Landscape of exosomes to modified exosomes: a state of the art in cancer therapy

Abstract

Exosomes are a subpopulation of extracellular vesicles (EVs) that naturally originate from endosomes. They play a significant role in cellular communication. Tumor-secreted exosomes play a crucial role in cancer development and significantly contribute to tumorigenesis, angiogenesis, and metastasis by intracellular communication. Tumor-derived exosomes (TEXs) are a promising biomarker source of cancer detection in the early stages. On the other hand, they offer revolutionary cutting-edge approaches to cancer therapeutics. Exosomes offer a cell-free approach to cancer therapeutics, which overcomes immune cell and stem cell therapeutics-based limitations (complication, toxicity, and cost of treatment). There are multiple sources of therapeutic exosomes present (stem cells, immune cells, plant cells, and synthetic and modified exosomes). This article explores the dynamic source of exosomes (plants, mesenchymal stem cells, and immune cells) and their modification (chimeric, hybrid exosomes, exosome-based CRISPR, and drug delivery) based on cancer therapeutic development. This review also highlights exosomes based clinical trials and the challenges and future orientation of exosome research. We hope that this article will inspire researchers to further explore exosome-based cancer therapeutic platforms for precision oncology.

Fig. 1. Timeline of exosome-based therapeutics (reproduced with permission under Creative Commons CC BY 4.0 license from ref. Copyright@2022 The Authors).

Fig. 2. Exosome biogenesis (reproduced with permission under Creative Commons CC BY 4.0 license from ref. Copyright@2020 The Authors).

Fig. 3. Exosome isolation and characterization. (a) Contact-free exosome sorting, (b) schematics of a microfluidic chip that enables continuous mixing and isolation of EVs using immunomagnetic beads, microscopy images of the device: (c) Y-shaped injector, (d) serpentine fluidic mixer for immunomagnetic binding, (e) magnetic aggregates, and (f) bound EVs on immunomagnetic beads. (g) Transmission electron microscopy (TEM) image of exosomes, (h) scanning electron microscopy (SEM) image of exosomes, (i) atomic force microscopy (AFM) image of exosomes, (j) cryo-electron microscopy (cryo-EM) image of exosomes, (k) western blotting based EVs protein expression analysis (figure (a) to (k) reproduced with permission from ref. Copyright@2018 American Chemical Society), and (l) single EV profiling approaches (reproduced with permission from ref. Copyright@2022 American Chemical Society.).

Fig. 4. Impact of exosomes in cancer. (reproduced with permission under Creative Commons CC BY 4.0 license from ref. Copyright@2020 Nature publisher).

Fig. 5. Therapeutic exosomes and modified exosomes in cancer therapy (created with https://www.Biorender.com).

Fig. 6. Immune cell-derived exosomes in cancer inhibition. Cytolytic activity of CAR exosomes in vitro. (a) Flow cytometry analyses of CAR exosomes linked to latex beads (4 mm diameter) or CAR-T cells stained with the indicated primary Abs. The histograms shown in black correspond to the isotype controls of the respective Abs, whereas the red histograms indicate positive fluorescence. (b) Immunoblots for perforin and granzyme B expression in CAR exosomes and CAR-T cells. (c) Killing activity of CAR exosomes in response to tumor cells. (d) Confocal microscopy analysis of MCF-7 EGFR cells (up) and MCF-7 HER2 cells (down) after incubation with NHS-Rhodamine (Rho)-labelled CAR-EXO-CTX for 2 h, CAR exosomes have notable anti tumor activity in vivo. (e) Tumor volumes of MDA-MB-231 (left), HCC827 (middle) and SK-BR-3 (right) tumor xenografts after treatment with the indicated treatment, (f) and (g) tumor volumes of MDA-MB-231 (b) and SK-BR-3 (c) tumor xenografts after treatment with the indicated CAR exosome treatment with or without blocking recombinant antigen, and (h) cancer cell lines or patient-derived tumour tissue fragments established as subcutaneous xenografts (reproduced with permission under Creative Commons CC BY 4.0 license from ref. Copyright@2019 The Authors).

