Journal: medRxiv

Article Title: Red Blood Cell-Derived Exosomal Oncogenic MicroRNA Promote Cancer Development and Progression

doi: 10.1101/2024.05.10.24307177

Figure Lengend Snippet: Analysis of miR-93-5p and miR-451 in lung cancer patients and cancer-free smokers (controls). (A) The expression of miR-93-5p is significantly higher in the RBCs and RBC-derived exosomes of lung cancer patients compared to controls (all p≤ 0.01, t-test). The expression of miR-93-5p is undetectable in plasma for both groups. (B) The correlation plot displays a positive relationship between miR-93-5p levels in RBCs and their derived exosomes in lung cancer patients, with a Pearson’s correlation coefficient of r=0.732 and p<0.01. (C) Kaplan-Meier survival curves for lung cancer patients categorized by levels of miR-93-5p in their RBCs and exosomes. The green line represents patients with low levels of miR-93-5p in both RBCs and exosomes, the red line corresponds to patients with high levels of miR-93-5p in RBCs, and the blue line is for patients with high levels of miR-93-5p in exosomes. Patients with higher miR-93-5p levels in RBCs or exosomes have reduced survival rates compared to those with lower levels. (*, all p<0.05). (D) The expression patterns for lung cancer patients and cancer-free controls show that miR-451 is detectable in all sample types with no significant difference between the patient and control groups. ns, not significant.

Article Snippet: To further validate the therapeutic targeting of miR-93-5p, twenty additional mice were subcutaneously injected with A549-Luc cells (CCL-185-LUC2™, ATCC) and treated with either a ASO-control or a ASO-miR-93-5p (AstraZeneca, Wilmington, DE).

Techniques: Expressing, Derivative Assay

Journal: medRxiv

Article Title: Red Blood Cell-Derived Exosomal Oncogenic MicroRNA Promote Cancer Development and Progression

doi: 10.1101/2024.05.10.24307177

Figure Lengend Snippet: miRNA transfer from RBCs to lung cancer cells via exosomes (EXO.). (A) Relative levels of miR-93-5p in cancer cells after exposure to RBCs from cancer patients (Can. Pat.) and healthy controls, in upper chamber (Up chamber), lower chamber (LO. chamber), and regular medium as negative control. (B) Expression of miR-451 in cancer cells under the same experimental conditions as A. (C-D) Electron microscopy image and Western blot analysis demonstrating the isolation of exosomes and presence of Ago2 proteins of lung cancer patinets and healthy controls, respectively. (E) Quantification of miR-93-5p in isolated exosomes and Ago2-immunoprecipitated complexes, indicating significant presence in exosomes but not in Ago2 complexes. (F) Analysis of miR-451 levels in the same isolated fractions as in E. G) Fluorescent microscopy images showing the uptake of exosomes (red) marked with CD63 (exosome marker) in lung cancer cells with nuclei stained in blue. (H) Similar to G, showing uptake in cells incubated with exosomes from healthy controls. (I) The percentage of cancer cells positive for CD63 following incubation with exosomes sourced from both cancer patients and healthy controls. The exosomes released by RBCs of healthy controls exhibit CD63 labeling on cancer cells, indicating a baseline level of RBC exosomal activity. (J) Bar graphs displaying the levels of miR-93-5p and miR-451 in cancer cells after treatment with exosomes from cancer patients and healthy controls. (K) Effects of the exosome release inhibitor GW4869 on miRNA transfer from RBC-derived exosomes to cancer cells. Fluorescence images display nuclei stained with DAPI (blue) and CD63 immunostaining (red) in cancer cells. The up panel depicts cells treated with GW4869, showing an absence of red fluorescence, indicating reduced exosome uptake. The low panel shows untreated cells, displaying prominent red fluorescence. (L) The levels of miR-93-5p and miR-451 in cancer cells are lower after GW4869 treatment compared to untreated cells. (M) Effects of the exosome uptake inhibitor Dynasore on miRNA transfer. Cancer cells treated with Dynasore show lower levels of miR-93-5p and miR-451 compared to those without Dynasore treatment. ns, not significant, **, p<0.01, ***, p<0.001.

