeding numbers of MCF10A cells (i.e., 1,000; 5,000; 10,000; 20,000; 50,000; 100,000, 250,000; day 0). Cell viability was measured using an MTT metabolic activity assay. The MTT assay was performed 24 hours (i.e., day 2) after the MW-treatment (day 1). The error bars represent the Standard Error of the Mean (SEM). Statistical significance, indicated by (*) was determined using a Student's unpaired t-test. Statistical significance levels are denoted as follows: * p < 0.05, ** p < 0.01, and **** p < 0.0001. Non-significant differences are denoted as "ns".

[0228] Figure 20 is an analysis of perinuclear F-actin ring formation in HCC38 cells upon MW-treatment, relative to the indicated microweight values. Statistical analysis of the impact of single 0.5-, 1-, 2-, 5-, and 10-gram microweight treatment lasting 10 minutes on the perinuclear F-actin cytoskeleton (%) in HCC38 cells. The data represent the mean ±SEM (Standard Error of the Mean), and were collected one day post seeding. Seeding density at day 0 was 20,000 cells. Statistical significance, indicated by (*) was determined using one-way ANOVA with a Dunnett's post-hoc test, comparing the data to the condition where no exogenous MWs (MW-0) were used (green bar). Statistical significance levels are denoted as follows: ** p < 0.01, and *** p < 0.001.

[0229] Figure 21 is a comparative analysis of the perinuclear F-actin cytoskeleton (%) in MCF10A (non-tumorigenic), MDA-MB-231 (highly metastatic and triple-negative), and HCC38 (non-metastatic basal-like) cells upon a single 0.5-, 1-, 2-, 5-, and 10-gram microweight (MW) 10-minute treatment (day 1). Seeding density at day 0 is 20,000 cells. The data represent the mean ±SEM (Standard Error of the Mean).

[0230] Figure 22 is a comparative analysis of the perinuclear F-actin cytoskeleton (%) in MCF10A, MDA-MB-231, and HCC38 cells upon a single 0.5 and 10-gram microweight (MW) 10-minute treatment (day 1) relative to control (MW-0). Seeding density at day 0 is 20,000 cells. The data represent the mean ±SEM (Standard Error of the Mean). Statistical significance was determined using one-way ANOVA for each condition (MW- 0, MW-0.5 and MW-10), followed by Tukey's post-hoc multiple comparisons test. Statistical significance levels are denoted as follows: *** p < 0.001, and **** p < 0.0001, and ns = not significant.

[0231] Figure 23 is an analysis of the microweight (MW) responsiveness factor (RF) for MCF10A, MDA-MB-231, and HCC38 cells. RF is defined as the ratio of the perinuclear F-actin ring response (%) at 10 g (MW-10) to the response at 0.5 g (MW-0.5) (RF = MW-10 I MW-0.5), providing a measure of how the cell lines adapt from low to high mechanical load. The data represent the mean ±SEM (Standard Error of the Mean). SEM was computed using error propagation rules. Statistical significance was determined using bootstrap-based ANOVA followed by Tukey's post-hoc test.

[0232] Statistical significance is: *** p < 0.00. Raw data source is Figure 22.

[0233] Mammalian cells are encapsulated by a lipid bilayer known as the plasma membrane, which acts as a selective barrier, protecting the cells from their surrounding environment. The inventors have surprisingly discovered that microweight-induced compression and consecutive relaxation of adherent cells (i.e., "mechanofection") enables the delivery of external molecules into the cell, using three different mammalian cell lines, including MCF10A, MDA-MB-231, and HCC38 cell lines. In doing so, the inventors have demonstrated that applying force onto the cells using "mechanofection" enables the successful introduction of a wide variety of molecules, including external bare DNA, bare RIMA, protein biomolecules, and chemical agents, into adherent cells. As shown in the following Examples, the inventors have succeeded in the delivery of exogenous green fluorescent protein (GFP)-encoding bare DNA and mRNA into cells, which resulted in the production of GFP protein. The successful delivery of protein macromolecules (>150 kDa), such as antibodies against nuclear envelope components, has also been demonstrated using immunofluorescence microscopy, which resulted in the collapse of nuclear structure. Moreover, the penetration of small toxins conjugated to small fluorescent dyes is documented both visually (using microscopy) and biologically, by examining the consequences on cell survival post mechanofection. The inventors have also demonstrated that the mechanical stimulation enabling mechanofection sensitises breast cancer cells to a chemotherapy drug. Further, the inventors have found that malignant and non- malignant cells behave differently upon mechanical stimulation, and that cell response to mechanical stimulation can distinguish between non-malignant, malignant, and non-tumorigenic cell lines in vitro.

[0234] Materials and Methods Cell culture

[0235] The non-malignant breast epithelia cell line MCF10A was cultured in phenol red-free DMEM / F-12 medium (Sigma-Aldrich, Gillingham, UK), supplemented with 100 ng / ml cholera toxin (Sigma-Aldrich, Gillingham, UK), 20 ng / ml epidermal growth factor (Peprotech, ThermoFisher Scientific, Cramlington, UK), 2 mM L-glutamine (Sigma- Aldrich, Gillingham, UK), 500 ng / ml hydrocortisone (Sigma-Aldrich, Gillingham, UK), 0.01 mg / ml insulin (Sigma-Aldrich, Gillingham, UK), 1% Penicillin-Streptomycin (Sigma-Aldrich, Gillingham, UK) and 5% Horse Serum (Invitrogen, Loughborough, UK).

[0236] The triple negative breast cancer cell lines MDA-MB-231 and HCC38 were cultured in phenol red-free media. MDA-MB-231 cells were grown in DMEM medium containing 4.5 g / L glucose (Corning, Flintshire, UK), whilst HCC38 cells were grown in RPMI 1640 media (Gibco, London, UK), with both mediums supplemented with 10% FBS (Sigma- Aldrich, Gillingham, UK), 2 mM L-Glutamine (Sigma-Aldrich, Gillingham, UK) and 1% Penicillin-Streptomycin (Sigma-Aldrich, Gillingham, UK). Cells were maintained as monolayers and incubated at 37°C and 5% CO2 in a humidified incubator (PHC Europe, Suffolk, UK).

[0237] Immunofluorescence and Microscopy Cells were plated onto 11 mm glass coverslips (No 1.0, 0.13-0.16 mm thick soda glass coverslips, LaboQuip, London, UK) at a density of 20,000 cells per coverslip in a volume of 50 ptL. The cells were incubated at 37°C and 5% CO2 in a cell incubator for at least 2 hours to allow adherence. Subsequently, the cells were topped up with complete media and further incubated for 24 hours under the same conditions (37°C, 5% CO2). After 24 hours, the coverslips were removed from the wells of the 24 well plate (Starlab, Milton Keynes, UK), and subjected to mechanofection, as outlined below. The coverslips were placed back into the wells of the plate and incubated for another 24 hours. After a 24 hour incubation, the media was removed, and the cells were washed twice with PBS (Phosphate Buffered Saline, Severn Biotech, Kidderminster, UK). The cells were then fixed in 3.7% formaldehyde (in PBS) for 20 minutes at room temperature (RT). Following fixation, the cells were rinsed with PBS and permeabilised with 0.5% Triton X-100 (ThermoFisher Scientific, Cramlington, UK) diluted in PBS for 10 minutes at RT. The cells were then blocked in PBG (0.1% bovine serum albumin (BSA) (Sigma-Aldrich, Gillingham, UK), 0.1% Fish gelatine (Sigma- Aldrich, Gillingham, UK; diluted in PBS) for 1 hour at RT. Subsequently, the cells were transferred to a humidified chamber and incubated with the primary antibody (diluted in PBG) for 1 hour at RT (outlined in Table 1). After the antibody incubation, the cells were washed with PBS (3x 10 minutes) and then incubated with the appropriate secondary antibodies (diluted in PBG), or with TRITC-phalloidin (outlined in Tables 2 and 3, respectively), for 1 hour at RT in the dark. Following this, the cells were washed with PBS (3x 10 mins). DNA staining performed using DAPI (diluted in PBS, 5 minutes at RT, in the dark) (outlined in Table 3). Finally, the cells were washed in PBS (2x 5 minutes) before being mounted onto glass slides (LaboQuip, London, UK) with the VECTASHIELD® mountant (H-1000, Vector Laboratories, Peterborough, UK), and sealed with nail varnish. Cells were imaged using the Axioskop 40 epifluorescent microscope (Carl Zeiss Ltd, Cambridge, UK) with ZeissEC Plan-Neofluar 20x / 0.5 Ph2, 40x / 0.75 Ph2, or 40x / 1.3 Oil Ph3 Objectives.