Fig. 7. Stem cell-derived exosomes in cancer therapy. (a) Schematic overview of the exosome production process, (b) western blot of cell lysate (cells) and exosomes (exo). To confirm the purity of the exosomes, positive exosomal markers CD63, syntenin and CD9 and negative exosomal markers vinculin (Vinc.) and calreticulin (Calr.) and β-Actin (actin) were analyzed, (c) scanning electron micrograph of purified exosomes, and (d) size distribution of exosomes determined by insights showing that the highest abundance of particles was below 200 nm. (e) Hoechst-stained MenSC on BioNOC II carrier, showing a typical confluence for exosome production. (f) Yield of purified exosomes in PBS as particles (part) per mL of initial cell culture supernatant. Tumor growth and angiogenesis is significantly reduced by exosome treatment. (g) Scheme of experimental design. Tumors were induced with four weeks of DMBA treatment and four injections of exosomes were administered every 3–4 days, (h) and (i). Tumor growth in mm³ tumor volume and relative tumor growth indicating days of exosome treatment. Control tumors are shown as triangles and exosome treated tumors as circles. (j) Histological sections of tumors at day 25 (end-point) with Hematoxylin and eosin stain (H&E), and (k) Dextran-Fitc (green), VE Cadherin (red) and Hoechst (blue) stained histological sections of tumors at day 25 (end-point) (Reproduced with permission under Creative Commons CC BY 4.0 license from ref. Copyright@2019 The Authors).

Fig. 8. Plant-derived exosomes in cancer inhibition. (a) Plant exosomes in the sucrose gradients after ultracentrifugation, (b) TEM imaging (scale bar: 100 nm), (c) AFM imaging, (d) hydrodynamic particle size distribution, (e) lipid compositions, (f) protein summary, (g) KEGG annotated statistical charts, (h) Go secondary classification statistical charts of plant exosomes, (i) flavonoids, and (j) polyphenols in plant exosomes. In vitro anti-tumor effects of plant exosomes, (k) cytotoxicity of plant exosomes against various tumor cell lines after co-incubation with plant exosomes, (l) pro-apoptotic properties of TLNTs after co-incubation with plant exosomes, (m) CLSM images of 4T1 cells stained with DCFH-DA after co-incubation with plant exosomes for 4 and 8 h, (n) ROS fluorescence intensity of 4T1 cells after co-incubation with plant exosomes for 4 and 8 h, respectively. (o) Mitochondrial membrane potential changes in 4T1 cells (scale bar: 50 μm), (p) TLNTs restrained cell cycle progression in 4T1 cells after co-incubation with plant exosomes for 12 and 24 h, respectively, (q) western blot analysis of 4T1 cells receiving the treatment of plant exosomes for 48 h. Cyclin A, cyclin B and cyclin D proteins were probed. GAPDH was probed to ensure the equal loading of total proteins in each lane (reproduced with permission under Creative Commons CC BY 4.0 license from ref. Copyright@2023 The Authors).

Fig. 9. Bacteria-derived exosomes in cancer inhibition. (a) Outer membrane vesicles (OMV) OMV-PD1 were obtained by engineering E. coli to stably express the mouse PD1 ectodomain fused with the surface protein ClyA and then purifying the vesicles from the parent bacteria by ultracentrifugation. OMV-PD1 accumulation at the tumor site increases the infiltration of immune cells, such as DCs and NK cells, and activates an immune response in vivo. At the same time, the PD1 ectodomain on the OMV-PD1 surface blocks the PD1/PD-L1 interaction and protects CD8+ T cells, which can then attack tumor cells. (b) A schematic illustration of the construct used to express ClyA-mPD1E-3HA, (c) western blot analysis of the expression of ClyA-mPD1E-3HA on the OMV, detected using an anti-HA and an anti-PD1 antibody, (d) and TEM images of OMV and OMV-PD1. Scale bar = 100 nm. (e) The size distribution of OMV and OMV-PD1 was measured by DLS. (f) Representative TEM image of OMV-PD1 immunostained with an anti-HA primary antibody and then with an immunogold-labeled secondary antibody. Red arrowheads indicate 5 nm gold particles. Scale bar 25 nm. Antitumor effects of OMV-PD1 in vivo. (g and h) Growth curves of subcutaneously implanted B16 tumors (g) and CT26 tumors (h) following treatment with saline, αPD-L1, OMV, OMV + αPD-L1, or OMV-PD1, (i and j) the final weight of excised B16 tumors (i) and CT26 tumors (j) from mice in each group at the end of the treatment, (k) representative H&E (upper) and fluorescent TUNEL (bottom; indicates apoptotic cells) stained sections of B16 and CT26 tumor tissue at the end of the experiments. (l and m) Statistical analysis of the number of TUNEL+ cells per field for B16 (l) and CT26 (m) tumor. (n) Survival curves of CT26 tumor-bearing mice treated with different formulations, and (o) tumor volumes of subcutaneous re-challenge with CT26 tumor cells at day 55 in OMV-PD1-cured CT26 tumor-bearing mice (reproduced with permission under Creative Commons CC BY 4.0 license from ref. Copyright @2020The Authors).