Article Snippet: To further validate the therapeutic targeting of miR-93-5p, twenty additional mice were subcutaneously injected with A549-Luc cells (CCL-185-LUC2™, ATCC) and treated with either a ASO-control or a ASO-miR-93-5p (AstraZeneca, Wilmington, DE).

Techniques: Negative Control, Expressing, Electron Microscopy, Western Blot, Isolation, Immunoprecipitation, Microscopy, Marker, Staining, Incubation, Labeling, Activity Assay, Derivative Assay, Fluorescence, Immunostaining

Journal: medRxiv

Article Title: Red Blood Cell-Derived Exosomal Oncogenic MicroRNA Promote Cancer Development and Progression

doi: 10.1101/2024.05.10.24307177

Figure Lengend Snippet: Effects of RBC-derived exosomal miR-93-5p on tumorigenicity of lung cancer cells. (A) Cancer cells, when incubated with RBC-exosomes from lung cancer patients with high levels of miR-95-3p, show increased proliferation compared to those incubated with RBC-exosomes from lung cancer patients with low miR-95-3p and healthy controls. (B) Invasion assays for cancer cells treated similarly demonstrate enhanced invasive capabilities with lung cancer patients RBC exosomal miR-95-3p. (C) Bars represent the invasive cells per field, confirming increased invasion with high miR-93-5p exosomes. (D) Migration assays for cancer cells treated with the same groups of exosomes. Cells exhibit increased migration capabilities when treated with exosomes from cancer patients with high miR-93-5p. (E) Quantification of migration assays shown in (D). Bars represent the migration cells per field, demonstrating enhanced migration with high miR-93-5p exosomal treatment. *, p<0.05., **, p<0.01.

Article Snippet: To further validate the therapeutic targeting of miR-93-5p, twenty additional mice were subcutaneously injected with A549-Luc cells (CCL-185-LUC2™, ATCC) and treated with either a ASO-control or a ASO-miR-93-5p (AstraZeneca, Wilmington, DE).

Techniques: Derivative Assay, Incubation, Migration

Journal: medRxiv

Article Title: Red Blood Cell-Derived Exosomal Oncogenic MicroRNA Promote Cancer Development and Progression

doi: 10.1101/2024.05.10.24307177

Figure Lengend Snippet: Impact of RBC-exosomal miR-93-5p on PTEN Regulation and Tumorigenicity in Lung Cancer Cells. (A) Sequence alignment highlighting the predicted binding sites of miR-93-5p on the 3’UTR of the PTEN gene, contrasting the wild type (WT) and mutant (MUT) versions. (B) Luciferase reporter assays displaying reduced activity in lung cancer cells co-transfected with WT PTEN 3’UTR, suggesting direct interaction and targeting. (C) Western blot analysis showing that lung cancer cells treated with RBC-exosomal miR-93-5p exhibit decreased PTEN protein levels compared to those treated with regular medium only. (D-E) The effect of PTEN overexpression on cell proliferation in cancer cells treated with miR-93-5p-rich exosomes, indicating a suppressive effect on proliferation due to PTEN. (F-G) The impact of PTEN on invasion and migration of cancer cells incubated with miR-93-5p-rich exosomes. *, p<0.05., **, p<0.01.

Article Snippet: To further validate the therapeutic targeting of miR-93-5p, twenty additional mice were subcutaneously injected with A549-Luc cells (CCL-185-LUC2™, ATCC) and treated with either a ASO-control or a ASO-miR-93-5p (AstraZeneca, Wilmington, DE).

