CD73 downregulation by EGFR-targeted liposomal CD73 siRNA potentiates antitumor effect of liposomal doxorubicin in 4T1 tumor-bearing mice

Abstract

Blocking CD73 ectonucleotidase has been proposed as a potential therapeutic approach for cancer treatment. The present study aimed to investigate the antitumor effect of a novel EGFR-Targeted liposomal CD73 siRNA formulation in combination therapy with liposomal doxorubicin in the 4T1 mouse model. CD73 siRNA was encapsulated into nanoliposomes by the ethanol injection method. After preparation, characterization, morphology, and stability evaluation of formulations, the toxicity was measured by MTT assay. Uptake assay and efficiency of the liposomal formulations were investigated on the 4T1 cell line. The liposomal formulation containing CD73 siRNA was targeted with GE11 peptide for in vivo evaluations. Following biodistribution analysis, the antitumor activity of prepared formulations in combination with liposomal doxorubicin was studied in mice bearing 4T1 metastatic breast cancer cells. Finally, the induction of immune response of formulations in concomitant treatment with liposomal doxorubicin was evaluated in the tumor microenvironment of a mouse model of breast cancer. The size of prepared liposomal formulations at N/P = 16 for the liposomal CD73 siRNA and GE11-liposomal CD73 siRNA groups were 89 nm ± 4.4 and 95 nm ± 6.6, respectively. The nanoparticle's PDI was less than 0.3 and their surface charge was below 10 mV. The results demonstrated that N/P = 16 yielded the best encapsulation efficiency which was 94% ± 3.3. AFM results showed that the liposomes were spherical in shape and were less than 100 nm in size. The results of the MTT assay showed significant toxicity of the liposomes containing CD73 siRNA during the 48-h cell culture. Real-time PCR and flow cytometry results showed that liposomes containing CD73 siRNA could effectively downregulate CD73 expression. Liposomal formulations were able to significantly downregulate CD73 gene expression, in vivo. However, CD73 downregulation efficiency was significantly higher for the targeted form compared to the non-targeted formulation (P value < 0.01). The combination showed maximum tumor growth delay with remarkable survival improvement compared to the control group. Studying the immune responses in the treatment groups which received doxorubicin, showed decreased number of lymphocytes in the tumor environment. However, this decrease was lower in the combination therapy group. Finally, our results clearly showed that CD73 downregulation increases the activity of CD8⁺ lymphocytes (IFN-ℽ production) and also significantly decreases the Foxp3 in the CD25⁺ lymphocytes compared to the control group. GE11-Lipo CD73 siRNA formulation can efficiently knockdown CD73 ectonucleotidase. Also, the efficacy of liposomal doxorubicin is significantly enhanced via the downregulation of CD73 ectonucleotidase.

Figure 1

Liposomal formulation containing CD73 siRNA were incubated in the PBS/FBS medium at 37 °C for 96 h. The amount of remained siRNA reached near to 50% after 24 h incubation in the PBS/FBS medium at 37 °C and it decreased to around 30% after 96 h incubation. Data are shown as mean ± standard deviation (n = 3).

Figure 2

Morphology of empty liposomes (A, B) and liposomes Containing CD73 siRNA (C, D) by atomic force microscopy (AFM). Both empty liposomes and liposomes containing CD73 siRNA have spherical shape with high resolution and an average size of less than 100 nm. Figure (A) and (C) show the two-dimensional (2D) image of empty liposome and liposomes containing CD73 siRNA respectively. Figure (B) and (D) show the Three-dimensional (3D) images of empty liposome and liposomes containing CD73 siRNA respectively.

Figure 3

Uptake assay of liposomes containing Cy3-siRNA and Lipofectamine Cy3-CD73 siRNA in the 4T1 cancer cells. (A) The untreated 4T1 cells. (B) Un-liposomal Cy3-siRNA (Naked Cy3-CD73 siRNA). (C) Liposomes containing Cy3-siRNA. (D) lipofectamine Cy3-siRNA transfected cells. (E) The histogram analysis of siRNA uptake in 4T1 cells with liposome and lipofectamine compared to Naked siRNA. (F) Geometric mean (geom. mean) analysis of Cy3-siRNA uptake in 4T1 cells with liposome and lipofectamine. Data are shown as mean ± standard deviation (n = 3). Statistically significant differences are represented as follows: ***P < 0.001, ****P < 0.0001. The images are taken at 40X magnification.

