(F-208) Optical metabolic signatures of extracellular vesicles from biofluids and ex vivo tissue in breast cancer using label-free multimodal multiphoton microscopy

Introduction:: Extracellular vesicles (EVs) are sub-micron particles released by all cell types that play a role in cell-to-cell communication. In cancer, EVs have been implicated in metastasis and proposed as biomarkers for screening and diagnostics. Barriers to characterization of EVs include their heterogeneity and small size, which makes assessments of inter-EV biochemical heterogeneity difficult [1]. Additionally, many methods are unable to access EVs in their native microenvironment, instead relying on isolation methods which can take hours and remove the EVs from their in situ locations and niche, losing spatial context and potentially altering their function.

One method that provides single-EV capabilities and can be used to characterize both isolated EVs and EVs in ex vivo and in vivo environments is label-free multimodal multiphoton microscopy. This method relies on endogenous contrast mechanisms, such as multiphoton autofluorescence intensity and lifetime of NAD(P)H and FAD. This has been extensively used to quantify metabolism in cells and tissues, but only recently applied for characterizing EVs in a limited number of studies [2-6]. Previous work has shown promise for using the optical metabolic signatures of NAD(P)H and FAD for differentiating cancerous EVs in ex vivo tissue [2,3] or isolated EVs from urine [4,5], but no prior study has compared the optical metabolic signatures from isolated biofluid-derived EVs to ex vivo tissue EVs. Thus, this work aims to examine and compare the capabilities of label-free multimodal multiphoton microscopy for extracting screening and diagnostic metrics from both biofluid-derived isolated EVs and ex vivo tissue EVs in breast cancer.


Materials and Methods:: Before surgery, fresh urine and blood serum samples were obtained from human breast cancer patients (n = 31; 18 with invasive tumors, 7 with DCIS, 6 with benign tumors) or healthy (control) breast reduction surgery patients with no history of cancer (n = 5). EVs were isolated from the biofluids by differential ultracentrifugation: samples were spun at 800×g for 10 min, 2000×g for 30 min, and 12000×g for 60 min; after the final spin, the EV pellet was resuspended in sterile PBS. Isolated EVs were stabilized in glycerol or agarose and imaged with custom multimodal multiphoton microscopy systems to extract the NAD(P)H and FAD multiphoton intensity and lifetimes with single-EV capabilities [2-4,6]. After surgery, tissue samples from tumor regions, normal-appearing regions adjacent to tumors, or normal tissue from breast reduction surgery patients was obtained. Fresh ex vivo tissue samples were imaged with the same systems as isolated EVs. EVs were segmented within images and their optical metabolic signatures were compared across sample origin and patient cancer status.

Informed consent was obtained from research participants. This study was approved by the Institutional Review Boards at Carle Foundation Hospital and the University of Illinois at Urbana-Champaign.


Results, Conclusions, and Discussions:: Preliminary results indicated significant differences based on EV origin, with urinary and serum-derived EVs showing lower autofluorescence intensities than ex vivo tissue EVs from the same patient. Additionally, urinary EVs had significant elevated NAD(P)H and FAD intensity compared to serum-derived EVs. This suggests that biofluid-derived isolated EVs do not functionally mirror EVs in the tissue microenvironment, even when subpopulation analysis is applied, and that EV origin plays a large role in the measured optical metabolic signature.

The fluorescence lifetime of NAD(P)H in urinary EVs was significantly elevated in cases of invasive cancer compared to patients without cancer, yet the origin of this difference is unknown (whether this is from EVs from the primary tumor or showing a secondary effect such as patient immune response). Furthermore, no significant differences were found between groups within serum-derived EVs. In ex vivo tissue EVs, the quantity of EVs in the tumor microenvironment was significantly elevated when compared to normal tissue from control subjects, confirming that EVs are elevated in the tumor microenvironment [2]. These data suggest that ex vivo tissue EVs and urinary isolated EVs may provide better diagnostic capabilities than serum-derived EVs and necessitate further research into the origins of biofluid-derived EVs and functional changes that occur in biofluid transport, collection, and isolation.


Acknowledgements (Optional): : JES was supported by UIUC Department of Bioengineering and an NIH/NIBIB training grant (T32EB019944). This work was supported in part by NIH (R01CA213149, P41EB031772). The authors thank Darold Spillman for his technical and administrative support.


References (Optional): : [1] SH Hilton, IM White, “Advances in the analysis of single extracellular vesicles: A critical review,” Sens. Actuators Rep., 3:100052 (2021).

[2] Y Sun, et al., “Intraoperative visualization of the tumor microenvironment and quantification of extracellular vesicles by label-free nonlinear imaging,” Sci. Adv., 4(12): eaau5603 (2018).

[3] S You, et al., “Label-free visualization and characterization of extracellular vesicles in breast cancer,” Proc. Natl. Acad. Sci., 116(48):24012-24018 (2019).

[4] J Park, et al., “Label-free optical redox ratio from urinary extracellular vesicles as a screening biomarker for bladder cancer,” Am. J. Canc. Res., 12(5): 2068-2083 (2022).

[5] AV Borisov, et al., “A Criterion of Colorectal Cancer Diagnosis Using Exosome Fluorescence-Lifetime Imaging,” Diagnostics, 12(8)L 1792 (2022).

[6] JE Sorrells, et al., “Label-free characterization of single extracellular vesicles using two-photon fluorescence lifetime imaging microscopy of NAD(P)H,” Sci. Rep., 11:3308 (2021).