PRISAR2 - Proactive monitoRIng of cancer aS An alteRnative to surgery 2
From 2016 -2019, the “first” PRISAR consortium implemented the first Dutch RISE project of H2020 to investigate the use of imaging technologies and hybrid fluorescence/radionuclide probes for the surgical intervention of cancer. This PRISAR project provided a career-enabling human resource plethora of inter-disciplinary and inter-sectoral secondments, which resulted in the development, of not only a hybrid probe for image-guided surgery, but also a variety of tools and instruments.
We have now been awarded of funding for a new project, PRISAR2, as coordinator of a consortium of 25 beneficiaries that will start next April 2020 and will last until March 2024.
Our aim is to implement the next logical phase of this work, which is to clinically translate new and innovative monitoring technologies from industry. This will provide better patient selection and increase the surveillance window to allow for better treatment options over time and avoid surgery. This will involve developing new ‘watch and wait’ strategies to study the behaviour of the cancer so that any risks can be minimized. This would lead to a better clinical outcome and quality of life (QOL) for the patient and maximize the benefits of an active monitoring policy for both the patient and the healthcare system. When a patient is discovered to have cancer, one of the primary goals is to have the tumour removed by surgery. Here, however, we aim to:
- develop new monitoring technologies from industry so that the surveillance window can be increased to allow for better treatment options over time and to avoid surgery,
- develop new ‘watch and wait’ strategies in order to study the behaviour of the cancer so that any risks to the patient can be minimised leading to a better clinical outcome and
- train a new generation of preclinical and clinical scientists to be able to implement this new concept as standard of care and to maximise the benefits of an active monitoring policy.
Official website: www.prisar2.eu
BESG - Bioengineered exosomes-based approaches for the effective treatment of non-small cell lung cancer
Lung cancer is the principal cause of cancer-related death around the world and has become more predominant among former than current smokers. Smoking, alcoholism, air pollution, occupational exposure are among the main causes of non-small cell lung cancer (NSCLCs). It is difficult to simultaneously deliver therapeutics and nucleic acid to the target site. With the advancement of nanotechnology, a delivery system composing of cellular proteins which can bypass the reticuloendothelial system (RES) and have the ability to release payload at the specific target site is the necessity of the current era. Exosomes (Exo) are clinically acceptable, having protein membrane composition and the ability to deliver payload at specific target sites. Having endogenous origin, Exo evade immune recognition and clearance compared to exogenous nanovesicles. Exo are natural carriers of nucleic acids and they can be engineered to deliver siRNA as well as anti-cancer drugs. In this we propose the conjugation or covering of mesenchymal stromal cells (MSCs) derived exosomes with HSP4 peptide (target specific to NSCLCs) and loading them with Gefitinib (GEF), an EGFR inhibitor and SPC24 siRNA (target for NSCLCs and metastasis). The delivery of GEF and SPC24 siRNA using bioengineered exosomes (BESG) will reduce their dosage; improve patient compliance and life expectancy of patients. BESG will be evaluated for their uptake mechanisms and their mechanism of action in cell culture models of NSCLCs. BESG will specifically target the NSCLCs and release its payloads at cancer site. The underlying mechanisms, pharmacokinetics, biodistribution and anti-cancer activity will be evaluated in xenograft model of NSCLCs using advance imaging technologies. The BESG developed during this program will have a clinical potential and the trainings obtained through this program will help in career advancement in the area of drug delivery and precision medicine. To reach the objectives of “BESG”, Dr Vijay Kumar will adopt a phased set of coherent approaches and methodologies, divided in 3 work packages. WP1 Formulation Development of peptide targeted loaded exosomes: In Task1.1, Dr Vijay Kumar will optimize the isolation of exosomes from MSCs and characterize them. These exosomes will be conjugated with peptide using biochemical conjugation approaches and will be loaded with SPC24 siRNA and GEF to exosomes (using electroporation /microinjection /transfection etc.) by FbD approaches. Task1.2 The loaded exosomes will be characterized for their charge, size, shape and loading efficacy as well as release kinetics mechanisms. WP2 In vitro anti-cancer activity: For this WP, In Task2.1, Dr Vijay Kumar will use the different NSCLC cell lines such as H460, A549 and SK-MES-1 cells to make 2D and 3D NSCLC models Task2.2. “BESG” will be evaluated for cellular uptake mechanisms using FACS, confocal laser scanning microscopy. In addition, in Task2.3, Cellular toxicity assays will be performed to evaluate the in vitro efficacy. The underlying mechanisms for efficacy will be evaluated by qPCR, western blotting and immunocytochemistry. WP3 In vivo Delivery to BESG to xenograft model of NSCLC: Task3.1 In vivo, pharmacokinetics, biodistribution will be evaluated in a xenograft model of NSCLC. Imaging strategies will be employed in this task. Task3.2 Therapeutic efficacy of “BESG” will be evaluated in xenograft model of NSCLC and the underlying mechanisms of anti-cancer and anti-metastatic effects will be evaluated by qPCR, western blot, FACS, histology and immunohistochemistry.
