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Novel precision technological platforms to promote non-invasive early diagnosis, eradication and prevention of cancer relapse: proof of concept in the bladder carcinoma.

Periodic Reporting for period 1 - EDIT (Novel precision technological platforms to promote non-invasive early diagnosis, eradicationand prevention of cancer relapse: proof of concept in the bladder carcinoma.)

Reporting period: 2018-10-01 to 2019-09-30

The unmet clinical needs in the management of bladder cancer (BCa) are the prevention of tumor onset/relapse/progression, and therapy of the aggressive bladder carcinoma in situ (Cis), requiring frequent and endless follow-up and weekly treatments, with a consequent poor quality of life and the highest cost per patient among all cancers.
EDIT will exploit the structural and mechanical properties of the bladder extracellular matrix (ECM) as a unique biomarker of the early onset/progression/relapse of carcinoma, through engineered gold nanorods (GNRs) used as intravesical photoacoustic antennas targeted at the neoplastic ECM and consequently generating an ad hoc visualization platform. GNRs will be further utilized as heat-releasing effectors for targeted cancer photothermal therapy. EDIT is designed to detect pre-neoplastic area and eradication of local areas at few cells resolution.
The main Objectives of EDIT are:
1) Early prognosis based on the recognition of fibrillary collagen rich matrix, predisposing a soil needed for the onset/progression/relapse of bladder cancer. EDIT will provide novel functionalized nanoparticles to be employed as intravesical source of ultrasound through photoacoustic effect.
2) Early eradication will exploit the superior targeting properties of locally delivered nanoparticles able to thermal ablating Cis cells upon near-infrared laser irradiation.
3) Novel biomarkers will assess sub-aims, implementing i) characterization of markers that identify pre-conditioning of the ECM, ii) novel targets for the identification of Cis cells, and iii) new ligands for expanding the armamentarium for targeted delivery of GNRs.
Exploitation of PAUS features of urine-stable GNRs@Chit-Dec and GNRs@Chit-Lig
CTAB-coated Gold Nanorods with absorption band at either 800 nm or 950 nm were prepared. The chitosan macromolecules were modified with thioglycolic acid to introduce the thiol groups required for the formation of GNRs@Chit. A complete characterization of GNRs has been performed, and the stability of GNRs@Chit in urine showed no signs of i) destabilization, ii) morphological transformation or iii) aggregation.
Decoys and peptides have been coupled to free amino groups of chitosan (3.3 nmol of CgA and 13.33 nmol of IsoDGR per µg of gold).
Head-to-tail cyclic isoAsp-Gly-Arg (isoDGR) and linear Arg-Gly-Asp (RGD) peptides from human chromogranin A (CgA) were prepared (purity >95%).
All GNRs@Chit-Lig induced cell adhesion more efficiently than control nanoparticles. The pro-adhesive effect of GNRs@Chit-CgA freshly prepared or stored for 4 months at 4°C (after resuspension) were similar. Similar results were also obtained GNRs@Chit-Iso4. The impact of urine on the binding of IsoDGR and CgA peptides to purified integrins showed that the overall recovery of binding was 32-66% in 90% natural urine suggesting that isoDGR peptides still bind their receptors in this very harsh condition. The capability of GNRs@Chit-Lig to bladder cancer cells was also confirmed by FACS analysis, indicating that isoDGR and CgA peptides can be useful for delivery to cancer cells.
Imaging platforms by mathematical models
-Identification of early tissue modification by Machine Learning Algorithms (MLA) were designed for classification of bladder tissue into malignant, benign or normal by utilising photoacoustic and ultrasound data. The model benefits from feature subset selection in order to increase the generalization of the model, shorten the training times of the MLAs and obtain faster models.
-A model of heat transfer in the bladder wall was prepared. Modelling the heat transfer during photothermal ablation is a two-step process that involves: 1) simulation of light propagation inside the tissue and 2) simulation of heat transfer inside the tissue due to absorption of light energy. Simulations of light propagation inside tissue were carried out using the Monte Carlo method, while simulations of heat transfer inside tissue were based on the Mie-Gans theory and carried out using the finite element method. In silico experiments were carried out analysing the influence of GNRs concentrations and laser power on the tissue heating.
Visualization platform for the overlay of 3D medical imaging was carried out for 3D reconstruction and visualization of the bladder and the tumour. The system architecture has been defined, which will be enhanced with the machine learning algorithms (MLA) for automatic detection of tumour at early stages. In addition to system architecture, technical specifications of components/subcomponents and modules have been outlined, and their interconnectivity was defined. Significant progress has been achieved towards the 2D bladder detection, by employing image pre-processing, processing and reconstruction techniques using Ultrasound and Photoacoustic images.
Early Prognosis - Markers that are shared between human and preclinical models of non-muscle invasive bladder carcinoma were identified and a protocol for measuring the mechanical properties of the tissue through atomic force microscopy was developed. Furthermore, a non-invasive strategy for distributing the intravesical therapy on the entire urothelium was also developed.
EDIT will pursue a three-fold transformational impact on Technology, Society and Economy.
Technology: EDIT will develop and provide radical innovation and impact in the management of solid tumors with a proof of concept in BCa by providing a new clinical platform based on a sophisticated imaging-guided-therapy. Increased spatial resolution and efficacy of therapy through non-invasive approach will offer unmet prognostic, diagnostic and therapeutic opportunities against BCa, and being unrelated to cellular metabolism these platforms will overcome bias identified in the gender medicine. The multidisciplinary expertise of the Consortium will allow for rapid delivery of the EDIT outcomes to clinical trials assessing for superior safety and efficacy vs current treatments.
Society: Considering that increased stiffness of ECM is also shared by tumors at breast, colon and prostate the novel and non-invasive approach of the EDIT platform could also be applied in these clinical and therapeutic applications, as well as visualization of deep anatomical targets or delicate organs such as brain, having a profound impact on the diagnosis, localized surgery and thermo-ablation of different malignancies.
Economic: By improving diagnosis and treatment the outcomes of EDIT will reduce the costs of the BCa management. Identification of the PAUS clinical setup in this study will contribute to the identification of the minimal requirements needed for upgrading the existing high-frequency ultrasound scans currently in use in thousands European hospital. Structural and mechanical modification of the ECM is a common denominator of different solid tumors; broading the range of EDIT applications will further contribute to the spread and lower costs of the technology, and increasing indirect jobs in the relevant industries, academies and in parallel in the indirect jobs.
The generated know-how will have deep impact at the society level and in related scientific fields, fostering i) combinatoric chemistry for engineering of nanoparticles adapting to specific microenvironments, ii) bladder cancer research, iii) mathematical modelling and iv) development of visualization algorithms.
EDIT project concept