Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Mar 15:25:57-68.
doi: 10.1016/j.omto.2022.03.005. eCollection 2022 Jun 16.

Combination microRNA-based cellular reprogramming with paclitaxel enhances therapeutic efficacy in a relapsed and multidrug-resistant model of epithelial ovarian cancer

Srujan K Gandham  1 , Mounika Rao  1 , Aayushi Shah  1 , Malav S Trivedi  2 , Mansoor M Amiji  1   3
Affiliations

Combination microRNA-based cellular reprogramming with paclitaxel enhances therapeutic efficacy in a relapsed and multidrug-resistant model of epithelial ovarian cancer

Srujan K Gandham et al. Mol Ther Oncolytics. .

Abstract

Most advanced-stage ovarian cancer patients, including those with epithelial ovarian cancer (EOC), develop recurrent disease and acquisition of resistance to chemotherapy, leading to limited treatment options. Decrease in Let7b miRNA levels in clinical ovarian cancer has been associated with chemoresistance, increased proliferation, invasion, and relapse in EOC. We have established a murine EOC relapsed model by administering paclitaxel (PTX) and stopping therapy to allow for tumor regrowth. Global microRNA profiling in the relapsed tumor showed significant downregulation of Let7b relative to untreated tumors. Here, we report the use of hyaluronic acid (HA)-based nanoparticle formulation that can deliver Let7b miRNA mimic to tumor cells and achieve cellular programming both in vitro and in vivo. We demonstrate that a therapeutic combination of Let7b miRNA and PTX leads to significant improvement in anti-tumor efficacy in the relapsed model of EOC. We further demonstrate that the combination therapy is safe for repeated administration. This novel approach of cellular reprogramming of tumor cells using clinically relevant miRNA mimic in combination with chemotherapy could enable more effective therapeutic outcomes for patients with advanced-stage relapsed EOC.

Keywords: IDG-VEGF; hyaluronic-acid-based nanoparticles; microRNA-Let7b; ovarian cancer; relapsed/MDR model.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent licensing arrangements) or non-financial interest (such as personal or professional relationships, affiliations, knowledge, or beliefs) in the subject matter or materials discussed in this manuscript.