[0238] Table 1. Primary antibodies used in immunofluorescence.

[0239] Table 2. Secondary antibodies used in immunofluorescence. Table 3. Stains used in immunofluorescence.

[0240] Mechanofection

[0241] Cells were plated onto glass 11 mm coverslips (No 1.0, 0.13-0.16 mm thick soda glass coverslips, LaboQuip, London, UK) at a density of 20,000 cells per coverslip in a volume of 50 ptL. The cells were incubated in a humidified cell incubator at 37°C and 5% CO2 for at least 2 hours to allow the cells to adhere. Once adherence was established, the wells were topped up with complete media and incubated for a further 24 hours (37°C, 5% CO2). After 24 hours, the coverslips were removed from the wells of the 24 well plate (Starlab, Milton Keynes, UK), and were subsequently subjected to mechanofection. For the latter, a piece of Bemis™ Parafilm™ (Thermofisher Scientific, Cramlington, UK) was placed on top of a glass slide. The parafilm was cleaned with 70% ethanol and left to dry. Subsequently, 25 pL of either DPBS (Dulbeccos's Phosphate Buffered Saline, Gibco, London, UK) or the relevant serum-free medium (medium not containing serum, all other components the same), either without (control), or including biological or chemical agents (i.e., primary rabbit antibody, TRITC-Phalloidin, eGFP-mRNA or DNA plasmid) was added (outlined in Table 4). The coverslip containing the cells was removed from the well of the 24 well plate and placed directly on top of the liquid (cells facing down), to which either; no weight (MW-0) or a microweight (MW-1 [1 gram], MW-10 [10 gram], MW-20 [20 gram]; purchased from Oakleyweight and Amazon) was placed directly on top for 10 seconds. After the incubation period, the coverslips were flipped (cells facing upwards) and placed directly back in the tissue culture plate well (lx treatment) that contained media. For experiments where the procedure was repeated, 10 seconds after submersion into the media, the coverslips were removed, flipped around, and the cells were exposed to the biological or chemical agents for 10 seconds for a second time (2x treatment), again in the absence (MW-0) or presence of microweights. Afterwards, the coverslips were placed back into the well of the 24 well plate (cells facing up), and the cells were incubated for 24 hours in a humidified cell incubator set at 37°C and 5% CO2. The next day the coverslips were either processed for immunofluorescence, as described above, or MTT measurements, as described below. The average weight of the glass coverslips based on five independent measurements is: 40.64mg + / - 1.467 SD. Control transfection experiments were performed with the transfection reagent Lipofectamine™ 3000 (ThermoFisher Scientific, Cramlington, UK) to deliver eGFP- mRNA (Genscript) into MDA-MB-231 and HCC38 cells. Cells were seeded onto coverslips and allowed to adhere for 24 hours, as outlined above. Following this, lipofectamine complexes were prepared. Specifically, to two separate Eppendorf tubes (Starlab, Milton Keynes, UK) 25 piL of serum free media was added to each. To the first tube, 1.5 piL Lipofectamine™ 3000 transfection reagent was added. To the second tube, 10 pig GFP-mRNA was added. The contents of each tube were mixed with the existing media solution. The contents of both tubes were combined and allowed to incubate at RT for 15 minutes. During incubation, the media of the cells within the 24 well plate was changed to serum free media (500 piL per well). Following incubation, the lipofectamine-RNA complexes were added directly to this medium, and cells were incubated for another 4 hours within a cell culture incubator (37°C and 5% CO2). After 4 hours, the medium was replaced with media containing serum "complete media", and the cells were incubated for a further 20 hours. Following incubation, the cells were processed for immunofluorescence, as described above.

[0242] Table 4. Materials used for mechanofection.

[0243] Cell viability

[0244] Cell viability was performed as an MTT assay. MDA-MB-231 cells were plated onto 11 mm (diameter) glass coverslips (No 1.0, 0.13-0.16 mm thick soda glass coverslips, LaboQuip, London, UK) in triplicate, with 20,000 cells per coverslip in 50 pL of medium. After incubating in the humidified cell incubator at 37°C and 5% CO2 for at least 2 hours to allow cell adherence, the wells were topped up with 450 pL of complete media, and further incubated for 24 hours (37°C, 5% CO2). After 24 hours, the coverslips were removed from the wells, and subjected to the microweight treatment, as outlined above and as shown in Figure 1, and then returned to the wells of a 24-well plate (Starlab, Milton Keynes, UK) for an additional 24 or 48 hours of incubation. Following the incubation period, a stock solution of MTT was prepared (5 mg / mL in PBS), and a working solution (1 mg / mL) was made in media. The working solution was mixed with the existing media (on the cells) in a 1: 1 ratio and incubated for an additional 3 hours. After incubation, the media was carefully removed from the coverslips to avoid disturbing the formazan crystals and replaced with DMSO. The absorbance at 540 nm was measured using a Promega GloMax plate reader (Promega, Southampton, UK).

[0245] Statistical analysis

[0246] Statistical analysis was conducted using either ANOVA (Analysis of Variance) for comparisons involving more than one treatment group or a Student's unpaired t-test for comparisons involving one treatment group against the control. The ANOVA was performed using Prism Graphpad (Version 10.2.3). When statistical significance was observed, a Dunnetts post hoc test was carried out to determine specific significant differences. The Student's unpaired t-test was performed using Microsoft Excel (version 16.66.1). Data are presented as mean ±SEM. Significance was determined when the p-value was < 0.05.

[0247] Example 1 - Delivery of molecules into biological cells using so-called "mechanofection" methodology

[0248] To exert mechanical pressure to adherent cells, calibrated microweights were used. To achieve this, adherent cells grown on a glass coverslip were inverted before mechanical pressure was exerted, as shown in Figure IB. The omission of microweights was considered the control setting, as shown in Figure 1A; MW-0. Nonetheless, it should be noted that for HCC38 cells, the weight of the glass coverslip itself (~40 mg) was sufficient to deliver exogenous materials into the cells, as shown in Figure 7. For MCF10A and MDA-MB-231 cells, the weight of the glass coverslip was not sufficient to deliver external molecules into cells, as shown in Figure 2, Figure 3B, Figure 4, and Figure 6. Biomolecules from an external source were positioned between the cells and the underlying hydrophobic parafilm surface. It is deduced that the mechanical pressure exerted by the microweights increases the permeability of cellular membranes, enabling the delivery of biomaterials into cells, as shown in Figure IB, inset.

