br model and demonstrate the potential use
model and demonstrate the potential use of this technology for bio-marker discovery in the presence and absence of a functional immune system.
Progenesis statistical analysis of the corona proteins revealed 328 proteins diﬀerentially expressed with statistical significance (p value < 0.05) between tumor-bearing and healthy animals, of which 178 were upregulated and 150 were downregulated (Fig. 5A and Table S9). In addition, disease and function IPA search demonstrated the association of 172 diﬀerent proteins with previously reported adeno-carcinoma pathways (with a p value of 2.6E-5); (Fig. S3), and the as-sociation of 43 proteins with lung cancer pathways, as shown in Fig. 5B. According to IPA, 8 out of the 43 lung cancer related corona proteins have been already described in the literature as potential biomarkers (3 for diagnosis, 1 for treatment eﬃcacy, 1 disease progression, 1 safety and 1 unspecified application) (Fig. 5B). It is becoming increasingly evident that tumor progression is determined by a number of complex interactions between malignant Paclitaxel (Taxol) and their surrounding endogenous host stromal cells . The identification of intracellular and lung-carcinoma associated proteins (Fig. 5B), suggests that the nanoscale liposomal corona contains host (murine) stromal released proteins.
Finally, we wanted to investigate whether any uniquely human proteins, released from the transplanted A549 cells into the blood cir-culation of SCID mice, were found in the liposomal corona. All corona proteins identified, were sorted into ‘human’, ‘mouse’, or ‘indis-tinguishable’ based on their species-specific sequences. To confirm the species assignment, human peptides were searched using BLAST against a mouse-only database (UniProt/SwissProt) to remove any human se-quences that exactly matched mouse sequences. When considered the detection of human-sourced protein material to be evidenced by the detection of two or more human specific peptides per protein identity (Fig. 5C), four human proteins were found to be surface-captured by liposomes. Among them, proliferation marker protein Ki-67 was pre-viously described as a potent biomarker with significant predictive and prognostic value in lung cancer . The identification of human proteins in the mouse serum of xenograft models has been scarcely attempted previously and required the extensive multidimensional fractionation of serum proteins prior to proteomic analysis . In this study, our results suggest that liposomes can act as carriers not only for low abundance blood circulating proteins released from the host cells of the tumor microenvironment, but also for highly specific biomarker proteins shed by tumor cells.
New molecular biomarkers are needed to improve cancer diagnosis and evaluate disease progression and response to treatment . The proteomic technologies available today are powerful tools for cancer biomarker detection. However, the discovery of blood biomarkers using proteomics is hampered by the wide dynamic range of blood proteins which spans more than ten orders of magnitude, with tumor-tissue derived proteins present at the lower end of this range . Robust high-throughput proteomic platforms to facilitate the identification of blood-buried molecules are of immediate importance. Nanotechnology-en-abled discovery of biomarkers, holds great promise but it is still in its
infancy. Previously described nanoparticle-based technologies have been mainly used to detect already known disease-specific molecules [26–29]. Even though NPs have been found to spontaneously interact with a wide range of diﬀerent protein molecules once in contact with biofluids, the surface-capture of disease-specific molecules by blood circulating NPs has never been attempted before .
The concept of utilizing the nanoparticle protein corona fingerprints for biomarker discovery has been only theoretically proposed and re-mains experimentally unexplored , with the exception of two re-cent reports that investigated protein corona formation after the ex vivo incubation of active maghemite nanoparticles (SMANS) in the milk of cows aﬀected by mastitis [31,32]. Whether the NPs employed in this study are able to harvest disease specific molecules from more complex and clinically-relevant biofluids, such as plasma, remains to be ex-plored. The idea of utilizing protein corona for biomarker discovery was further reinforced by preliminary ex vivo studies suggesting that changes in the plasma proteome, induced by diﬀerent disease pheno-types, may be reflected in the protein corona [33,34].