Szymon Prauzner-Bechcicki

Cancer cell recognition--mechanical phenotype.

The major characteristics of cancer metastasis is the ability of the primary tumor cells to migrate by way of the blood or lymph vessels and to form tumors at multiple, distant sites. There are evidences that cancer progression is characterized by disruption and/or reorganization of cytoskeleton (i.e. cellular scaffold). This is accompanied by various molecular alterations influencing the overall mechanical resistance of cells. Current approach in diagnosis focuses mainly on microbiological, immunological, and pathological aspects rather than on the biomechanics of diseases. The determination of mechanical properties of an individual living cell has became possible with the development of local measurement techniques, such as atomic force microscopy, magnetic or optical tweezers. The advantage of them lies in the capability to measure living cells at a single cell level and in liquid conditions, close to natural environment. Here, we present the studies on mechanical properties of single cells originating from various cancers. The results show that, independently of the cancer type (bladder, melanoma, prostate, breast and colon), single cells are characterized by the lower Youngs modulus, denoting higher deformability of cancerous cells. However, the obtained Youngs modulus values were dependent on various factors, like the properties of substrates used for cell growth, force loading rate, or indentation depth. Their influence on elastic properties of cells was considered. Based on these findings, the identification of cancerous cells based on their elastic properties was performed. These results proved the AFM capability in recognition of a single, mechanically altered cell, also in cases when morphological changes are not visible. The quantitative analysis of cell deformability carried out using normal (reference) and cancerous cells and, more precisely, their characterization (qualitative and quantitative) can have a significant impact on the development of methodological approaches toward precise identification of pathological cells and would allow for more effective detection of cancer-related changes.

Cancer cell detection in tissue sections using AFM.

Currently, cancer diagnosis relies mostly on morphological examination of exfoliated, aspirated cells or surgically removed tissue. As long as standard diagnosis is concerned, this classical approach seems to be satisfactory. In the recent years, cancer progression has been shown to be accompanied by alterations in mechanical properties of cells. This offers the detection of otherwise unnoticed cancer cell disregarded by histological analysis due to insignificant manifestations. One of techniques, sensitive to changes in mechanical properties, is the atomic force microscopy, which detects cancer cells through their elastic properties. Such measurements were applied to tissue sections collected from patients suffering from various cancers. Despite of heterogeneity and complexity of cancer cell sections, the use of the Youngs modulus as an indicator of cell elasticity allow for detection of cancer cells in tissue slices.

PDMS substrate stiffness affects the morphology and growth profiles of cancerous prostate and melanoma cells.

A deep understanding of the interaction between cancerous cells and surfaces is particularly important for the design of lab-on-chip devices involving the use of polydimethylsiloxane (PDMS). In our studies, the effect of PDMS substrate stiffness on mechanical properties of cancerous cells was investigated in conditions where the PDMS substrate is not covered with any of extracellular matrix proteins. Two human prostate cancer (Du145 and PC-3) and two melanoma (WM115 and WM266-4) cell lines were cultured on two groups of PDMS substrates that were characterized by distinct stiffness, i.e. 0.75 ± 0.06 MPa and 2.92 ± 0.12 MPa. The results showed the strong effect on cellular behavior and morphology. The detailed analysis of chemical and physical properties of substrates revealed that cellular behavior occurs only due to substrate elasticity.

Nano-characterization of two closely related melanoma cell lines with different metastatic potential.

Cutaneous malignant melanoma is one of the most lethal types of skin cancer. Its progression passes through several steps, leading to the appearance of a new population of cells with aggressive biological potential. Here, we focused on the nano-characterization of two different melanoma cell lines with similar morphological appearance but different metastatic potential, namely, WM115 from vertical growth phase (VGP) and WM266-4 derived from metastasis to skin. The first cell line represents cells that progressed to the VGP, while the WM266-4 cell line denotes cells from the metastasis to skin. Exploring with a combination of atomic force and fluorescence microscopes, our goal was to identify cell surface characteristics in both cell lines that may determine differences in the cellular nano-mechanical properties. Cell elasticity was found to be affected by the presence of F-actin-based flexible ridges, rich in F-actin co-localized with β1 integrins in the studied cell lines. These results point out how progressive changes in the surface structure of melanoma cells can affect their bionanomechanical properties.

Precise positioning of cancerous cells on PDMS substrates with gradients of elasticity.

In this work the novel method to create PDMS substrates with continuous and discrete elasticity gradients of different shapes and dimensions over the large areas was introduced. Elastic properties of the sample were traced using force spectroscopy (FS) and quantitative imaging (QI) mode of atomic force microscopy (AFM). Then, fluorescence microscopy was applied to investigate the effect of elastic properties on proliferation of bladder cancer cells (HCV29). Obtained results show that cancerous cells proliferate significantly more effective on soft PDMS, whereas the stiff one is almost cell-repellant. This strong impact of substrate elasticity on cellular behavior is driving force enabling precise positioning of cells.

