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Description
The idea our project is to combine microfluidic approach with selective capturing of cells for enhanced identification of mechanically altered cells. Custom designed microfluidic devices will be used to sort cells floating through the channels, based on mechanical deformability generated during floating. Furthermore, cells will be captured on modified microarray surfaces prepared using lectins/antibodies against the most characteristics changes in surface antigen/glycans expression. Next, their mechanical and microrheological properties will be measured by AFM. In microfluidics, floating cells are round, therefore, it is essential to understand the relation between mechanical properties of adherent cells and cells in suspension. So far, there is no direct proof showing to what extent initially larger cellular deformability is preserved in floating cancer cells. Understanding this relation is essential for enhancing the identification of cells by microfluidic devices. To test the effectiveness of the developed microfluidic device, a few groups of cancer cell lines will be applied, namely human bladder cancer cells characterized by a large deformability, human melanoma cells with smaller deformability differences and pancreatic cancer cells of unknown mechanical relation. Based on the obtained results, we expect that already reported limitations in quantification of cellular deformability, helpful in the comparison of healthy and pathologically altered cells, will be overcome. Through this, a developed microfluidic device will be capable to differentiate/separate specific cancer cell populations in suspension composed of cells characterized by a small deformability difference, thus could be translated and applied in cancer diagnosis, delivering a tool for high throughput and quantitative identification of mechanically altered cells.
Summary of project results
The main obstacles during project realization were the Covid-2019 pandemic/post-pandemic time and war-related constraints. First, national COVID restrictions in Poland and Norway were different, which impacted the number of visits between project partners. Common experiments were conducted with all accessible means (online meetings, Whatsup messages). The late delivery of consumables needed to perform experiments (e.g., blebbistatin used to assess the role of actomyosin motors in melanoma cell elasticity ordered in October 2022, arrived in March 2023) was another obstacle to be overcome. This created the need to work in parallel at IFJ PAN and NTNU on the same materials (leading to the increase in project costs and increased prices of compounds needed for cell growth). As a consequence, we decided to realize all foreseen tasks in parallel to minimize the delays in the project realization. As a consequence, most of our manuscripts were prepared and submitted in the second half of 2023 and 2024. Also, the presentation of the results to NGOs and other public organizations was delayed until 2023.
The BioMechCanDet project aimed to combine a microfluidic approach with selective cell capturing and quantitative determination of cell nanomechanical fingerprints to enhance the identification of mechanically altered cells. Custom-designed microfluidic devices were used to capture cells floating through the channels on lectin-coated surfaces. Lectins (plant glycoproteins) were chosen due to their binding to specific elements of the sugar coat present on the cell surface (called glycosylation pattern). Such glycosylation patterns are different in various cancers. Next, the mechanical and microrheological properties were measured by various approaches, namely, directly in the microfluidic device during flow or after using an atomic force microscope (AFM), working in both indentation and microrheological modes. The work carried out focused on gathering data linked with project objectives by completing project tasks, which led to (i) designing, producing, and optimizing microfluidic devices, (ii) biologically active lectin-coated surfaces for selective capture of cells, (iii) defining the experimental conditions for selective capture of cancer cells, (iv) determining biomechanical fingerprints of adherent and suspended cells and alginate microbeads with stiff core, (v) biomechanics-related characteristics of cell invasiveness, and (vi) identification of criteria for selective cell sorting.
1) The developed microfluidic devices used to measure cell deformability or adhesive properties of cancer cells. Outcome: results comparing the adhesive properties of bladder cancer cells and deformability of melanoma cells affected by inhibitors of specific cytoskeleton elements. Impact: Identification of small cell populations deviating from a mean that, combined with data on cell invasiveness, will enhance the identification process and affect the diagnosis of cancer. Beneficiary: researchers (within such domains as biophysics, cell biology, and medicine) and clinicians.
2) The use of lectins (plant proteins) that bind to cell surface glycans (oligosaccharide moieties attached to surface proteins or lipids) as biosensors, enabling selective capturing of specific cancer cells. Outcome: Lectin-dependent specific cell adhesion for melanoma, bladder, and pancreatic cancer cells. Impact: Lectin-based selective capture of cancer cells offers a practical guide. Encouraging studies on detailed identification of glycans and using them as sensors in various lab-on-a-chips to produce biosensors used in cancer diagnosis. Beneficiary: researchers (within such domains as biosensing, surface physics, biophysics, cell biology, and medicine), clinicians, and biosensors engineers.
3) Detailed control of surface properties is essential in providing specific adhesion signatures of cancer cells. Outcome: Surface functionalization with lectins. Fabricated lectin microarrays for selective capture of bladder cancer cells. Identified leakage of silanes from PDMS devices (stamps, microfluidic channels). Impact: Attention to the preparation of surfaces and device materials is paramount in this field to achieve specificity and avoid adverse effects. Our findings represent guidelines that can inspire researchers in the field. Beneficiary: researchers (within such domains as surface physics, biophysics, cell biology, and medicine) and engineers.
4) Biomechanical fingerprints of cancer cells. Outcome: Understanding cell mechanics in AFM and microfluidic devices and their relation to the internal structure of both cells and alginate microbeads. Impact: Increased confidence of researchers in applying microfluidic-based constriction devices to provide information related to differences in the mechanical properties of suspended cells. Beneficiary: researchers (within such domains as biosensing, surface physics, biophysics, cell biology, and medicine), clinicians, and biosensors engineers.
Summary of bilateral results
The bilateral collaboration between IFJ PAN and NTNU was highly productive, resulting in four joint scientific publications in journals such as International Journal of Molecular Sciences, Gels, ACS Applied Materials & Interfaces, and Biosensors and Bioelectronics. The cooperation also extended beyond publications, as joint research efforts were critical in designing, producing, and optimizing the microfluidic devices used in the experiments. NTNU played a pivotal role in the fabrication of these devices, while IFJ PAN led the characterization of cell biomechanics. This collaboration was further solidified by multiple meetings, both virtual and in-person, such as the midterm meeting organized by NTNU in Trondheim and the end-of-project meeting hosted by IFJ PAN.