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Specific Fluorogenic Peptides for Imaging Metastasis-associated Macrophages

Periodic Reporting for period 1 - MACROIMAGING (Specific Fluorogenic Peptides for Imaging Metastasis-associated Macrophages)

Reporting period: 2015-10-01 to 2017-09-30

Cancer is one of the leading causes of death worldwide, accounting for more than 8 million deaths in 2012 and with predictions of over 20 million new cancer cases each year by 2030. Cancer is also one of the public health priorities for the EU2 as it is necessary to create effective patient-oriented therapies. The metastatic potential of tumours is defined by the tumour microenvironment (TM), where immune cells are the most abundant cells. There is a need for new imaging probes that can selectively report cellular events with high sensitivity and spatiotemporal resolution in real-time. MACROIMAGING is a highly interdisciplinary project encompassing the design, preparation and biological characterization of innovative fluorogenic tools by playing at the interface between peptides, immunology and real-time optical imaging. The two main scientific objectives are:

1. Immune cells (e.g. macrophages) are fundamental in the development of the tumour microenvironment and metastasis. However, the role of macrophages in the TM remains elusive mainly because of the lack of technologies to target these unique populations of cells in vivo. MACROIMAGING aimed at developing the first generation of chemical probes that enable us to visualize with high resolution and target with high selectivity subpopulations of immune cells, in order to maximize therapeutic effect and reduce side effects. The project has provided optical probes that can selectively light up inside specific subpopulations of macrophages and, if conjugated to drugs, also elicit a selective cytotoxic response.
2. Peptides are biopolymers with outstading target specificity and low biotoxicity, properties that have boosted its use as marketed drugs in the past decades. In example, there are several peptide sequences that have been identified as potential cancer biomarkers. However, there is a lack of suitable fluorescent labelling methods that allow for real-time imaging without hampering peptide affinity. MACROIMAGING delivers Trp-BODIPY as an exceptional fluorogenic amino acid with outstanding optical properties that can be incorporated into any peptide sequence with minimal impact on its binding properties. As proof of concept, Trp-BODIPY has been applied to report fungal infection in real-time and to selectively visualize apoptotic vesicles, which play a role in the formation of the pre-metastatic niche.
"One of the main achievements of MACROIMAGING is the generation of profluorophore-drug conjugates that are activated inside specific macrophage subpopulations and suitable for in vivo imaging. As silent profluorophore we selected PhagoGreen, which shows good discrimination of macrophages over other cells in tumours. We further derivatized PhagoGreen with the cytotoxic drug doxorubicin through a cleavable hydrazone bond without compromising the physical and optical properties of the profluorophore (5-fold quantum yield increase at phagosomal pH). Specific cleavage of the prodrug conjugates within the acidic phagosomes of M1 macrophages led to the intracellular release of pH-activatable PhagoGreen as well as doxorubicin for in situ cell tracking and subpopulation-specific macrophage depletion. We have proved the applicability of these conjugates in vivo using regeneration models in zebrafish to image and deplete phagocytic M1 macrophages. Notably, we have observed a proregenerative role for TNF-α expressing M1 macrophages in vivo. Our work has been published in ACS Central Science, one of thetop journals in multidisciplinary chemistry.
Another hallmark of MACROIMAGING has been the optimisation of a protocol to prepare Trp-BODIPY, an exceptional fluorogenic amino acid that can be incorporated into peptides to produce optical probes with minimal interference in the peptide physicochemical properties. Aside from the excellent photophysical properties of the BODIPY fluorescent scaffold, Trp-BODIPY includes a molecular rotor that is non-fluorescent in aqueous media and highly fluorescent in hydrophobic environments (30-fold quantum yield increase), making it an optimal reporter for imaging biological processes in intracellular hydrophobic granules or cell membranes. We have proven that the coupling of BODIPY to the C2 position of Trp via a spacer-free C-C linkage does not impair the molecular interactions of Trp. We have conjugated the amino acid to an antifungal peptide (BODIPY-cPAF26) for wash-free imaging of fungal pathogens, including real-time visualization of Aspergillus fumigatus (published in Nature Protocols). In addition, we prepared a fluorescent version of a small phosphatidylserine(PS)-binding peptide (cLacBODIPY) to perform imaging of apoptotic bodies. We demonstrated specific binding to PS in Giant Unilamellar Vesicles as well as imaged apoptotic vesicles from BL2 cells (published in Chemical Communications). The IP of this work has been also protected (WO2016207626). License agreements have been signed with different companies to commercialise Fmoc-Trp-BODIPY-OH (Sigma-Aldrich, Cat # 900606) and Trp-BODIPY-labelled antimicrobial peptides (Cambridge Research Biochemicals, Cat # Discovery® Peptides). In addition, the fellow has disseminated the work in national (RSC Chemical Biology 2016, and RSC Bioorthogonal&Bioresponsive 2017) and international conferences (European Peptide Symposium 2016).
Therapies targeting immune cells are promising approaches for intractable tumours, but unfortunately there are very limited tools to visualise and understand how they stop cancer progression in vivo. Current fluorescent methods to image macrophages rely on technologies that are not compatible with in vivo imaging or have limited clinical translation. Small fluorophores stand out as an optimal tool for biomedical imaging, due to high sensitivity, low cost and labelling without genetic manipulation. However, to date, activatable fluorophores mostly discriminate largely different cells and cannot discriminate close subpopulations of immune cells. In MACROIMAGING, we have delivered the first fluorophore-drug conjugates that can selectively modulate the function of M1macrophages in vivo without affecting other macrophage subpopulations. This discovery paves the way for future selective treatments to increase prognosis and facilitate clinical translation. Our ongoing collaborations with world-leaders in cancer biology will produce second-generation probes for in vivo imaging in relevant animal models and ex vivo in resections of human cancer tissue.

Most peptides cannot be directly visualized using fluorescence imaging as they lack proper reporter groups. A critical step in peptide labelling is the incorporation of fluorophores without impairing the peptide properties. This has been achieved by introducing unnatural amino acids with chemical groups reactive to fluorophores, although this two-step approach often includes washing steps that impede real-time imaging. In MACROIMAGING we have optimized the use of an environmentally-sensitive amino acid (Trp-BODIPY) for labelling peptides and allow real-time monitoring of cellular events. This methodology embraces potential for future projects in chemical biology and medicinal chemistry connected with protein recognition, drug discovery and theranostics.