[0249] 2: Bare DNA is delivered into adherent mammalian cells mechanical

[0250] The inventors have demonstrated the delivery of exogenous bare DNA, which encodes for green fluorescent protein (GFP) as an exemplar, into cells. As shown in Figures 2 and 3, the delivery of DNA into MDA-MB-231 cells and MCF10A is indicated upon mechanofection, respectively. The delivery of the DNA is demonstrated by a green signal in cells, indicating GFP protein expression (see Figure 2B and Figure 3A), which were processed for immunofluorescence microscopy. The use of the F-actin counterstain TRITC-phalloidin (red channel) highlights the flattened shape of the cells and confirms their adherence to the substrate.

[0251] The expression of GFP protein indicates that the exogenous bare DNA has been delivered into the nucleus, where mRNA synthesis occurs before being translated into a protein in the cytoplasm. This indicates that mechanofection can deliver bare DNA into both the cytoplasm and the nucleus.

[0252] Example 3: The successful delivery of exogenous material into cells is directly linked to the mechanical pressure exerted on cells

[0253] A coverslip with an area of 0.9503 cm2was seeded with 20,000 MDA-MB-231 cells. One day post seeding, the cells were exposed to 0 gram (MW-0), 5 gram (MW-5) and 10 gram (MW-10) in the presence of GFP-encoding plasmid (DNA). As the glass coverslip itself has a weight (40.64 mg) and an area of 0.9503 cm2, the pressure ranges that have been examined are 0.0004191 Pascal for MW-0, 516 Pascal for MW- 5, 1,031 Pascal for MW-10 (Figure 2C), and 2,064 for MW-20 (Figure 2D). As indicated in Figure 2C, the highest transfection percentages (>8.5%) for a single MW-treatment (lx 10 seconds) are established with MW-10. MW-20 also yields to a statistically increase of GFP-expressing cells when compared to MW-0 (Figure 2D), however, the % of GFP-expressing cells is lower (5.6%) compared to MW-10 treated cells. Without microweights (MW-0), GFP-expression is absent in MDA-MB-231 cells (Figures 2A, 2C and 2D).

[0254] Example 4: Cycles of mechanofection involving MW-5 or MW-10 increase the DNA- transfection rates

[0255] The inventors have shown how additional rounds of mechanofection, i.e., cycles of mechanical pressure and relaxation, affect the delivery of GFP-encoding DNA plasmids into MDA-MB-231 cells. Specifically, two rounds of MW-treatments (2 xlO seconds) were administered, with a 10 second relaxation step between the treatments. Figure- 2C indicates that successive rounds of mechanofection using MW-5 and MW-10 enhances the transfection efficiency, leading to higher percentages of GFP-expressing cells. In contrast to MW-5 and MW-10, two cycles of mechanofection (2 xlO seconds) using MW-20 resulted in a reduction of GFP-expressing cells (Figure 2D). Specifically, a single treatment with MW-20 for 10 seconds yields in 5.6% of cells expressing GFP, which is reduced to 2.6% if the MW-20 treatment is repeated. 5: The mechanofection buffer affects the DNA transfection results

[0256] Two solutions were evaluated for the mechanofection method: a) serum-free media (Figures 2C and 2D) and b) DPBS (Figure E). As can be seen in Figure 2E, the highest transfection rates (18.6%) for MDA-MB-231 cells are obtained for a single mechanofection treatment (lx 10 seconds) using MW-10 and DPBS. : The transfection rates for DNA mechanofection are cell

[0257] Referring to Figure 4, the percentages of GFP-expressing cells are examined after a MW-10 single mechanofection treatment (1 x 10 seconds) for MCF10A and MDA-MB- 231 cells, when 2 pg of GFP-encoding plasmid are diluted in DPBS. Figure 4 indicates that the metastatic cell line (MDA-MB-231) is more easily transfected with DNA using MW-10 than the non-metastatic cell line (MCF10A). GFP-encodinq RNA is delivered into adherent mammalian cells treatment

[0258] The inventors have demonstrated the delivery of exogenous mRNA, which encodes for green fluorescent protein (GFP) into cells. Referring to Figures 5 and 8, the delivery of mRNA into MDA-MB-231 and HCC38 cells is indicated upon mechanofection, respectively. The delivery of the mRNA is demonstrated by the green signal in cells, indicating GFP protein expression (see Figure 5C, and Figure 8B-E), which were processed for immunofluorescence microscopy. The use of anti-GFP counterstain (red channel) was performed to enable the identification of cells with low GFP expression levels. Lipofectamine is used as a control (Figure 5A and 8A), to indicate the successful transfection of cells using a conventional chemical transfection method.

[0259] 8: Transfection rates for RNA are cell

[0260] Figure 5D indicates that a single treatment with MW-10 is sufficient to deliver GFP- encoding mRNA in ~20% of MDA-MB-231 cells. Importantly, without microweights (MW-0), the external mRNA is not delivered into cells. Notable is the lack of green cells in Figures 5B and 5D for MW-0 treated cells. In sharp contrast to MDA-MB-231 cells, the MW-0 treatment is sufficient to deliver mRNA into HCC38 cells (Figure 8B and 8F). Transfection rates above 20% involving MW-10, which are statistically significant when compared to MW-0 treated cells, are obtained only when cells are subjected to two rounds of mechanofection (2x 10 seconds; Figure 8F).

[0261] Example 9: Proteins are delivered into adherent mammalian cells with mechanofection To establish the delivery of macromolecular proteins (>150 kDa) into cells, the inventors performed experiments using exogenous antibodies. The inventors used rabbit polyclonal antibodies (pAbKl) directed against a protein that is positioned at the outer nuclear membrane, namely nesprin-2. Nesprin-2 is a member of the spectrin family of proteins that binds to the cytoskeleton, including F-actin, microtubules, and intermediate filaments. The functionality of the pAbKl antibody is established in Figure 6A, using conventional immunofluorescence microscopy. As indicated in the inset, the pAbKl staining results in a nuclear rim pattern, which encapsulates the nucleus (denoted by the DAPI staining; blue channel). Without mechanofection (MW-0 treatment; Figure 6B), the cells lack a nuclear rim staining, after they were processed for immunofluorescence. In contrast with mechanofection (MW-10 treatment), and concomitant immunofluorescence examination, the nuclei are positive for nesprin-2 staining (red channel; Figure 6C). Moreover, from the inset in Figure 6C it is observed that the nesprin-2 pAbKl antibody results in a strong nuclear rim staining. Referring to Figure 6D, it is shown that two consecutive rounds of mechanofection (2x 10 seconds) using MW-10 are more efficient in delivering exogenous proteins into cells than a single mechanofection round (lx 10 seconds).

[0262] Example 10: Delivery of exogenous anti-Nesprin-2 pAbKl antibody affects the function of nesprin-2 within cells

[0263] Nesprin-2 is a structural protein of the nucleus which regulates nuclear shape through its interaction with the cytoskeleton and cytoskeleton-associated motor proteins (Lu et al., 2012). Referring to Figure 6C and 6E, it is shown that the delivery of nesprin-2 into the cells with mechanofection results in nuclear shrinking. This highlights that mechanofection does not affect the biological properties of the delivered proteins, and that the present method enables the study of the function of internal cellular proteins. The inventors assume that the binding of pAbKl to nesprin-2 sterically affects the interactions of nesprin-2 to the cytoskeleton which results in the collapse of the nucleus. In conclusion, the successful delivery of large proteins into cells using mechanofection is demonstrated visually (e.g., detection of the delivered protein;

[0264] Figure 6C) and functionally (Figure 6E), as it compromises the function of the targeted protein.

