In this work, we focused on testing the efficacy of a neutral DOPC-liposomal system for the delivery of siRNAs administered in a non-invasive way. This was performed by treating mice that were induced with melanoma that ultimately metastasized to the lungs. The lungs are one of the organs most affected by metastatic cells, as it offers plenty of space to grow, high blood irrigation, and access to nutrients and oxygenation. All these characteristics make this organ an ideal target for tumors.
Being the lung the second most affected organ by tumoral metastases, we proposed a feasible therapy using L-siRNAs as a treatment of lung metastases. For this purpose, we designed a system to deliver encapsulated siRNA in neutral DOPC-liposomes, administered by respiratory nebulization. Our L-siRNA could be delivered to the lungs with simple administration, achieving the silencing of the WT1 oncogene, and obtaining a significant therapeutic effect over the melanoma metastases on the lungs of our animal model. This modality of therapy results in a very comfortable non-invasive high levels of stress derived from frequent visits to the hospital and intravenous administrations.
This work offers an expanded vision of the diverse mechanisms that participate in the process of malignancy of the melanoma cell line B16F10 that are dependent on WT1. We describe the effects of the silencing of this protein by quantifying its expression level, migration and invasion, viability, progression in the cell cycle, and apoptosis. We also propose a new administration system that is efficient, safe, non-invasive, comfortable, and of a low cost to deliver siRNA encapsulated in neutral liposomes. We further describe the biodistribution, advantages and limitations, as well as the therapeutic effects of this treatment in the short and long term in a model of metastatic lung cancer in mice. Looking ahead of our work, we propose that there is strong evidence pointing to the belief that the efficacy of our treatment can be widely improved by the use of concomitant therapies. It is feasible to think that silencing the oncogene of these tumoral cells makes them more sensitive to chemotherapy or radiotherapy and could imply the use of lower doses minorizing the appearance of adverse effects, greatly improving the quality of life of the patients and possibly achieving a complete remission of the tumors. Additionally, the application of respiratory L-siRNA is not limited to the treatment of lung cancer; it can also be effective to control the expression of inflammatory factors in pneumonia, slow the progression of hereditary diseases, and even prevent the spread of respiratory viruses by silencing important proteins involved in their replication.
4. Materials and Methods
4.1. Cell Line and Culture Conditions
The murine melanoma B16F10 cell line was acquired from the ATCC (Manassas, VA, USA), catalog CRL-6475. The cells were nurtured with Gibco’s DMEM/F12 medium (Thermo Fisher, Waltham, MA, USA, Cat. 11320033) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO, USA, Cat. F0926-500ML) and maintained in a humidified incubator at 37 °C with 5% CO2. The number of passages did not exceed 30 after initial stock was recovered. The cell line was confirmed as mycoplasma negative through a PCR analysis using the Universal Mycoplasma Detection Kit (ATCC, Cat. 30-1012K).
4.2. siRNA and Transfection
4.3. Western Blot
Total protein was extracted from cultured cells after transfection using a NP-40 lysis buffer (150 mM NaCl, 50 mM Tris and 1% Triton X-100, pH 8.0) containing protease-inhibitor 1× (Promega, Fitchburg, WI, USA, Cat. G6521). A total of 50 µg of protein was loaded on an 8% SDS-PAGE gel; after electrophoresis, the resolved proteins were transferred to a PVDF membrane. Antibodies for WT1 (Santa Cruz Biotechnology, Paso Robles, CA, USA, Cat. sc-192) and β-actin (Sigma, Cat. A2228) detection were utilized as detailed by the manufacturer. β-actin was detected after membrane stripping and used as a loading control. As a chemiluminescent substrate, we used Pierce ECL for the WB (Thermo Fisher, Cat. 32209).
4.4. Migration and Invasion Assay
For the evaluation of the migration capacity of the cells, 24-well transwell inserts with 8-µm pores (Corning Life Science, Tewksbury, MA, USA, Cat. 3530097) were used, and for invasion assay a coat of Matrigel (Corning, Cat. 354248) at 1.2 mg/mL over the insert was applied. B16F10 cells were cultured in a serum starving condition using DMEM/F12 with 1% FBS for 24 h. After this time, cells were transfected corresponding to the treatment group. At the bottom of the transwell, 500 µL of DMEM/F12 with 10% FBS were added, and at the top of the insert, 2.5 × 104 cells were seeded in 200 µL of DMEM/F12 with 1% FBS to produce a serum chemotaxis gradient. After 48 h the transwell inserts are collected for migration, and after 72 h for the invasion assay. All non-migrating cells were scrapped from the top of the insert and the remaining cells at the bottom were gently washed with PBS and fixed with 4% paraformaldehyde for 15 min. Transwell membranes were stained with nuclear dye DAPI (Vector Laboratories, Burlingame, CA, USA, Cat. H-1200) and mounted over a slide to allow its visualization and counting on a fluorescent microscope.