Fig. 10. Advantages of exosome-based drug delivery (reproduced with permission under Creative Commons CC BY 4.0 license from ref. Copyright 2022 The Authors).

Fig. 11. Exosome-based drug delivery. (a) The main composition of the EGFP-C1-iRGD-Tyr7-Lamp2b plasmid and an image of iRGD/blank-Tyr7-EGFP-293T cells using fluorescence microscopy (scale bar = 100 μm). Representative TEM images and particle size distribution of (b) blank-Exos, (c) iRGD-Exos, (d) western blotting analysis of exosome marker proteins (TSG101, CD9 and Alix) of blank-Exos and iRGD-Exos, (e) blank-Exos-131I, (f) iRGD-Exos-131I and (g) Dox@iRGD-Exos-131I, in vitro targeting of iRGD-Exos. (h) Confocal microscopy images of 8505C cells incubated with PKH26-blank-Exos and PKH26-iRGD-Exos at 4 h. Nuclei were stained with DAPI (blue). Fluorescence from PKH26 (red) and DAPI (blue) was observed. The scale bar is 10 μm. (i) Flow cytometric analysis of PKH26-iRGD-Exos binding to 8505C cells. Exosomes were labelled with PKH26 and incubated with 8505C for different lengths of time, viability of (j) Nthy-ori 3-1 and (k) 8505C cells treated with different concentrations of iRGD-Exos, (l) 8505C and (m) Hth7 cells were incubated with control medium, iRGD-Exos, Na131I, blank-Exos-131I, iRGD-Exos-131I, Dox, Dox@ iRGD-Exos, or Dox@iRGD-Exos-131I. A CCK-8 assay was used to assess cell viability in each group (reproduced with permission under Creative Commons CC BY 4.0 license from ref. Copyright @ 2022 The Authors).

Fig. 12. Exosome surface modification (reproduced with permission under Creative Commons CC BY 4.0 license from ref. Copyright @ 2021 The Authors).

Fig. 13. Chimeric exosomes in tumor inhibition. (a) A schematic illustration of the design of biomimetic ACEs for anti-phagocytosis and targeted cancer therapy. (b) TEM images of ACEs, (c) hydrodynamic diameter and, (d) zeta potential of liposomes, AREs, AMEs and ACEs. AFM images of (e) liposomes and (f) ACEs reveal the presence of hinged structures on the surface of ACEs. (g) Protein content visualization of (1) RBCs, (2) AREs, (3) ACEs, (4) AMEs and (5) MCF-7 cells. (h) Membrane protein characterization by western blotting analysis of (1) RBCs, (2) AREs, (3) ACEs, (4) AMEs and (5) MCF-7 cells. (i) In vitro DOX release behaviour at 37 °C, intracellular uptake and cytotoxicity of ACEs. (j) In vitro DOX fluorescence imaging of ACEs in MCF-7 cells, HeLa cells, and RAW264.7 cells after 2 h incubation. The nucleus was stained with Hoechst 33342 (blue). The vesicles were loaded with DOX (red), (k) semiquantitative intracellular uptake of ACEs determined by the averaged DOX fluorescence intensity of each cell, in vitro cytotoxicity of different nanovesicles (l) without DOX and (m) with various concentrations of DOX after 24 h incubation with MCF-7 cells. In vivo antitumor efficacy of ACEs to tumor-bearing nude mice. (n) Tumor growth curves of different groups after treatments. (o) Changes of body weight with increasing time. (p) Representative tumor photos and (q) H&E stained tumor sections from tumor-bearing mice after treatment with (1) PBS, (2) liposomes, (3) AREs, (4) AMEs and (5) ACEs (reproduced with permission under Creative Commons CC BY 4.0 license from ref. Copyright @ 2018 The Authors).