[0265] 11: Delivery of TRITC- toxic bicyclic peptides into adherent cells using Mechanofection

[0266] Referring to Figure 7, the delivery of TRITC-phalloidin, a toxic bicyclic peptide (~ 1233 g / mol) that binds filamentous actin (F-actin) in cells, using mechanofection was evaluated. In Figures A and A', the functionality of TRITC-phalloidin is demonstrated using conventional immunofluorescence. As can be seen, TRITC-phalloidin detects filamentous actin-based structures, which are enriched at the cortex of MDA-MB-231 cells, when the cells are permeabilised with detergent. Without mechanofection (MW-0 treatment), the exogenously added TRITC-phalloidin cannot enter the cells, which is demonstrated by the lack of staining (Red channel; Figure 7B'). Upon mechanofection using a single (lx 10 second) MW-10 treatment, some cells are positive for TRITC- phalloidin (red channel), while others either lack (highlighted with asterisks, Figure 7C') or are weakly stained for TRITC-phalloidin. Figure 7D demonstrates that a single MW-10 treatment is more efficient in delivering TRITC-phalloidin into cells (37.1% phalloidin positive cells) than a double MW-10 treatment (15.2%). The entry of phalloidin into cells affects cell viability, as the binding of phalloidin to F-actin stabilises actin filaments and prevents their depolymerisation. A dynamic actin cytoskeleton is essential to accomplish vital cellular processes such as cell division, movement and intracellular trafficking of molecules. One day post-mechanofection, cell viability is reduced in TRITC-phalloidin mechanofected cells. However, the established changes are not significant when compared to MW-0 treated cells (Figure 7E). A major impact on cell viability is established 2 days post-mechanofection for TRITC-phalloidin treated cells (Figure 7F). Collectively, the detection of TRITC- phalloidin microscopically within cells (Figure 7C), and the impact on cell viability two days after mechanofection (Figure 7E), establishes that the mechanofection method successfully delivered TRITC-phalloidin into cells.

[0267] Example 12: Mechanofection delivers different biomolecules into adherent mammalian cells

[0268] The delivery of DNA is demonstrated in Figures 2 and 3, for MDA-MB-231 and MCF10A cells, respectively. The delivery of mRNA is demonstrated in Figures 5 and 8, for MDA- MB-231 and HCC38 cells, respectively. Delivery of proteins into MDA-MB-231 cells is demonstrated in Figure 6. The delivery of small peptides into MDA-MB-231 cells is shown in Figure 7.

[0269] Example 13: Mechanical stimulation sensitises MDA-MB-231 cancer cells to doxorubicin chemotherapy treatment

[0270] Referring to Figure 9, the inventors have found that mechanical stimulation sensitises MDA-MB-231 cancer cells, which are doxorubicin-resistant, to doxorubicin chemotherapy treatment. As such, the methods of the invention provide advantageous synergistic effects (i.e., synthetic lethality) if mechanical stimulation and doxorubicin chemotherapy is combined. Furthermore, the inventors envisage that mechanical stimulation may advantageously enable the usage of lower cytotoxic doses of doxorubicin, since more of the external drug can enter a cancer cell if mechanically stimulated. The inventors have also found that the cytotoxic effects of mechanical stimulation (i.e., compressive forces) are pronounced in dense cell cultures, as shown in Figure 9.

[0271] Example 14: MW-treatment induces peri-nuclear F-actin rings

[0272] Referring to Figure 10, the inventors have discovered that MW-treatment induces perinuclear F-actin rings. MW-value is shown to affect the percentage of F-actin rings, the optimal value for MDA-MB-231 cells being MW-10, since these cells did not react to MW-0, MW-05, and MW-1. Also referring to Figure 10, the inventors found that MW- treatment affects cell viability, with MW-value differentially affecting cell viability percentage.

[0273] The inventors could also conclude that MW-induced peri-nuclear ring formation requires F-actin, since no rings were observable with latrunculin B treatment (latrunculin B being an inhibitor of actin polymerisation).

[0274] The inventors further noted that MW-induced F-actin rings are structurally distinct from cytoplasmic F-actin structures; Cytoplasmic F-actin structures (cell periphery) post-Lat B treatment were found, but no F-actin ring structures are observed around the nucleus. Referring to Figure 10 (H), the inventors found that MW-induced F-actin rings restrict nuclear shrinkage upon LatB-treatment and thus may play protective roles. Also referring to Figure 10 (H), the inventors noted that prolonged MW-20 treatment (for 10 minutes) enlarged the MDA-MB-231 nuclei.

[0275] Referring to Figure 10 (I), the inventors advantageously identified synergistic cell viability effects of combined LatB and MW-treatment on cell viability. Latrunculin B mechanofection is inferred, since the combined MW and LatB treatment produced more severe cytotoxic effects than either MW- or LatB-treatments alone.

[0276] Example 15: Prolonged MW-10 treatment significantly enlarges the cancer cell nuclear area

[0277] Referring to Figure 11 (A, B), the inventors have found that prolonged MW-10 treatment (10 minutes) significantly enlarges the MDA-MB-231 nuclear area.

[0278] Example 16: MW-induced peri-nuclear F-actin rings are dynamic structures

[0279] Referring to Figure 12, the inventors have found that MW-induced peri-nuclear F-actin rings are dynamic structures, and are disassemble within minutes once the MW- stimulus is absent. The inventors hypothesise, therefore, that recovery of those healthy cells that neighbour cancer cells to their physiological state after microweight treatment may be faster than that for cancer cells.

[0280] Example 17: MW-treatment induces F-actin rings around the nucleus within seconds Referring to Figure 13, the inventors have found that MW-treatment induces F-actin rings around the nucleus within seconds. Further, the inventors noticed that MW- induced cell toxicity is time-dependent, and that consecutive rounds of MW-treatment reduce cell viability.

[0281] Example 18: MW-effects on nuclear area are cell number dependent

[0282] Referring to Figure 14, the inventors have found that MW-effects on nuclear area are cell number dependent for the MDA-MB-231 cell line. Their results suggested that the maximum effects of MW- 10 induced nuclear expansion are generated with 1,000 or 5,000 cells, and that no effects on nuclear area are induced with > 20,000 cells.

[0283] Referring to Figure 14(1), the inventors found that the effects on nuclear area with 20,000 cells are time-dependent. Indeed, a single 10 second MW0-=-10 treatment did not impact nuclear area, as shown in Figure 14 (I), while a 10-minute treatment does, as shown in Figure 11.

[0284] However, MW-induced effects on cell viability are cell number independent, as show in Figure 14. r F-actin rings in MCF10A cells

[0285] Referring to Figure 15, the inventors have found that MW-treatment induces perinuclear F-actin rings in MCF10A (non-tumorigenic) cells. The inventors also noted that, in these cells, light MWs (MW-0.5 and MW-1) more efficiently induce perinuclear actin rings than heavier MWs.