Elasticity patterns induced by phase-separation in polymer blend films

Systematical studies on the impact of the thickness of thin films composed of polystyrene (PS) or poly(ethylene oxide) (PEO) on the effective elasticity of polymer-decorated soft polydimethylsiloxane substrate were performed. For both investigated polymer films, elasticity parameter was determined from force-displacement curves recorded using atomic force microscopy. Effective stiffness of supported film grows monotonically with film thickness, starting from the value comparable to the elasticity of soft support and reaching plateau for polymer layers thicker than 200 nm. In contrary, for films cast on hard support no significant thickness dependence of elasticity was observed and the value of elasticity parameter was similar to the one of the substrate. Based on these results, non-conventional method to produce elasticity patterns of various shapes and dimensions induced by phase-separation process in symmetric and asymmetric PS:PEO blend films on soft support was demonstrated. Elevated PS domains were characterized by elasticity parameter 2 times higher than lower PEO matrix. In contrary, adhesion force was increased more than 3 times for PEO regions, as compared to PS areas.

Discrimination between HCV29 and T24 by controlled proliferation of cells co-cultured on substrates with different elasticity

In this work the impact of substrate elasticity on the proliferation of two cell lines, a non-malignant transitional epithelium HCV-29 and a bladder transitional cell carcinoma T24 cultured individually and in co-culture was analyzed. A significantly stronger, highly cell-dependent impact of mechanical properties on cellular behavior was shown for cells co-cultured from the mixture. A more effective proliferation process observed for T24 cells was analyzed quantitatively for asymmetric HCV29: T24 mixtures co-cultured on soft substrate. The obtained results suggest that the proliferation of T24 cells is even 4 times more effective as compared to HCV29 cells and confirm strong invasiveness of metastatic T24 cells. The high adaptiveness of T24 cells to adverse environmental conditions enables easy and accurate discrimination between not isolated healthy and cancer cells.

Physicochemical properties of PDMS surfaces suitable as substrates for cell cultures

T study reports the preparation of the new drug carrier gel system based on poly (itaconic anhydride-co-3, 9-divinyl-2, 4, 8, 10-tetraoxaspiro (5.5) undecane) (PITAU) copolymer and hyaluronic acid (HA-PITAU). In relation with its composition PITAU has specific conformational structure owing to the unsaturated double bond of 3, 9-divinyl-2, 4, 8, 10-tetraoxaspiro (5.5) undecane comonomer and the spiroacetal moiety, giving macromolecular chains with network type structures. PITAU is biocompatible and biodegradable and present pH and temperature sensitivity. Itaconic anhydride (ITA) has been considered as an alternative to maleic anhydride for introducing polar functionality into polymers. Polymers based on ITA have not received as much attention as lactic acid derived materials. The composition was confirmed by FTIR spectra, evidencing the cross linking bridges between copolymer and hyaluronic acid. SEM microscopy and chemical imagining evidence the homogeneous porous structure of the new 3D network. NIR-chemical imaging technique proves the successful preparation of polymeric drug delivery system by using indomethacin (IND) as bioactive model substance. The dissolution data revealed the interdependence of the ratio between the two compounds and attaining optimum loading capacity. In vivo study demonstrated that HA_ PITAU and HA_ PITAU_IND determined similar blood parameters modifications and biochemical responses with distilled water, after intraperitoneal administration in mice. Systemic administration of the tested substances in mice did not modify their immune reactivity comparing with control group. All these results reveal a good in vivo biocompatibility. The bioactive compound caused a significant antinociceptive effect occurring after 60 minutes and lasts about 3 hours in tail flick test.R dystrophic epidermolysis bullosa (RDEB) has been defined as severe chronic skin fragility and caused by mutations in COL7A1, which encodes for the elastic structural protein type VII collagen (C7). The 8.9 Kb COL7A1 transcript is particularly a large sequence with many repeating units which makes it difficult to manipulate and package into viral systems. Therefore, the minicircle system is ideal for use with COL7A1, firstly to minimize the overall DNA construct size while secondly increasing the safety profile of the gene therapy. We successfully inserted COL7A1 into the parental plasmid MN512A1 and combined it with our highly efficient a non-viral vector (HPAE). HPAE-MC-COL7A1 polyplexes successfully produced significant levels of recombinant C7 with negligible cytotoxicity in RDEB-TA4 keratinocytes. Minimal effect was seen on primary keratinocyte metabolic health, even after multiple applications of HPAE polyplexes. Furthermore, in vivo transfection studies revealed that HPAE carrying MC-COL7A1 restores the expression of C7 along the basement membrane zone in a human RDEB graft mouse model after intradermal injection and topical application. Of the 7 animals treated, 5 were positive for recombinant C7, with all animals receiving 2 or more applications having strong positive signal. While further assessment is required to prove this approach is safe and well tolerated in the long term, HPAE-MC-COL7A1 polyplexes showed great promise as a potential therapeutic for RDEB.M play important roles to repair human tissue defect. In this work, human amniotic membrane (HAM) was decellularized and explored the efficacy as an implantable biological mesh. Surfactant, hypertonic saline, lipase and DNAase were used individually or collectively to remove all cell components and remain the extracellular matrix. Results of H&E and DAPI staining demonstrated that the method of surfactant and lipase combining with DNAase is the most effective treatment for HAM decellularization. Primary smooth muscle cells were seeded to evaluate the decellularized HAM’s (dHAM) in vitro cytocompatibility. The in vivo test was performed via implantation at rabbits’ uterus with clinic polypropylene mesh (PP) as the control. The results indicated that dHAM possessed good biocompatibility and will be a potential candidate for biological mesh.