Figure 4

Evaluation of siRNA-mediated downregulation efficiency and toxicity of CD73 siRNA in 4T1 cell line. (A) Cytotoxicity of formulations including liposomes containing CD73 siRNA (100, 50, and 25 nM), naked CD73 siRNA (25 and 100 nM), non-targeting siRNA (25 nM), and empty liposomes was evaluated by MTT assay. The data are the mean ± SD of triplicate experiments. (B) After 48-h incubation of 4T1 cell with different formulations, the impact of liposomal CD73 siRNA and lipofectamine-CD73 siRNA (positive control) on the level of CD73 mRNA were assessed by real time PCR method. Both liposomes containing CD73 siRNA (25 nM) and lipofectamine CD73 siRNA complex (25 nM) can inhibit CD73 mRNA expression level up to 51%, but the naked form of CD73 siRNA and non-targeting siRNA could not affect CD73 expression effectively. (C, D) The results of flow cytometry analysis also demonstrated that liposomes containing CD73 siRNA and lipofectamine transfected group decrease the CD73 expression as compared with the empty liposome group.

Figure 5

Tissue biodistribution of GE11-targeted, non-targeted liposomal formulation containing Cy3-siRNA, and Naked Cy3 siRNA at three-time intervals (4, 24 and 48 h post injection) in tumor site, heart, liver, spleen, lung, and kidney tissues. (A) Tissue biodistribution of different formulations at 4 h time interval. (B) Tissue biodistribution of different formulations at 244 h time interval. (C) Tissue biodistribution of different formulations at 48 h time interval. The intensity of Cy3 fluorescence dye indicates drug accumulation in different tissues. Data are expressed as mean ± standard deviation (n = 3). Statistically significant differences are represented as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 6

Therapeutic efficacy of liposomal CD73 siRNA, GE11-liposomal CD73 siRNA, liposomal doxorubicin, and GE11-liposomal CD73 siRNA+ liposomal doxorubicin combination in female BALB/c mice bearing 4T1 tumor. (A) Mean tumor volume in all groups. (B) Survival analysis of therapeutic groups (The log-rank test was used to analyze the survival). Data are expressed as mean ± standard deviation (n = 5). Statistically significant differences are represented as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 7

Efficacy of liposomal formulations on both CD73 gene expression and CD73 ectonucleotidase level by RT-PCR and flow cytometry, respectively, in the tumor tissue of mice inoculated with 4T1 cell line. (A) The result of RT-PCR showed that the targeted form of liposome containing CD73 siRNA (GE11-Lipo CD73 siRNA) alone and in combination with Liposomal doxorubicin could efficiently reduce the expression of CD73 gene (****: P value < 0.0001). (B) Decreased fluorescence intensity indicates decreased CD73 ectonucleotidase level. Histogram shows the intensity of PE fluorescence in the treatment groups. CD73 PE G mean stands for geometric mean of fluorescence intensity of CD73 PE. Data are shown as the mean ± SD (n = 3). Statistically significant differences are shown as follow: **P < 0.01 and ****P < 0.0001.

Figure 8

Frequency of tumor-infiltrated lymphocytes (TILs) and T cell subpopulations in the tumor environment of different groups. Two days after the last injection at the fourth week, three mice from each treatment group were sacrificed and cells from tumor tissue were isolated. Isolated cells were stained with conjugated antibodies against surface markers (CD3, CD4, CD8, and CD25 FoxP3). FACS analysis was carried out to determine the frequency of lymphocytes secreted into the tumor site. (A) The frequency of TIL following injection of mice with different formulations. (B) Liposomal formulation alone or in combination with GE11-Lipo CD73 siRNA decreased the frequency of CD3/CD4 positive lymphocytes. (C) Lipo Dox formulation group significantly decreased the frequency of CD3/CD8 positive lymphocytes (P value < 0.01). (D) Lipo Dox formulation negatively affected the frequency of CD8⁺, INF⁺ lymphocytes. In combination group, the negative effect of Lipo Dox formulation is attenuated by GE11-Lipo CD73 siRNA formulation. (E) Frequency of CD4⁺, INF⁺ in CD3 gated lymphocytes. (F) Effect of different formulation on the frequency of Treg CD25⁺, Foxp3⁺ lymphocytes. Data are reported as mean ± SD (n = 3). Statistically significant differences are represented as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 9

Intracellular cytokine profile of tumor-infiltrated lymphocytes (TILs). Two days after the last injection at the fourth week, three mice from each treatment group were sacrificed and tumor tissue cells were isolated. Isolated cells were stained with intracellular markers. Fluorescence intensity (geometric mean) of cytokines including IL-4, IFN-γ, and FoxP3 were assessed by flow cytometry. (A, C) Different formulations had not any significant differences on the geometric mean of IFN and IL-4 in the CD4⁺ gated cells. (B) Injection of Liposomal siRNA, GE11targeted siRNA-loaded liposomes and the combination group (GE11-Lipo CD73 siRNA+ Lipo Dox) markedly increased the geometric mean of IFN-γ in CD8⁺ T cells. (D) On the other hand, these treatments significantly decreased the florescent intensity of FoxP3 compared to control (PBS) and Liposomal doxorubicin groups. Data are reported as mean ± SD (n = 3). Statistically significant differences are represented as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.