Somatostatin receptor type 2-based reporter expression after plasmid-based in vivo gene delivery to non-small cell lung cancer

Abstract

Plasmids tend to have much lower expression than viruses. Gene expression after systemic administration of plasmid vectors has not been assessed using somatostatin receptor type 2 (SSTR2)-based reporters. The purpose of this work was to identify gene expression in non-small cell lung cancer (NSCLC) after systemic liposomal nanoparticle delivery of plasmid containing SSTR2-based reporter gene. In vitro, Western blotting was performed after transient transfection with the plasmid cytomegalovirus (CMV)-SSTR2, CMV-TUSC2-IRES-SSTR2, or CMV-TUSC2. SSTR2 is the reporter gene, and TUSC2 is a therapeutic gene. Mice with A549 NSCLC lung tumors were injected intravenously with CMV-SSTR2, CMV-TUSC2-IRES-SSTR2, or CMV-TUSC2 plasmids in DOTAP:cholesterol-liposomal nanoparticles. Two days later, mice were injected intravenously with 111In-octreotide. The next day, biodistribution was performed. The experiment was repeated including single-photon emission computed tomography/computed tomography (SPECT/CT). Immunohistochemistry was performed. In vitro, SSTR2 expression was similar in cells transfected with CMV-SSTR2 or CMV-TUSC2-IRES-SSTR2. TUSC2 expression was similar in cells transfected with CMV-TUSC2 or CMV-TUSC2-SSTR2. Biodistribution demonstrated significantly greater 111In-octreotide uptake in tumors from mice injected with CMV-TUSC2-IRES-SSTR2 or CMV-SSTR2 than the control plasmid, CMV-TUSC2 (p < .05). Gamma-camera and SPECT/CT imaging illustrated SSTR2 expression in tumors in mice injected with CMV-TUSC2-IRES-SSTR2 or CMV-SSTR2 versus background with control plasmid. Immunohistochemistry corresponded with imaging. SSTR2-based reporter imaging can visualize gene expression in lung tumors after systemic liposomal nanoparticle delivery of plasmid containing SSTR2-based reporter gene or SSTR2 linked to a second therapeutic gene, such as TUSC2.

Figure 1. Western blot and immunofluorescence results demonstrating SSTR2 and TUSC2 expression after plasmid-based gene transfection

Western blot results in HT1080 cell lines transfected with CMV-TUSC2-IRES-SSTR2, CMV-SSTR2 and CMV-TUSC2 plasmid DNA with anti-SSTR2 antibody for the expressed SSTR2 protein (A). The blot was stripped and then probed with anti-TUSC2 antibody for the expressed TUSC2 protein (B). Immunofluorescence stain results showed the fluorescent labeling in HT1080 cell lines transfected with CMV-TUSC2-IRES-SSTR2, CMV-SSTR2 and CMV-TUSC2 plasmid DNA with anti-SSTR2 antibody for the expressed SSTR2 protein (C) and with anti-TUSC2 antibody for the expressed TUSC2 protein (D). Western blot results in A549-luc cell lines transfected with CMV-TUSC2-IRES-SSTR2, CMV-SSTR2 and CMV-TUSC2 plasmid DNA with anti-SSTR2 antibody for the expressed SSTR2 protein (E). The blot was stripped and then probed with anti-TUSC2 antibody for the expressed TUSC2 protein (F).

Figure 1. Western blot and immunofluorescence results demonstrating SSTR2 and TUSC2 expression after plasmid-based gene transfection

Western blot results in HT1080 cell lines transfected with CMV-TUSC2-IRES-SSTR2, CMV-SSTR2 and CMV-TUSC2 plasmid DNA with anti-SSTR2 antibody for the expressed SSTR2 protein (A). The blot was stripped and then probed with anti-TUSC2 antibody for the expressed TUSC2 protein (B). Immunofluorescence stain results showed the fluorescent labeling in HT1080 cell lines transfected with CMV-TUSC2-IRES-SSTR2, CMV-SSTR2 and CMV-TUSC2 plasmid DNA with anti-SSTR2 antibody for the expressed SSTR2 protein (C) and with anti-TUSC2 antibody for the expressed TUSC2 protein (D). Western blot results in A549-luc cell lines transfected with CMV-TUSC2-IRES-SSTR2, CMV-SSTR2 and CMV-TUSC2 plasmid DNA with anti-SSTR2 antibody for the expressed SSTR2 protein (E). The blot was stripped and then probed with anti-TUSC2 antibody for the expressed TUSC2 protein (F).

