D IKK Results were calculated as protein actin ratio and
(D) IKKγ. Results were calculated as protein/β-actin ratio and presented as mean percent of the CL-siScr with SD. Statistical analysis: student t test: (treatment vs. siScr: ****P b 0.0001, treatment vs. cisplatin: ##P b 0.01; ###P b 0.001; ####P b 0.0001, and treatment vs. CL-siSOX2: ††††P b 0.0001).
and charge characteristics (b100 nm and ~15 mV respectively) of the lipoplexes. The precedent for this assumption is based on related studies that have demonstrated that cationic nanoparticles through electrostatic interactions are 15–30 times more efficient in targeting the luminal tumor vasculature subsequently resulting in extravasation compared to normal tissues.35–37 Moreover, surface functionalization of the lipoplex by pegylation was
incorporated in the formulation process as a safeguard against potential degradation during plasma transit. Subsequently, the capacity of the lipoplexes to ensure lung exposure for subsequent tumor uptake was demonstrated by ex vivo fluorescence imaging (Figure 2). Tissue uptake of lipoplexes were observed in lung, spleen, heart, kidney and liver. Notably, uptake was higher in the liver compared to other tissues, which is supported by previous
Figure 8. Cationic lipoplex loaded with siSOX2 (CL-siSOX2) inhibits expression of markers of tumor growth in mice xenograft tumors. C.B.17 SCID mice with H1650 SP G-418 as tumor xenografts received treatment as described. (A) Immunoblotting of tumor lysates shows protein expression of (B) Smad5, (C) TGFβ, (D) Bcl-2 and (E) Survivin. Results were calculated as protein/β-actin ratio and presented as mean percent of the CL-siScr with SD. Statistical analysis: student t test: (treatment vs. siScr: ****P b 0.0001, treatment vs. cisplatin: ###P b 0.001; ####P b 0.0001, and treatment vs. CL-siSOX2: ††††P b 0.0001).
observations that initial uptake of nanoparticles, and subsequent saturation of phagocytic cells in the liver precede uptake by other tissues/organs.38 We also observed variable exposure of the lipoplexes to the lung; an initial increase in lung exposure (1 h) was followed by a decrease over the course of 2 h and 3 h, followed by an increase at 4 h and 5 h. This variable exposure kinetics may be explained by models of in vitro cellular uptake of nanoparticles that show initial saturation at the cell surface due to adsorption, and preceding endocytosis, where adsorption has been shown to occur at rates greater than three orders of magnitude faster than endocytosis.38 Subsequent increase in the rate of accessibility and recycling of surface receptors may have contributed to the clearance of surface adsorbed lipoplexes at 2 h and 3 h. Thus, the increased exposure at 5 h may be interpreted as intracellular accumulation rather than surface saturation by lipoplexes.
The H1650 lung cancer cell line has been validated in many studies as exhibiting resistance phenotypes to different treatments including small molecule chemo- and targeted therapies, as well as large molecules such as monoclonal antibodies.39–41 H1650 has also been characterized to demonstrate resistance to epidermal growth factor receptor (EGFR)-targeted therapies which is attributed to the presence of activating somatic mutations including EGFR-T790 M, KRAS and PTEN loss.42–44 Further complicating the therapeutic targeting of EGFR-enriched H1650 cells is the inherent heterogeneity of tumor cells typified by such subpopu-lations as the highly resistant, SOX2-overexpressing H1650 CSCs.7 Altogether, these characteristics present H1650 CSCs as a suitable cancer model for investigating the efficacy of a targeted
therapy approach for sensitizing and/or treating resistant cancers.1,45,46
Many cancer treatment protocols comprise a combination treatment regimen as a strategy for enhancing efficacy, overcoming acquired resistance and improving the safety profile of treatment. For example, the monoclonal antibody cetuximab used in combination with cisplatin/vinorelbine as first-line therapy demonstrated superior efficacy compared to cisplatin/ vinorelbine alone in a phase II clinical trial of patients with EGFR-positive advanced NSCLC.47 However, the incidence of hematologic, dermatologic, and respiratory toxicities was higher in the combination group compared to the chemotherapy group. Our study, while precluding the investigation of these toxicities showed evidence of distress of mice to cisplatin treatment, resulting in drastic loss in body weight, which necessitated the termination of the cisplatin group at day 8. The animals were, however, tolerant to CL-siSOX2 and CL-siSOX2 + cisplatin. Importantly, significant reduction in (xenograft) tumor volume and weight, and decreased number of (orthotopic) tumor nodules and areas of dissemination were observed with treatment with cisplatin, CL-siSOX2 and CL-siSOX2 + cisplatin. Additionally, semi-quantitative assessment of tissue sections showed de-creased expression of SOX2 and reversal of EMT in the CL-siSOX2 and CL-siSOX2 + cisplatin groups; and histologic staining of tissue sections showed less tissue remodeling in the treatment groups.