MechanoFluorescence

Cancer progression is closely related to changes in mechano-cellular phenotype and in the structure and mechanical properties of the tumor microenvironment-TME in a complex and not well-understood manner. Desmoplasia, a tumour-associated fibrotic reaction, is responsible for tumor stiffening, poses a major barrier to effective drug delivery and has been associated with poor prognosis. Although novel therapeutic strategies that target cancer cells (chemotherapy), or the TME (TME normalization) are emerging, their efficacy varies due to intra- and inter-patient variability as well as tumor heterogeneity. Given that only some patients respond to a particular treatment, a new era of personalized, patient-specific treatments has been initiated, the basis of which is the identification of biomarkers that characterize a particular tumor. Our work so far has demonstrated that Atomic Force Microscopy-AFM techniques can be used for identifying unique nanomechanical fingerprints that can be used as treatment monitoring and prognostic predictive biomarkers. Furthermore, we have shown that the nanomechanical fingerprints are closely related with collagen content alterations. Consequently, the combination of AFM nanomechanical fingerprints and collagen-based optical characterization can lead to the development of novel MechnanoOptical signatures that can be used as biomarkers. However, a number of drawbacks limit the possible clinical used of the MechanoOptical signatures in real clinical practice. First of all, AFM nanomechanical characterization of tissue requires the experiments to be conducted within the same day of tissue harvest. Also, the majority of the techniques for assessing the collagen-based signatures require quite complex tissue preparation, which is a time consuming procedure that also alters tissue mechanical properties. Tissue preparation techniques prevent the simultaneous optical and mechanical characterization of tissue samples. What is more, so far for combining collagen-based optical characterization with AFM mechanical signatures, expensive bio-AFM systems (AFM&fluorescence microscopes) were used or the characterization was performed on two different microscopes. In order to overcome these bottlenecks in this project we proposed the development of the appropriate protocols for the simultaneously nanomechanical and fluorescence characterization of fresh tissue biopsies. A combination of cutting-edge experimental approaches in desmoplastic breast tumors will be used. Firstly, we will optimize the protocol for collecting, transferring and sectioning (with a vibratom) fresh tissue biopsies. Then we will develop a protocol a protocol for staining and measuring fluorescence intensity (with low-cost fiber-optic based fluorescence measurements techniques) from fresh tissue sections mounted on AFM system. Finally, we will optimize the protocol for simultaneously measurements of nanomechanical and fluorescence properties of fresh tissue specimens and we will test it on a short pilot study. Successful completion of the project will enable assessing the MechanoOptical signatures of fresh tissue specimens within the same day of tissue harvest leading to the possible clinical use these novel biomarkers.