Figure 2. Biodistribution demonstrating SSTR2 expression in NSCLC lung tumor metastases after systemic plasmid SSTR2-based gene transfer

Biodistribution analysis illustrates significantly greater uptake of ¹¹¹In-octreotide in lung metastases of mice injected (iv) with CMV-SSTR2 (*p<0.05, n=6) or CMV-TUSC2-IRES-SSTR2 (**p<0.001, n=6) plasmid DNA as compared to control plasmid DNA, CMV-TUSC2. There was no significant difference between CMV-SSTR2 and CMV-TUSC2-IRES-SSTR2 (A). Graph including organs and tumors shows no significant difference in biodistribution among organs (B) of mice injected (iv) with CMV-SSTR2, CMV-TUSC2-IRES-SSTR2, or CMV-TUSC2 plasmid DNA, whereas, differences among tumors is as above in A.

Figure 3. Gamma-camera images demonstrating SSTR2 expression in NSCLC lung tumor metastases after systemic SSTR2-based plasmid gene transfer. Representative bioluminescence luciferase imaging for monitoring tumor development demonstrated lung tumors in groups (A-C) that were then injected with plasmid nanoparticles and subsequently ¹¹¹In-octreotide

Representative planar images, demonstrate increased uptake (arrows) in the thorax of mice with NSCLC lung metastases injected systemically with CMV-SSTR2 (D) or CMV-TUSC2-IRES-SSTR2 (E) compared with control CMV-TUSC2 plasmid DNA (F). Mice bearing lung tumors and exposed to plasmid systemically two days earlier were injected intravenously with ¹¹¹In- octreotide (11MBq) and imaged 24 hours later.

Figure 4. SPECT/CT images demonstrating SSTR2 expression in NSCLC lung tumor metastases after systemic SSTR2-based plasmid gene transfer

Representative slices of three dimensional, tomographic SPECT/CT images demonstrate increased uptake in the lung tumors of mice with NSCLC lung metastases injected systemically with CMV-SSTR2 (A) or CMV-TUSC2-IRES-SSTR2 (B) compared with CMV-TUSC2 plasmid DNA (C). Top: lung windows demonstrating lung tumors (arrows); Bottom: soft tissue windows with SPECT overlay. Biodistribution analysis (D) illustrates significantly greater uptake of ¹¹¹In-octreotide in lung metastases of mice injected (iv) with CMV-SSTR2 (**p<0.001, n=3) or CMV-TUSC2-IRES-SSTR2 (**p<0.001, n=3) plasmid DNA as compared to control plasmid DNA, CMV-TUSC2. There was no significant difference between CMV-SSTR2 and CMV-TUSC2-IRES-SSTR2. Mice bearing lung tumors and exposed to plasmid systemically two days earlier were injected intravenously with ¹¹¹In-octreotide (13MBq) and imaged 24 hours later.

Figure 4. SPECT/CT images demonstrating SSTR2 expression in NSCLC lung tumor metastases after systemic SSTR2-based plasmid gene transfer

Representative slices of three dimensional, tomographic SPECT/CT images demonstrate increased uptake in the lung tumors of mice with NSCLC lung metastases injected systemically with CMV-SSTR2 (A) or CMV-TUSC2-IRES-SSTR2 (B) compared with CMV-TUSC2 plasmid DNA (C). Top: lung windows demonstrating lung tumors (arrows); Bottom: soft tissue windows with SPECT overlay. Biodistribution analysis (D) illustrates significantly greater uptake of ¹¹¹In-octreotide in lung metastases of mice injected (iv) with CMV-SSTR2 (**p<0.001, n=3) or CMV-TUSC2-IRES-SSTR2 (**p<0.001, n=3) plasmid DNA as compared to control plasmid DNA, CMV-TUSC2. There was no significant difference between CMV-SSTR2 and CMV-TUSC2-IRES-SSTR2. Mice bearing lung tumors and exposed to plasmid systemically two days earlier were injected intravenously with ¹¹¹In-octreotide (13MBq) and imaged 24 hours later.

Figure 5. Immunohistochemical (IHC) staining demonstrating SSTR2 expression in NSCLC lung metastases after systemic SSTR2-based plasmid gene transfer

Representative images show expression of SSTR2 (brown staining) in NSCLC metastases from mice injected systemically with CMV-SSTR2 (A) or CMV-TUSC2-IRES-SSTR2 (B), but not control TUSC2 (C) plasmid DNA. Images do not show expression of TUSC2 (brown staining) in NSCLC metastases from mice injected systemically with CMV-SSTR2 (D) plasmid DNA as expected, but do show TUSC2 expression in NSCLC metastases from mice injected systemically with CMV-TUSC2-IRES-SSTR2 (E) or CMV-TUSC2 (F) plasmid DNA. Images do not show expression of apoptosis (brown nuclear staining) in NSCLC metastases from mice injected systemically with CMV-SSTR2 (G) plasmid DNA as expected, but do show TUSC2 expression in NSCLC metastases from mice injected systemically with CMV-TUSC2-IRES-SSTR2 (H) or CMV-TUSC2 (I) plasmid DNA.