[0286] 20: MW-treatment affects the vi of MCF10A cells

[0287] Referring to Figure 16, the inventors have found that MW-treatment affects the viability of MCF10A (non-tumorigenic) cells. r F-actin rings at the of MCF10A cell colonies

[0288] Referring to Figure 17, the inventors have found that MW-treatment preferentially induces perinuclear F-actin rings at the periphery of MCF10A (non-tumorigenic) cells. As such, the inventors' methods provides a mechanistic link between cell position, mechanistic environment (i.e., peripheral cells are more spread and under greater tension when compared to central cells), and phenotype. Thus, the methods of the invention advantageously disentangle inherent spatial heterogeneity within a colony.

[0289] Example 22: MCF10A nuclei are resilient to MW-induced nuclear shape changes Referring to Figure 18, the inventors have found that MCF10A (non-tumorigenic) nuclei are resilient to MW-induced nuclear shape changes. Advantageously, the MW- treatment effects on the MCF10A nucleus are notably distinct from those observed in MDA-MB-231 (highly metastatic and triple-negative) cells.

[0290] Example 23: MW-treatment affects the MCF10A cell viability

[0291] Referring to Figure 19, the inventors noted that MW-treatment affects MCF10A (non- tumorigenic) cell viability. In addition, MW-treatment was found to affect the MCF10A cell viability in a less pronounced manner under high cellular confluence. r F-actin rings in HCC38 metastatic basal-li cells

[0292] Referring to Figure 20, the inventors found that MW-treatment also induces perinuclear F-actin rings in HCC38 cells, and that such perinuclear F-actin ring formation depends on the value of the MW.

[0293] Example 25: Malignant and non-malignant cells behave differently upon MW- treatment

[0294] Referring to Figures 21 and 22, the inventors found that malignant and non-malignant cells behave differently upon MW-treatment. The profile of perinuclear F-actin ring formation upon MW-treatment was found to be different for MCF10A (non- tumorigenic), HCC38 (non-metastatic basal-like), and MDA-MB-231 (highly metastatic and triple-negative) cells.

[0295] MCF10A shows high sensitivity at low weights, peaking around 0.5-1 g, however, the response diminishes at higher weights. In contrast, MDA-MB-231 shows minimal response at very low weights, but exhibits a strong, sustained increase at higher weights. Thus, MCF10A behaviour suggests a switch-like response, compared to the gradual adaptation seen in MDA-MB-231. These results highlight differences in responses, with cancer cells (MDA-MB-231) showing resilience and altered force adaptation. Further, HCC38 cells display a very strong response at MW-1, but this response was not sustained. Fluctuating responses were noted at higher weights, but never reaching the high levels seen in MDA-MB-231 cells. In addition, HCC38 cells exhibit more "burst-like" sensitivity rather than a smooth, dose-response.

[0296] Example 26: MW-responsiveness factor (RF) can distinguish between non-maliqnant, malignant, and non-tumorigenic cell lines in vitro

[0297] Referring to Figure 23, the inventors found that MW-responsiveness factor (RF) can distinguish between non-malignant, malignant, and non-tumorigenic cell lines in vitro, where RF is defined as the ratio of the perinuclear F-actin ring response (%) at 10 g (MW-10) to the response at 0.5 g (MW-0.5) (RF = MW-10 I MW-0.5), providing a measure of how the cell lines adapt from low to high mechanical load.

[0298] 27: Effects of MW-treatment

[0299] Referring to the results discussed in Examples 13-26, the inventors conclude that the profile of nuclear area alteration upon MW-treatment advantageously infers data about the biomechanical properties of the nucleus and the cell in general.

[0300] The inventors have also discovered that MDA-MB-231 (highly metastatic and triplenegative) cells harbour softer nuclei than MCF10A (non-tumorigenic) cells, as MW- treatment induces significant effects on nuclear area.

[0301] Further, the profile of perinuclear F-actin ring formation upon MW-treatment advantageously infers data about the biomechanical properties of the cells. Additionally, MW-induced F-actin rings separate and analyse subpopulations within the same colony, showing how spatial context drives cell behaviour. of metastatic propensity built on a microweiqht-based approach; standalone and combination approaches

[0302] Based on the data disclosed herein, the inventors envisage a prognostic assay of metastatic propensity build on a microweight-based approach. For such an assay, cells may either be extracted or grown from a biopsy in vitro, or the mechanical pressure may be applied directly on an ex vivo specimen). This prognostic assay may involve analysing any of the following four aspects:

[0303] (i) Uptake of external substances into cells. Cancer cells more readily take up exogenous material than healthy, non-tumorigenic cells; (ii) Responsiveness Factor (RF) ratio. High RF for metastatic cells, and very low RF for non-metastatic cells;

[0304] (iii) MW-induced nuclear expansion. The nucleus of metastatic cells expands more easily, while the nucleus of a healthy, non-tumorigenic cell does not; and / or

[0305] (iv) MW-induced cell viability relative to cell density.

[0306] The above-described prognostic assay could be used as a standalone approach, or in combination with a molecular biomarker-based approach, that uses primary antibodies to elucidate biomarker presence and organisation (nuclear / cell architecture) to increase specificity and sensitivity.

[0307] Example 29: Additional future approaches

[0308] Based on the data disclosed herein, the inventors envisage prognostic and therapeutic approaches built on mammography, notably without the X-ray imaging modality. Instead, the procedure would be performed with Adaptive kPa-controlled Cyclic Compression (ACC), which may comprise stepwise pressure plateaus, followed by brief relaxation intervals, followed by final target compression, with imaging at plateaus. Imaging would be based on the mechanofection concept.

[0309] Therapy for tumours that are positive (i.e., at the histology and imaging level) may comprise chemotherapy combined with Adaptive kPa-controlled Cyclic Compression (ACC), based on the inventors' finding that in vitro mechanical stimulation can overcome drug resistance, restore chemosensitivity, and enable cytotoxic effects at a lower drug concentration.

[0310] Conclusions

[0311] As described in detail above, the inventors have surprisingly discovered that mechanical stimulation of adherent biological cells enables the successful delivery of a wide variety of exogenous molecules into the cells. As such, the inventors' discovery provides a novel and improved methodology enabling the delivery of foreign genetic material, gene products, proteins, and chemical agents into mammalian cells, without causing adverse immune reactions and / or compromising the viability of treated cells.

[0312] In addition, the inventors have shown that cancer cells are surprisingly mechanofected more efficiently than normal, healthy cells, and therefore suggest that the microweight technology and methods described herein may be used to distinguish normal and cancerous cells through the distinction between cancer cell and healthy cell stiffness. Accordingly, the inventors have also devised a novel method of determining whether a cell is cancerous, and thereby diagnose cancer. References

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Claims

1. Claims1. A method of introducing at least one exogenous molecule into at least one biological cell, the method comprising:(i) contacting the at least one biological cell with the at least one exogenous molecule; and(ii) exerting force on the at least one biological cell, to thereby introduce the at least one exogenous molecule into the at least one biological cell.

2. The method according to claim 1, wherein the at least one biological cell comprises at least one mammalian cell, optionally wherein the at least one biological cell is selected from a group of cell types consisting of: epithelial cell, muscle cell, nerve cell, blood cell, connective tissue cell, cancer cell, and stem cell.

3. The method according to either claim 1 or claim 2, wherein the at least one cell comprises: (i) at least 50, 100, 250, 500 or 1000 cells; (ii) at least 1500, 2000, 2500, 3000 or 3500 cells; or (iii) at least 4000, 4500, 5000, 7500 or 10,000 cells.

