Periodic Reporting for period 1 - BrEXo-Apt (Non-invasive and accurate diagnosis and treatment of breast cancer.)
Reporting period: 2019-04-01 to 2021-03-31
“BrEXo-Apt” is about non-invasive and accurate diagnosis and treatment of breast cancer (BC). Early screening methods for BC are mainly based on instrumental tests, that may cause in many cases a failure to reach particular categories of patients. This failure could be overcome with the availability of specific and predictive circulating biomarkers allowing non-invasive earliest tumour detection on a larger scale of patients. Recent evidences indicate that the release of a great numbers of exosomes by tumour cells play a key role in tumour progression, drug resistance, immune surveillance escape, angiogenesis, tumour invasion and metastasis. For this reason, cancer-derived exosomes are emerging as very interesting targets for cancer therapy and diagnosis. BC is the most common tumour type and one of the leading causes of cancer mortality in women with more than 450,000 women die annually. The disease stage at diagnosis greatly influences patient survival. Although traditional diagnostic methods, such as mammography, are effective, they are limited by the minimum tumour size required for detection. Therefore, the research of circulating biomarkers is a fundamental challenge that can allow to find out tumours early, when they are non-invasive and most treatable, consequently improving disease outcome and health costs. Aptamers are high affinity ligands of disease-associated proteins and possess many advantages for diagnostic and therapeutic applications including low toxicity, cost-effectiveness, easy synthesis and modification, associated with no immunogenicity. The specific targeting ability of aptamers has ensured their efficient use for biomarker discovery, detection and profiling. In recent years, extracellular endosome-derived vesicles called exosomes are attracting growing interest as promising cancer biomarkers. Exosomes are vesicles of 50–150 nm diameter secreted by cells into circulation and containing nucleic acids and proteins. It has been demonstrated that tumour cells, including BC cells, release excessive amounts of cell specific exosomes that, being stable and easily accessible from body fluids, may be used for cancer specific detection. In addition, cancer exosome involvement in tumour progression and dissemination, drug resistance and immune surveillance. “BrEXo-Apt” took advantage of a novel class of nucleic acid-based molecules named aptamers to specifically identified and inhibit BC exosome and permit early BC detection and early therapeutic treatment. “BrEXo-Apt” used BC aptamers that specifically recognize BC-released exosomes to an early BC diagnosis and treatment. The rationale of our study was that in BC field, non-invasive modality of tumours early diagnosis and target therapy are urgently needed. Aptamers show proprieties that can be easily be applied to this issue. The objective was to develop an innovative, non-invasive approach for early BC detection and its treatment. Towards this question Dr Quintavalle proposed to develop an aptamer-based sandwich assay able to recognize BC-derived exosomes in blood and an aptamer-based affinity plasmapheresis system able to specifically remove BC-derived exosomes.
This is the final period for Dr Quintavalle. The project was concluded in December 2019. Dr Quintavalle started the characterization of one out of three identified aptamers specific for breast cancer exosomes, named ex-50.T. To better characterized ex-50.T aptamer she measured the apparent dissociation constant (Kd) of ex-50.T on BC-derived exosomes by ELONA-based assay. Plates left uncoated or coated with MDA-MB-231 derived exosomes and incubated with increasing amount of biotinylated ex-50.T or CtrlApt. Specific binding analyses revealed ex-50.T high affinity for BC exosomes with a Kd of about 7 nM. Further, she evaluated the aptamer binding to HSA that is the most enriched protein in human plasma that could bind nucleic acids due to its charge, thus limiting their applicability for patient sample analyses. To this purpose, the biotinylated ex-50.T or CtrlApt aptamers were incubated at increasing concentrations on plates previously coated or not with HAS. No aptamer binding was measured up to 1000 nM concentration, thus indicating that ex-50.T does not react with HSA. Ex-50.T aptamer contains 2’F-Py in order to increase the resistance to enzymatic degradation, providing a stable and easy to handle tool. She thus analyzed ex-50.T serum stability by incubating the aptamer in 85% human serum at 37 °C for increasing times. RNA samples were then analyzed by denaturing polyacrylamide gel electrophoresis. She found that the ex-50.T aptamer remains almost stable up to 24 hours and then is gradually degraded, showing an approximate half-life of about 40 hours in our experimental conditions.
In recent years, Dr Quintavalle and colleagues have developed several differential selection strategies successfully isolating aptamers able to discriminate between even related cell types. They have recently developed a novel differential SELEX (Systematic Evolution of Ligands by Exponential enrichment) strategy8 by using exosomes purified from epithelial BC primary cells in the positive selection step and exosome-derived from primary normal epithelial breast cell lines in the negative selection step (manuscript in preparation). Following eight rounds of SELEX, they found that the final pool of aptamers can specifically recognize BC-derived exosomes, discriminating them from those derived from normal breast epithelial cells. Further, the pool has been cloned and individual aptamers sequences enriched, as well as structural elements indicative of potential binding sites, have been identified. Three aptamers have been shown to be enriched in different families, and their ability to selectively bind BC cells-derived exosomes has been proven. Interestingly, two of them can selectively bind triple negative-derived exosomes, compared to more differentiated BC cells. To optimize the best aptamers, shortened version of aptamer (about 85mer) have been generated and their binding ability have been tested. Moreover, the aptamers showed no binding affinity for lung cancer and glioblastoma derived exosomes. The socio-economic impact and the wider societal implications of this project came out of the immunofluorescent experiments. Those showed that the short aptamers can block the up-take of MDA-231-derived exosomes on MDA-231 cell lines. All these encouraging preliminary results in in vitro models indicate a wide range of success for developing new strategies for BC diagnosis and treatment opening a new path towards clinical translation and will represent a proof of concept (POC) for the diagnosis of other cancer types.