Fig. 14. Exosome-based CRISPR transport. (a) A schematic illustration of exosome for in vivo delivery of Cas9 RNP for the treatment of liver disorders, (b) and exosomeRNP complexes (c), (d) biomarkers of exosome by western blotting. (e and f) DLS and TEM images of purified exosome. The arrows show the typical exosome nanoparticles. (f and G) DLS and TEM images of exosomeRNP complexes. The arrows show the typical exosomeRNP nanoparticles. (h and i) Cytosolic delivery of Cas9-FITC into LX-2 (h) and Huh-7 (i) cells by exosomes for 4 hours. The red arrows point at the efficient translocation of RNP into the nuclei. Scale bars, 25 μm. DAPI, 4′,6-diamidino-2-phenylindole. (j) Exosome-mediated Cas9 RNP delivery for genome editing. (k) Frequency of PUMA indel mutation detected by T7E1 assay from AML-12 cells after the specified treatments. (l) Frequency of CcnE1 indel mutation detected by T7E1 assay from AML-12 cells after the specified treatments. (m) Frequency of KAT5 indel mutation detected by T7E1 assay from LX-2 cells after the specified treatments. (n) In vivo distribution of DiR-labeled exosomes in whole mice (top) or in the organs of the mice (bottom). H, heart; Lu, lung; Li, liver; K, kidney; and S, spleen. (o) A schematic illustrating the procedure to isolate different hepatic cell types and determine exosomeRNP biodistribution. (p) Percentage of each hepatic cell type that is DiI-labeled exosomeRNP-positive. (q) Relative MFI of each hepatic cell type. (r and s) Mechanism of cellular uptake of exosomeRNP nanocomplexes in LX-2 (r) and Huh-7 (s) cells by the addition of different inhibitors (reproduced with permission under Creative Commons CC BY 4.0 license from ref. Copyright @ 2022 The Authors).

Fig. 15. Liposome hybrid in cancer therapy (a) representative image of exosomes captured by TEM at different magnifications. (b) The size distribution of exosomes and (c) the particle size distribution range of exosomes as measured by NTA. (d) The morphology of HENPs detected by TEM. (e) Size distribution of liposomes and HENPs. (f) The FRET assay showed the successful fusion of exosomes and liposomes. (g) Protein expression of exosomes and HENPs nanovesicles. (h and i) The nanoparticle size and PDI over time, used to assess the stability of the nanoparticles. (j) Zeta potential distribution of exosomes, liposomes and HENPs. (k and l) Release profiles of miRNC and TP at pH values of 5.5 and 7.4 at 37 °C. The targeting and antitumor activity of miR497/TP-HENPs in vivo. (m) In vivo imaging to observe the tumor targeting ability of different nanoparticles. (n) Ex vivo fluorescence images of the main organs and tumors isolated from mice bearing subcutaneous SKOV3-CDDP tumors. (o) Quantitative analysis of Dir distribution in the tumor site postinjection elevated by the fluorescence intensity measured in (m). (p) Quantitative assessment of the mean fluorescence intensity in major organs and isolated subcutaneous tumors. (q) Representative photographs of subcutaneous tumors harvested from all treatment groups. (r) Growth record curves of subcutaneous tumors in nude mice during the experiment. (s) The inhibition rate of OC treated with various drugs. (t) The H&E staining and TUNEL staining. (u) Immunohistochemical detection of ki67, p-PI3K, p-AKT, and p-mTOR (reproduced with permission under Creative Commons CC BY 4.0 license from ref. Copyright @ 2022The Authors).

Divya Mirgh

Swarup Sonar

Srestha Ghosh

Manab Deb Adhikari

Vetriselvan Subramaniyan

Sukhamoy Gorai

Krishnan Anand