4. The method according to any preceding claim, wherein the at least one biological cell comprises a monolayer comprising a plurality of biological cells.

5. The method according to claim 4, wherein the plurality of cells comprises a cell density of between 2 and 2,000,000 cells per cm2, between 10 and 1,000,000 cells per cm2, between 100 and 100,000 cells per cm2, between 1,000 and 500,000 cells per cm2, between 2,000 and 250,000 cells per cm2, between 3,000 and 200,000 cells per cm2, between 4,000 and 150,000 cells per cm2, or between 5,000 and 100,000 cells per cm2.

6. The method according to claim 4 or claim 5, wherein the plurality of cells comprises a cell density of between 2 and 1,909,000,000 cells per cm3, between 100 and 1,800,000,000 cells per cm3, between 1,000 and 1,700,000,000 cells per cm3, between 10,000 and 1,600,000,000 cells per cm3, or between 100,000 and 1,500,000,000 cells per cm3.

7. The method according to any preceding claim, wherein the at least one biological cell comprises a healthy cell.

8. The method according to any preceding claim, wherein the at least one biological cell comprises an unhealthy cell, optionally a cancer cell.

9. The method according to any preceding claim, wherein the at least one biological cell comprises an adherent cell.

10. The method according to any preceding claim, wherein the method comprises a step of adhering the at least one biological cell to a surface before the step of contacting the at least one biological cell with the at least one exogenous molecule and / or the step of exerting force on the at least one biological cell.

11. The method according to claim 10, wherein the surface is selected from a list of surfaces consisting of: glass, polystyrene, plastic, extracellular matrix coated, fibrin, silicon, ecoflex, hydrogel, spun silk fibroin, thermoplastic elastomer, polymeric film, polyurethane, polydimethylsiloxane, polyacrylamide, polyethylene terephthalate, polycarbonate, polylactic acid, poly(lactic-co- glycolic acid), polycaprolactone, chitosan, poly(N-isopropylacrylamide), graphene, graphene oxide, hydroxyapatite, carbon nanotube, polyethylene glycol, and alginate.

12. The method according to either claim 10 or claim 11, wherein the at least one biological cell is incubated to allow the or each cell to substantially adhere to the surface.

13. The method according to claim 12, wherein the at least one biological cell is adhered to the surface by incubation at a temperature of between 20°C and 50°C, or between 30°C and 35°C, for between 5 and 100 hours, between 10 and 50 hours, between 15 and 35 hours, or between 20 and 30 hours.

14. The method according to any one of claims 10-13, wherein once adherence of the cells to the surface is established, the method comprises subsequently contacting the or each cell with complete media and at a temperature of between 20°C and 50°C, or between 30°C and 35°C, for between 5 and 100 hours, between 10 and 50 hours, between 15 and 35 hours, or between 20 and 30 hours.

15. The method according to claim 14, wherein the complete media comprises:(i) DMEM / F-12 media, DMEM high glucose media, and / or RPMI 1640 media; and / or(ii) cholera toxin, epidermal growth factor, L-Glutamine, hydrocortisone, insulin, Penicillin-Streptomycin, Horse Serum, and / or FBS.

16. The method according to either claim 14 or claim 15, wherein the at least one biological cell is removed from the complete media prior to the step of exerting the force on the at least one cell.

17. The method according to any one of claims 10-16, wherein the at least one biological cell comprises a free apical cell surface.

18. The method according to any one of claims 10-17, wherein subsequent to incubating the at least one biological cell with complete media, the method comprises a step of inverting the surface on which the at least one biological cell is adhered.

19. The method according to any one of claims 10-18, wherein the surface on which the at least one cell is adhered is contacted with a medium comprising the at least one exogenous molecule, such that the at least one cell is contacted with the at least one molecule.

20. The method according to claim 19, wherein the medium comprises a solid, semi-solid, semi-liquid, or liquid.

21. The method according to any preceding claim, wherein the at least one exogenous molecule comprises a biological molecule selected from a group of biological molecules consisting of: protein, nucleic acid, nucleotide, carbohydrate, lipid, DNA, RIMA, antibody, antibody fragment, peptide, enzyme, hormone, growth factor, neurotransmitter, cytokine, antibiotic, oligonucleotide, synthetic or bioengineered pharmaceutical drug, chemotherapy agent, nutraceutical, and toxin.

22. The method according to any preceding claim, wherein the at least one exogenous molecule is not complexed with a carrier.

23. The method according to any preceding claim, wherein the at least one exogenous molecule comprises a protein-encoding DNA or RNA.

24. The method according to any preceding claim, wherein the at least one exogenous molecule is selected from a group of molecules consisting of: small molecule, nanoparticle, liposome, exosome, quantum dot, polymer, dendrimer, ion, nanomaterial, and antioxidant.

25. The method according to any preceding claim, wherein the force that is exerted on the at least one biological cell is a contact force applied to the at least onebiological cell, optionally wherein the force is selected from a group of contact forces consisting of: applied force, spring force, air resistance force, normal force, tension force and frictional force.

26. The method according to any preceding claim, wherein the at least one biological cell is contacted with the at least one molecule before, after, or at the same time the force is exerted on the at least one biological cell.

27. The method according to any preceding claim, the force is exerted on the at least one biological cell by a weight (W) and / or a microweight (MW).

28. The method according to claim 27 , wherein the weight weighs between 0.01 g and 50 g between 0.5 g and 40 g, between 1 g and 30 g, or between 2 g and 25 g, optionally between 5 g and 20 g.

29. The method according to any preceding claim, wherein a pressure is exerted on the at least one biological cell, and wherein the pressure exerted on the at least one biological cell is between 0.0001 Pascal and 10,000 Pascal, between 0.001 Pascal and 8,000 Pascal, between 0.01 Pascal and 7,000 Pascal, between 0.1 Pascal and 6,000 Pascal, or between 1 Pascal and 5,000 Pascal.

30. The method according to any preceding claim, wherein the force is exerted on the at least one biological cell for between 1 second and 10 minutes, between 2 seconds and 9 minutes, between 3 seconds and 8 minutes, or between 4 seconds and 7 minutes.

31. The method according to any preceding claim, wherein the force is exerted on the at least one biological cell for between 5 seconds and 6 minutes, between 6 seconds and 5 minutes, between 7 seconds and 4 minutes, between 8 seconds and 3 minutes, or between 9 seconds and 2 minutes.

32. The method according to any preceding claim, wherein the method comprises a second incubation period comprising contacting and incubating the at least one biological cell with media, but does not comprise exerting force on the at least one cell.

33. The method according to claim 32, wherein the second incubation period comprises incubating the at least one biological cell with media for between 1 second and 10 minutes, or between 4 seconds and 7 minutes.

34. The method according to any preceding claim, wherein the method comprises cycles of compression and consecutive relaxation of cells.

35. The method according to any preceding claim, wherein subsequent to one or more treatments, the method comprises a post-treatment incubation step comprising contacting and incubating the at least one biological cell with media at between 20°C and 50°C, or between 30°C and 35°C, for between 5 and 100 hours, between 10 and 50 hours, between 15 and 35 hours, or between 20 and 30 hours.

36. An apparatus for introducing at least one exogenous molecule into at least one biological cell, the apparatus comprising :(i) means for contacting the at least one biological cell with the at least one exogenous molecule; and(ii) means for exerting force on the at least one biological cell, to thereby introduce the at least one exogenous molecule into the at least one biological cell.