4.5. Cell Cycle Cytometry
Transfection and seeding of 1 × 105 cells were performed in a 6-well culture plate with 2 mL of DMEM/F12 with 10% FBS. Treated cells were collected after 72 h and stained with ice-cold propidium iodide for 5 min before being analyzed via flow cytometry (BD Bioscience, San Jose, CA, USA, FACSCanto II). An unstained control was used to determine basal fluorescence. All data analyses were performed utilizing the ModFit LT v5 software.
4.6. Cell Viability
For the cell viability assay the AlamarBlue (Thermo Fisher, Cat. DAL1100) method was used. For each well (6 wells per group), 1 × 104 cells were transfected and seeded in 96-well culture plates with 100 µL of DMEM/F12 with 10% FBS. After 72 h, plates were collected and read using an ELISA plate reader at a 570-nm wavelength. The experiment was conducted using a triplicate method.
4.7. Apoptosis TUNEL
A total number of 2.5 × 104 cells were seeded over a round coverslip placed in 12-well plates with 1 mL of DMEM/F12 with 10% FBS. After 72 h, the coverslips were washed with PBS and stained with the kit DeadEnd Fluorometric TUNEL System (Promega, Cat. G3250) following the merchant’s instructions. Lastly, the coverslips were mounted over a slide using VectaShield with DAPI (Vector Laboratories, Cat. H-1300) and visualized in a fluorescent microscope.
4.8. Animal Model of Melanoma Metastasis
A pulmonary melanoma metastatic model was stablished on C57BL/6 mice via inoculation via the tail vein with 5 × 105 cells of the syngeneic melanoma cell line B16F10. The cells were administered on 200 µL of physiological saline solution (NaCl 0.9%). This model develops visible tumoral nodules on the lungs between 7–15 days after I.V. administration.
4.9. Nebulization of Liposomal siRNA Treatment
For this study, two groups of 3 mice each were inoculated with B16F10 melanoma cells via the vein of the tail and the metastases left to implant for 15 days. A control group received the L-siRNA control, and a second group received the L-siRNA control with the fluorescent marker Alexa fluor 488 (L-siRNA Alexa 488). Both groups were administered L-siRNA via nebulization as described before and left to rest for 1 h to allow the systemic distribution of the L-siRNA. After that time, the animals were euthanized and samples from multiple organs were recovered to be embedded in Tissue-Tek (VWR, Radnor, PA, USA, Cat. 25608). Frozen sections were processed using a Leica Cryostat (Mod. CM1100), and a fluorescent signal was observed in a confocal microscope (Leica Biosystems, Buffalo Grove, IL, USA, Mod. TCS SP5).
4.11. Survival Study and Animal Termination Criteria
To evaluate the long-term efficacy of our L-siRNA treatment, we implemented a survival study inducing melanoma lung metastases as detailed before with 10 animals assigned to each treatment group. The animals received nebulized therapy with L-siRNA twice per week, and their general condition and behavior was carefully followed daily to detect animals that started to show signs of physical affectation caused by the growth of the tumors in the lung. Endpoint criteria included respiratory distress, reduced movement, general weakness, and slow reaction times after a touch stimulus.
4.12. Tumors Recovery and WT1 Expression Analysis
Animals reaching endpoint criteria were euthanized in a CO2 chamber and a full set of organs were recovered and stored for further analysis. We paid special attention to the lung since it was the primary organ affected by metastases, and it was weighted and separated in different fragments for study. One of the analyses we performed in the tumor-bearing lungs was the detection of the WT1 protein via a western blot, and for that we recovered the whole-lung protein extract using RIPA Buffer (Thermo Fisher, Cat. J62524.AD).
4.13. Statistical Analysis
All analysis were performed using GraphPad Prism 9.0.2. On graphs, bars represent the mean ± SDs of at least 3 independent experiments. For the comparison of the multiple groups’ comparison, a one-way ANOVA was used, and for the survival study, Kaplan–Meier analysis was compared with Log-rank. A value of p < 0.05 was considered to be statistically significant.
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