Dr. Apurba Dev
Associate Prof., Uppsala University, Sweden
Decoding the heterogeneity of nanoscale extracellular vesicles
Abstract: Nanoscale extracellular vesicles (EVs), capable of transmitting biologically active macromolecules, are emerging as key players in intercellular communication demonstrating their potential as diagnostics markers as well as therapeutic agents/targets1. Cells, however, release a variety of these vesicles which vary in size, compositions and biomechanical properties. EV phenotypes are known to depend on their cellular origin and the activation status of their parental cells. Besides, EV cargoes may also act in a combinatorial manner to communicate cellular directives making their functions multifaceted and contextually complex. As a result, an explicit understanding
of their physiological/pathological relevance and spatiotemporal fate, requires the capacity to elucidate their phenotypic variations in relation to their function. However, high heterogeneity of EV subtypes even from a single cell type means that an averaging analysis is largely ineffective. Instead a multiparametric analysis of single EVs is necessary in order to decipher their heterogeneity and consequently identify their function. If realized, such a system holds a great promise to advance our understanding of these biological nanoparticles and at the same time offer to improve and widen the prospect of EV-based diagnostics and therapeutics.
In this talk, I shall present a method which combines atomic force and fluorescence microscopy to characterize individual nanoscale vesicles by their size, membrane protein composition and mechanical properties2. We show that, the membrane protein composition does show high heterogeneity but also reveals interesting correlation depending on their parental cells. Further, the study demonstrate that in contrast to the common belief, the larger vesicles do not carry more proteins indicating that the cells may preferentially load proteins in certain vesicles. The method, when applied to a lung-cancer model system, was able to make a distinction between the effect of
two different precision cancer medicines. The induced changes in membrane protein expression of EVs as a result of the medicines was found to resemble the observation made on cells having the same mutations. The results show a clear prospect for early prediction/monitoring of cancer-drug efficacy using liquid biopsies since the EVs can be captured from circulations in body fluids. However, an accurate measurement of elastic properties of these EVs appeared to be more challenging due to their small size and thermal fluctuations, which necessitates the application of stochastic thermodynamics.
We first demonstrate that the thermal fluctuations play a major role leading to a large spread in the force-distance curves. Following that, we argue how Crooks fluctuations theorem3 can in principle be applied to generate equilibrium free energy-distance plot from a set of non-equilibrium force-distance measurement4. Finally, I shall briefly present the development of a microchip-based technology for the analysis of EVs in clinical settings5 or for home-based health care.
References:
(1) Pegtel, D. M.; Gould, S. J. Exosomes. In Annual Review of Biochemistry, Vol 88, Kornberg, R. D. Ed.; Annual Review of Biochemistry, Vol. 88; 2019; pp 487-514.
(2) Cavallaro, S.; Pevere, F.; Stridfeldt, F.; Gorgens, A.; Paba, C.; Sahu, S. S.; Mamand, D. R.; Gupta, D.; El Andaloussi, S.; Linnros, J.; et al. Multiparametric Profiling of Single Nanoscale Extracellular Vesicles
by Combined Atomic Force and Fluorescence Microscopy: Correlation and Heterogeneity in Their Molecular and Biophysical Features. Small 2021, 17, e2008155. DOI: 10.1002/smll.202008155.
(3) Crooks, G. E. Nonequilibrium measurements of free energy differences for microscopically reversible Markovian systems. Journal of Statistical Physics 1998, 90 (5-6), 1481-1487. DOI: 10.1023/a:1023208217925.
(4) Hartmann, C.; Schutte, C.; Zhang, W. Jarzynski's Equality, Fluctuation Theorems, and Variance Reduction: Mathematical Analysis and Numerical Algorithms. Journal of Statistical Physics 2019, 175 (6), 1214-1261. DOI: 10.1007/s10955-019-02286-4.
(5) Cavallaro, S.; Haag, P.; Sahu, S. S.; Berisha, L.; Kaminsky, V. O.; Ekman, S.; Lewensohn, R.; Linnros, J.; Viktorsson, K.; Dev, A. J. b. Multiplexed electrokinetic sensor for detection and therapy monitoring
of extracellular vesicles from liquid biopsies of non-small-cell lung cancer patients. Biosensors and Bioelectronics 2021, 193, 113568-113579. DOI: 10.1016/j.bios.2021.113568