37. The apparatus according to claim 36, wherein the apparatus is configured to perform the method according to any one of claims 1-35.

38. The apparatus according to either claim 36 or claim 37, wherein the means for contacting the at least one biological cell with the at least one molecule comprises a surface onto which the at least one cell is adhered or dispersed.

39. The apparatus according to any one of claims 36-38, wherein the means for contacting the at least one biological cell with the at least one molecule is configured to lower the surface onto which the at least one cell is adhered such that the at least one cell contacts the at least one exogenous molecule.

40. The apparatus according to any one of claims 36-39, wherein the means for exerting force on the at least one biological cell comprises a weight placed adjacent to the surface onto which the at least one cell is adhered.

41. A method of diagnosing or prognosing cancer in a subject, the method comprising, either:(A)(i) contacting at least one biological test cell obtained from a test subject with at least one exogenous molecule;(ii) contacting at least one biological non-cancerous control cell with at least one exogenous molecule;(iii) exerting force on the at least one biological test cell and the at least one biological control cell, to thereby introduce the at least one exogenous molecule into the at least one biological test cell and the at least one biological control cell;(iv) (a) detecting the amount of the at least one molecule which has been introduced into the at least one biological test cell and the at least one biological control cell, (b) determining the status of at least one organelle in the at least one biological test cell and the at least one biological control cell, and / or (c) determining the cell viability of the at least one biological test cell and the at least one biological control cell; and(v) (a) comparing the amount of the at least one molecule which has been introduced into the at least one biological test cell to the amount of the at least one molecule which has been introduced into a control cell, (b) comparing the status of the at least one organelle in the at least one biological test cell to the status of the at least one organelle in the at least one biological control cell, and / or (c) comparing the cell viability of the at least one biological test cell to the cell viability of the at least one biological control cell, to thereby diagnose or prognose cancer in the subject; and / or(B)(i) (a) determining the size and / or shape of the nuclei of at least one biological test cell and at least one biological control cell and / or (b) determining the cell viability of at least one biological test cell and at least one biological control cell;(ii) exerting force on the at least one biological test cell and the at least one biological control cell;(iii)subsequent to exerting force on the cells, (a) determining the size of the nuclei of the at least one biological test cell and the at least one biological control cell, and / or (b) determining the cell viability of the at least one biological test cell and the at least one biological control cell; and(iv)(a) comparing the sizes and / or shapes of the nuclei of the at least one biological test cell and the at least one biological control cell prior to and subsequent to exerting force on the cells; and / or (b) comparing the cell viabilities of the at least one biological test cell and the at least one biological control cell prior to and subsequent to exerting force on the cells, to thereby diagnose or prognose cancer in the subject.

42. A method of determining the likelihood of a cell to metastasize, the method comprising, either:(A)(i) contacting at least one biological test cell obtained from a test subject with at least one exogenous molecule;(ii) contacting at least one biological non-cancerous control cell with at least one exogenous molecule;(iii) exerting force on the at least one biological test cell and the at least one biological control cell, to thereby introduce the at least one exogenous molecule into the at least one biological test cell and the at least one biological control cell;(iv) detecting the amount of the at least one molecule which has been introduced into the at least one biological test cell and the at least one biological control cell; and(v) comparing the amount of the at least one molecule which has been introduced into the at least one biological test cell to the amount of the at least one molecule which has been introduced into a control cell, to thereby determine the likelihood of a cell to metastasize; and / or(B)(i) (a) determining the Responsiveness Factor (RF) ratio of at least one biological test cell and at least one biological control cell and / or (b) determining the cell viability of at least one biological test cell and at least one biological control cell;(ii) exerting force on the at least one biological test cell and the at least one biological control cell;(iii) subsequent to exerting force on the cells, (a) determining the Responsiveness Factor (RF) ratio of the at least one biological test cell and the at least one biological control cell, and / or (b) determining the cell viability of the at least one biological test cell and the at least one biological control cell; and(iv) (a) comparing the Responsiveness Factor (RF) ratios of the at least one biological test cell and the at least one biological control cell prior to and subsequent to exerting force on the cells; and / or (b) comparing the cell viabilities of the at least one biological test cell and the at least one biological control cell prior to and subsequent to exerting force on the cells, to thereby determine the likelihood of a cell to metastasize.

43. The method according to either claim 41 or 42, wherein steps (i) and (ii) are as defined with respect to the method according to any one of claims 1-35.

44. The method according to any one of claims 41-43, wherein the at least one molecule results in a signal in the cell into which the at least one molecule has been introduced.

45. The method according to any one of claims 41-44, wherein the at least one molecule is selected from a list of molecules consisting of: fluorescent protein, fluorescent protein-encoding DNA, fluorescent-encoding mRNA, fluorescent dye and conjugate, luciferase protein, luciferase-encoding DNA, luciferase-encoding mRNA, luminescent protein, chromogenic molecule, siRNA, chemical probe, nanoparticle (e.g., nanoparticle with optical properties), fluorescently-tagged antibody, labelled peptide, chromobody, labelled protein, quantum dot, and near- infra red dye.

46. The method according to any one of claims 41-45, wherein analysing the amount of the at least one molecule which has been introduced into the at least one mammalian cell comprises detecting the at least one molecule in the at least one cell.

47. The method according to claim 46, wherein detecting the at least one molecule in the at least one cell comprises a method of detection selected from the list of methods of detection consisting of: fluorescence microscopy, immunofluorescence, bioluminescence assay, flow cytometry, quantitative PCR, western blotting, northern blotting, fluorometric assay, radiolabelling, electron microscopy, mass spectrometry, positron emission tomography, magnetic resonance imaging, computer tomography, and single-photon emission computed tomography.

48. The method according to any one of claims 41-47, wherein the control comprises at least one healthy or normal cell, optionally wherein the control cell is obtained from a healthy subject.

49. The method according to any one of claims 41-48, wherein comparing the amount of the at least one molecule which has been introduced into the at least one mammalian cell to a control comprises identifying whether the same amount or a greater amount of the at least one molecule has been introduced into the at least one mammalian cell compared to the control, optionally wherein whether the same amount or a greater amount of the at least onemolecule that has been introduced into the at least one biological test cell compared to the at least one biological control cell is determined using statistical analysis.

50. The method according to any one of claims 41 or 43-49, wherein the organelle is selected from a group of organelles consisting of: nucleus, mitochondrion, smooth endoplasmic reticulum, rough endoplasmic reticulum, centrosome, cilium, cell membrane, golgi apparatus, peroxisome, lysosome, transport vesicle, ribosome, and cytoskeleton.

51. The method according to any one of claims 41 or 43-50, wherein determining the status of the at least one organelle comprises determining the size, shape, and / or function of the at least one organelle, optionally wherein determining the size, shape, and / or function of the at least one organelle comprises immunofluorescence examination, electron microscopy, confocal microscopy, live cell imaging, Western blotting, mitochondrial membrane potential assay, electron paramagnetic resonance, metabolomics, proteomics, gene expression profiling (e.g. RNA-seq), or flow cytometry.

52. The method according to any one of claims 41 or 43-51, comprising identifying whether the status of the at least one organelle in the at least one biological test cell is different to the status of the at least one organelle in the at least one biological control cell, optionally wherein the status of the at least one organelle in the at least one biological test cell is identified as different to the status of the at least one organelle in the at least one biological control cell using statistical analysis.