The project will start in October 2020.
BARRICADE - evolutionary platform to predict breast cancer (BC) patients’ outcome and response to therapy
“BARRICADE” is about an evolutionary platform to predict breast cancer (BC) patients’ outcome and response to therapy in a faster and specific manner. Breast cancer is one of the leading causes of cancer mortality among women and is characterized by a high interindividual variability in response to cancer treatment, and often resistance to therapy arise over the time or after first line of treatment. For this reason, the therapeutic approach of each patient has to be set up during its management causing some gaps, useless toxicities and a losing of time useful for patient’s cure. In cancer research, cells are usually grown in a monolayer on dishes of polystyrene plastic to perform experiment or toxicity studies. Alternatively, researchers use animal models that are able to mimic a more physiological growth of tumour cells and they take in account also the microenvironmental influences. Unfortunately, animal models do not recapitulate the human complexity, and sometimes they do not predict tumour behaviour and clinical response. For this reason, organoids are emerging as new more complex system that may overcome the limitations of traditional cancer model. “BARRICADE” will contribute to taking BC cancer therapy to beyond state-of-art, developing personalized anticancer therapies and giving useful information for cure setting up. Indeed, progress in developing in vitro 3D organoid culture system using primary human breast cancer tissues have attracted great interest as an emerging tool for precision medicine. BARRICADE will help to overcome some of those barriers by giving preclinical information for future therapy settings.
To this end, Dr Giuseppina Roscigno will establish a flexible platform of cutting-edge technologies for drug screening and drug penetration based on cancer organoids. To develop a new therapeutic strategy, Dr Roscigno will aim to generate a biobank of BC patients-derived organoids that can be propagated in three-dimensional cultures and she will verify ability of organoid platform to predict in vitro tumour response before patient undergoes to blind therapy. In addition, BARRICADE will give the possibility to Dr Roscigno to characterize molecules able to improve the drug penetration in 3D structures, aiming to increase the delivery efficiency of therapeutic compounds in BC. Therefore, Dr Roscigno aims to generate high-scale organoid cultures and subsequently to develop a 3D platform as a model. The project will start in September 2020.
GI.EXO - Innovative tools to detect Glioblastoma (GBM) exosomes
“Gl.EXO” is about innovative tools to detect Glioblastoma (GBM) exosomes and to have a timely, accurate and non-invasive diagnosis of GBM. Several studies have underlined the role of exosome cell–cell communication in different tumour types, including GBM, suggesting their potential use as diagnostic/prognostic/predictive biomarkers and therapeutic agents. In fact, evidences have demonstrated that the release of GBM exosomes plays a key role in cancer growth and progression by modulating tumour microenvironment and affecting angiogenesis, tumour migration, immune escape and drug resistance. However, GBM exosomes-enriched proteins characterization started only since few years and studies still lack information on GBM exosomes cargo and their impact on cancer progression. Moreover, the identification of specific biomarkers able to discriminate between cancer exosomes from normal exosomes and tools for their detection are necessary and still unknown. Therefore, by inhibiting GBM exosomes uptake, “Gl.EXO” will contribute to hamper GBM progression in many cancer aspects. To address this issue, “Gl.EXO” aims to use nucleic acid-based aptamers as innovative tools to detect specifically GBM exosomes.
Aptamers are selected by an in vitro combinatorial chemistry approach, named Systematic Evolution of Ligands by Exponential enrichment (SELEX), and they can bind with high affinity and specificity virtually any given molecule. Aptamers have many advances over antibodies. They have a small size, resulting in a better tissue penetration, a low toxicity, and no immunogenicity. Moreover, aptamers are chemically synthesised and easily modified to obtain required adjustment. Given the crucial role that GBM stem cells (GSCs) proved to have in tumour maintenance and sustenance, a specific aptamer for GSCs was selected and characterised by Dr Alessandra Affinito. This aptamer demonstrated to be able not only to recognise GSCs but also to bind to exosomes released by GSCs released exosomes. Dr Affinito intends to generate a novel aptamer-based sandwich assay usable as an easy and timely tool for GBM detection through biological fluids (blood, urine or cerebrospinal fluid). Here, this aptamer will be used to develop a non-invasive diagnostic assay for the detection of GSC-derived exosomes detection. Moreover, the pivotal role of exosomes in cancer progression opens great scenarios for GBM therapy. Thanks to the selected aptamer Dr Affinito wants to generate an exosomes-plasmapheresis system able to remove GBM exosomes from fluids biological fluids. Indeed, the exosomes’ uptake hampering and reduction provide a new therapeutic tool for cancer treatment. Thus, the obtained results will represent a fundamental milestone to develop new early diagnostic approach able to ultimately improve early tumour detection and will constitute an important step in the treatment of GBM. The project will start in July 2020.