Techniques: Sequencing, Binding Assay, Mutagenesis, Luciferase, Activity Assay, Transfection, Western Blot, Over Expression, Migration, Incubation

Journal: medRxiv

Article Title: Red Blood Cell-Derived Exosomal Oncogenic MicroRNA Promote Cancer Development and Progression

doi: 10.1101/2024.05.10.24307177

Figure Lengend Snippet: Effects of miR-93-5p-rich RBC-exosomes and ASO-miR-93-5p on tumorigenicity of cancer cells. (A) Cells treated with cancer-derived exosomes enriched in miR-93-5p (Cancer-Exo-miR-93-5p) have increased proliferation over time, compared to the negative control. The ASO-miR-93-5p treatment reduces cell numbers compared to cells treated with miR-93-5p-rich exosomes, approaching the levels of regular medium. B) Invasion assay results for cancers cells treated with RBC-exosomes rich in miR-93-5p from lung cancer patients, ASO-miR-93-5p, and control conditions, represented as the fold change relative to the negative control. In both cell lines, RBC-exosomes rich in miR-93-5p from lung cancer patients enhance invasion capabilities, which are significantly decreased by ASO-miR-93-5p treatment. C) Migration assay results for cancer cells under the same treatment conditions as in (B), showing a similar trend where miR-93-5p increases cell migration, and this effect is mitigated by ASO-miR-93-5p. Error bars indicate standard deviation. *, p<0.05, **, p<0.01.

Article Snippet: To further validate the therapeutic targeting of miR-93-5p, twenty additional mice were subcutaneously injected with A549-Luc cells (CCL-185-LUC2™, ATCC) and treated with either a ASO-control or a ASO-miR-93-5p (AstraZeneca, Wilmington, DE).

Techniques: Derivative Assay, Negative Control, Invasion Assay, Migration, Standard Deviation

Journal: medRxiv

Article Title: Red Blood Cell-Derived Exosomal Oncogenic MicroRNA Promote Cancer Development and Progression

doi: 10.1101/2024.05.10.24307177

Figure Lengend Snippet: Effects of RBC-Derived miR-93-5p on Tumor Development in Lung Cancer Xenograft Mouse Models and the Therapeutic Potential of RBC-Derived miR-93-5p Inhibition. (A) On the left, extracted tumors at the end of week 4 from each group are displayed, demonstrating visibly larger tumors in the high miR-93-5p group. The graph on the right quantifies this difference, showing a significant increase in tumor volume in the high miR-93-5p group (p=0.046). (B) Immunohistochemical staining for PTEN (tumor suppressor) and KI-67 (proliferation marker) in tumors from mice injected with low vs. high miR-93-5p RBC-derived exosomes. The images were shown at 20x magnification. Tumors with high miR-93-5p show reduced PTEN expression and increased KI-67 levels. (C) Mice with tumors derived from cells treated with high miR-93-5p exosomes were given direct tumor injections with ASO-miR-93-5p or ASO-control. The graph indicates a significant reduction in tumor volume in the ASO-miR-93-5p treatment group (p=0.032). (D) Kaplan-Meier survival curves for mice treated with ASO-miR-93-5p (blue line) versus ASO-control (red line). Mice receiving the miR-93-5p-targeting ASOs demonstrated significantly extended survival (p=0.018).

Article Snippet: To further validate the therapeutic targeting of miR-93-5p, twenty additional mice were subcutaneously injected with A549-Luc cells (CCL-185-LUC2™, ATCC) and treated with either a ASO-control or a ASO-miR-93-5p (AstraZeneca, Wilmington, DE).

Techniques: Derivative Assay, Inhibition, Immunohistochemical staining, Staining, Marker, Injection, Expressing

Journal: medRxiv

Article Title: Red Blood Cell-Derived Exosomal Oncogenic MicroRNA Promote Cancer Development and Progression

doi: 10.1101/2024.05.10.24307177

Figure Lengend Snippet: Mice were injected with H460-Luc cells pre-treated with RBC-derived exosomes containing either low (left) or high (right) levels of miR-93-5p. Bioluminescence Imaging at week 1 and week 4 shows increased tumor growth in mice receiving cells with high miR-93-5p exosomes.

Article Snippet: To further validate the therapeutic targeting of miR-93-5p, twenty additional mice were subcutaneously injected with A549-Luc cells (CCL-185-LUC2™, ATCC) and treated with either a ASO-control or a ASO-miR-93-5p (AstraZeneca, Wilmington, DE).

Techniques: Injection, Derivative Assay, Imaging