53. The method according to any one of claims 41 or 43-52, wherein determining the cell viability of the at least one biological test cell and the at least one biological control cell comprises examination of the metabolic activity of and / or performing a live / dead cell counting assay on the at least one biological test cell and the at least one biological control cell.

54. The method according to any one of claims 41 or 43-53, comprising identifying whether the cell viability of the at least one biological test cell is equal to or less than the cell viability of the at least one biological control cell, optionally wherein the cell viability of the at least one biological test cell may be identified as equal to or less than the cell viability of the at least one biological control cell using statistical analysis.

55. The method according to any one of claims 41 or 43-54, wherein if more of the at least one molecule has been introduced into the at least one biological cell than the control, a diagnosis of cancer in the subject is made.

56. The method according to any one of claims 42-49, wherein comparing the amount of the at least one molecule which has been introduced into the at least one biological test cell to the amount of the at least one molecule which has been introduced into a control cell comprises comparing the amounts of the at least one molecule that have been introduced into the at least one biological test cell and the control cell to a mechanofection standard.

57. The method according to claim 56, wherein the mechanofection standard comprises a set of molecules and corresponding mechanofection values for said molecules, optionally wherein the mechanofection values comprise a number and / or percentage, and / or a range of numbers and / or percentages, which represents a known amount and / or amounts of the at least one molecule which is introduced into a control cell.

58. The method according to either claim 56 or claim 57, wherein comparing the amounts of the at least one molecule that have been introduced into the at least one biological test cell and the control cell to a mechanofection standard comprises deriving a mechanofection score for the at least one biological test cell, optionally wherein the mechanofection score is determined by the difference between the amount of the at least one molecule that has been introduced into the at least one biological test cell and the mechanofection standard for the at least one molecule, optionally wherein the difference between the amount of the at least one molecule that has been introduced into the at least one biological test cell and the mechanofection standard for the at least one molecule is determined by statistical analysis.

59. The method according to claim 58, wherein if the difference between the amount of the at least one molecule that has been introduced into the at least one biological test cell and the mechanofection standard for the at least one molecule is statistically significant, there is a high likelihood that the cell will metastasize, optionally wherein the greater the statistically significant difference between the amount of the at least one molecule that has been introduced into the at least one biological test cell and the mechanofection standard for the at least one molecule, the higher the mechanofection score and / or the greater the likelihood that the cell will metastasize.

60. The method according to any one of claims 41 or 43-59, wherein determining the size and / or shape of the nuclei comprises immunofluorescence examination, electron microscopy, confocal microscopy, live cell imaging, Western blotting, mitochondrial membrane potential assay, electron paramagnetic resonance, metabolomics, proteomics, gene expression profiling (e.g. RNA-seq), or flow cytometry of the at least one biological test cell and the at least one biological control cell.

61. The method according to any one of claims 41 or 43-60, wherein the method comprises identifying whether the size and / or shape of the nucleus in the at least one biological test cell is different to the size and / or shape of the at least one organelle in the at least one biological control cell, optionally wherein the size and / or shape of the nucleus of the at least one biological test cell is identified as different to the size and / or shape of the nucleus of the at least one biological control cell using statistical analysis.

62. The method according to any one of claims 41 or 43-61, wherein determining the cell viability of the at least one biological test cell and / or the at least one biological control cell comprises determining the cell viability of the at least one biological test cell and / or the at least one biological control cell relative to cell density, optionally wherein determining the cell viability of the at least one biological test cell and the at least one biological control cell comprises examination of the metabolic activity of and / or performing a live / dead cell counting assay on the at least one biological test cell and the at least one biological control cell.

63. The method according to any one of claims 41 or 43-62, wherein the cell viability of the at least one biological test cell and the at least one biological control cell is determined prior to and subsequent to exerting force on the at least one biological test cell and the at least one biological control cell.

64. The method according to any one of claims 41 or 43-62, wherein the method comprises identifying whether the cell viability of the at least one biological test cell is equal to or less than the cell viability of the at least one biological control cell, optionally wherein, if the cell viability of the at least one biological test cell is less than the cell viability of the at least one biological control cell, a diagnosis or prognosis of cancer in the subject is made, or wherein, if the cell viability of the at least one biological test cell is less than the cell viability of the at least one biological control cell, the greater the likelihood of the biological test cell to metastasize.

65. The method according to any one of claims 42-59, wherein determining the Responsiveness Factor (RF) ratio of the at least one biological test cell and the at least one biological control cell comprises determining the presence and / or amount of F-actin rings around the nuclei of the cells.

66. The method according to any one of claims 42-59 or 65, wherein the Responsiveness Factor (RF) ratio of the at least one biological test cell and the at least one biological control cell is determined prior to and subsequent to exerting force on the at least one biological test cell and the at least one biological control cell.

67. The method according to any one of claims 42-59, 65, or 66, wherein the method comprises identifying whether the Responsiveness Factor (RF) ratio of the at least one biological test cell is equal to or greater than the cell viability of the at least one biological control cell, optionally wherein, if the Responsiveness Factor (RF) ratio of the at least one biological test cell is greater than the cell viability of the at least one biological control cell, the greater the likelihood of the biological test cell to metastasize, or wherein, if the Responsiveness Factor (RF) ratio of the at least one biological test cell is high, the greater the likelihood of the biological test cell to metastasize, or wherein, if the Responsiveness Factor (RF) ratio of the at least one biological test cell is low, the lesser the likelihood of the biological test cell to metastasize.

68. The method according to claim 67, wherein: (i) a high RF ratio comprises a RF ratio of >5.0, >10.0, or >12.0, and / or wherein a low RF ratio comprises a RF ratio of <10.0, <5.0, or <2.0; and / or (ii) a high RF ratio indicates that a cell is cancerous metastatic, and / or a low RF ratio indicates that a cell is normal, or non-cancerous.

69. The method according to any one of claims 42-59 or 65-68, wherein exerting force on the at least one biological test cell and the at least one biological control cell comprises exerting force for one or more periods, optionally wherein the one or more periods comprise exerting varying amounts of force on the cells.

70. The method according to any one of claims 41-49, wherein the method is combined with a biomarker-based and / or cell and / or nuclear organisationbased diagnostic or prognostic assay.

71. A method of:(i) sensitising, to an anti-cancer drug, a subject that is suffering from cancer that is insensitive to the anti-cancer drug; or(ii) enhancing the sensitivity, to an anti-cancer drug, a subject that is suffering from cancer that is sensitive to the anti-cancer drug, the method comprising exerting force on an area of the subject comprising a cancerous tumour.

72. The method according to claim 71, further comprising administering, to the subject, the anti-cancer drug.

73. The method according to claim 72, wherein the anti-cancer drug comprises doxorubicin.

74. The method according to any one of claims 71-73, wherein the at least one biological cell is obtained from a resected tissue or biopsy sample from the subject.

75. The method according to any one of claims 71-74, wherein, it is determined that the subject has been sensitised to the anti-cancer drug, or that the sensitivity of the subject to the anti-cancer drug has been enhanced, by determining the response of the cancerous tumour to the treatment.

76. The method according to claim 75, wherein the response of the cancerous tumour is determined by a radiologic response, tumour biomarkers, and / or a pathologic response.

77. The method according to any one of claims 71-76, wherein the method comprises administering, to the subject, the anti-cancer drug (i) prior to exerting force on the area of the subject comprising the cancerous tumour, (ii) subsequent to exerting force on the area of the subject comprising the cancerous tumour, or (iii) concurrent to exerting force on the area of the subject comprising the cancerous tumour.

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