Figures

None
Graphical abstract
Figure 1
Figure 1
Development of the ID8-PTX-MDR/relapse model of ovarian cancer Time line showing tumor development and relapse. ID8-VEGF cells were inoculated in female C57BL/6 mice, followed by three doses of paclitaxel treatment (8 mg/kg i.p.) on day 14, 21, and 28 (as indicated by the arrows), and therapy was stopped for development of relapse and naive mice ID8-VEGF ovarian cancer cell line. (A) Graph showing tumor growth (ascitic fluid buildup) for the control group (n = 8) after day 35 and relapse of tumor growth in the PTX groups (n = 8) after day 49. (B) Representative images of mice from the naive (no abdominal swelling) group; control group, showing abdominal swelling due to buildup of ascitic fluid; and PTX group, showing ascites in the peritoneal cavity.
Figure 2
Figure 2
Molecular profiling in tumor cells and nodules from ID8-PTX-dosed ovarian cancer mouse model qPCR data showing upregulation of gene expression for MDR and other genes normalized to β-actin. (A) ID8 cells sorted from ascitic fluid of tumor bearing mice ID8-VEGF control and ID8-VEGF-PTX-dosed animals (n = 6). (B) Tumor nodules derived from diaphragm of ID8-VEGF control and ID8-VEGF-PTX-dosed animals. (C) Heatmap with unsupervised hierarchical clustering (columns) dendrograms show clustering of control and PTX groups-based miRNA expression levels (rows) using NanoString analysis. Scale showing fold changes in miRNA expression. (D) Differentially expressed miRNA in ID8 tumor cells derived from ascitic fluid using NanoString analysis of tumor cells derived from control and PTX-treated ID8-VEGF tumor bearing mice (n = 6). p values obtained (A and B) with Student’s t test: ∗∗p < 0.001 or ∗∗∗∗p < 0.0001, compared with the control group; bars represent fold changes; adjusted p values was obtained using Bonferroni correction method. Upregulated miRNA was defined as log2 fold >1 (fold change >2) and p < 0.01 and downregulated miRNA was defined log2 fold change <−1 (fold change <−2) and p < 0.01.
Figure 3
Figure 3
In vitro evaluation of combination therapy of HA-PEI-let7b and PTX for assessing improvement in potency of PTX and reprogramming of target mRNA levels ID8-VEGF were dosed with PTX for development of resistance (day 1, 3, and 5) and allowed to recover, followed by HA-PEI let7b nanoparticle dosing and MTS assay readout. (A) Graph % cell viability of various combinations of Let7b (0.1–100 nM) with PTX (0.001–100 μM) measured at 72 h time point. scRNA and untreated control were used for calculating relative cell viability. (B) Graph showing the IC50 values calculated from the nonlinear curve fitting of % cell viability (y axis) and log10 PTX concentration (μM) on x axis. All conditions were performed with n = 4 wells in a 96-well plate format with 1,500 cells/well. (C) Evaluation of knockdown of Let7b target mRNA in ID8-control cells 48 h post HA-PEI-Let-7b transfection at 1, 10, and 100 nM Let7b concentrations using qPCR. Scrambled RNA control (scRNA) was used as negative control. Untransfected controls were for normalizing the relative gene expression. B-actin was used as a housekeeping gene. All experiments were performed with n = 3 biological replicates and n = 3 technical/assay replicates. Student's t test was used for statistical significance (∗p < 0.05, ∗∗∗p < 0.001).
Figure 4
Figure 4
Restoration of Let7b levels in tumor nodules and downregulation of oncogenes using HA-PEI-let7b nanoparticles Time line showing tumor development in the ID8-VEGF mouse model and dosing schedule for HA-PEI-Let7b. (A) Quantification of absolute copies of Let7b in tumor nodules 48 h post 3 doses Let7b dosing (i.p.) (n = 4 per group). (B) Relative quantification of Let-7b mediated target gene expression in changes 48 h post 3 doses of HA-PEI-Let7b/scr (n = 4 per group). (C) Biodistribution of Let7b in liver, spleen, and lung 48 h post 3 doses of HA-PEI-Let7b dosing. (D) Relative quantification of HMGA2 mRNA levels in liver, spleen, and lung tissues 48 h post 3 doses of HA-PEI-Let7b. Statistical significance was evaluated using Student’s t test by comparing ID8 tumor relapse control or naive (no tumor) with HA-PEI-Let-7b groups (∗p < 0.05,∗∗p < 0.01, ns = not statistically significant).
Figure 5
Figure 5
Improved efficacy of combination of HA-PEI-Let7b and PTX in the ID8-PTX-MDR/relapse model of ovarian cancer Timeline for ID8-VEGF relapse development using repeat dosing of PTX (3 doses, 8 mg/kg, i.p.) on day 14, 21, and 28, followed by dosing regimen for combination therapy (PTX, 20 mg/kg, and HA-PEI-Let7b, 1 mg/kg, i.p.) (A) Graph showing tumor growth using body weight change on y axis as a measure of ascitic fluid buildup post tumor inoculation for various control and treatment groups. (B) Graph showing % body weight change normalized to naive (no tumor) on the y axis group on day 55 (euthanasia) for various control and treatment groups (n = 8 per, n = 4 for naive). (C) Measurement of anti-tumor efficacy of combination therapy using VEGF ELISA.VEGF levels (pg/mL) measured from supernatants of ascitic fluid on day 55 (n = 6–8 per group). (D) Representative images of ID8-VEGF-resistant tumor bearing mice on day 55 post tumor inoculation. Untreated control group (resistant/relapse) showing ascitic fluid buildup leading to abdominal bloating, PTX group only with ascites in the peritoneal cavity, and PTX + HA-PEI-Let7b group with no ascites formation (p values for A, B, and C: ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, and∗∗p < 0.01 were obtained from Student’s t test by comparing PTX + HA-PEI- Let7b with other groups)
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Siegel R.L., Miller K.D., Jemal A., Jemal A. Cancer statistics, 2017. CA Cancer J. Clin. 2021;67:7–30. - PubMed
    1. Howlader N., Noone A.M., Krapcho M., Miller D., Bishop K., Kosary C.L., Yu M., Ruhl J., Tatalovich Z., Mariotto A., et al. National Cancer Institute; 2017. SEER Cancer Statistics Review, 1975-2014.
    1. Patch A.M., Christie E.L., Etemadmoghadam D., Garsed D.W., George J., Fereday S., Nones K., Cowin P., Alsop K., Bailey P.J., et al. Whole-genome characterization of chemoresistant ovarian cancer. Nature. 2015;521:489–494. - PubMed
    1. Robey R.W., Pluchino K.M., Hall M.D., Fojo A.T., Bates S.E., Gottesman M.M. Revisiting the role of ABC transporters in multidrug-resistant cancer. Nat. Rev. Cancer. 2018;18:452–464. - PMC - PubMed
    1. Ahmed N., Stenvers K.L. Getting to know ovarian cancer ascites: opportunities for targeted therapy-based translational research. Front Oncol. 2013;3:256. - PMC - PubMed

LinkOut - more resources