Stearoyl gemcitabine nanoparticles overcome obesity-induced cancer cell resistance to gemcitabine in a mouse postmenopausal breast cancer model

Abstract

Obesity is associated with increased breast tumor aggressiveness and decreased response to multiple modalities of therapy in postmenopausal women. Delivering cancer chemotherapeutic drugs using nanoparticles has evolved as a promising approach to improve the efficacy of anticancer agents. However, the application of nanoparticles in cancer chemotherapy in the context of obesity has not been studied before. The nucleoside analog gemcitabine is widely used in solid tumor therapy. Previously, we developed a novel stearoyl gemcitabine solid-lipid nanoparticle formulation (GemC18-NPs) and showed that the GemC18-NPs are significantly more effective than gemcitabine in controlling tumor growth in mouse models. In the present study, using ovariectomized diet-induced obese female C57BL/6 mice with orthotopically transplanted MMTV-Wnt-1 mammary tumors as a model of postmenopausal obesity and breast cancer, we discovered that obesity induces tumor cell resistance to gemcitabine. Furthermore, our GemC18-NPs can overcome the obesity-related resistance to gemcitabine chemotherapy. These findings have important clinical implications for cancer chemotherapies involving gemcitabine or other nucleoside analogs in the context of obesity.

Keywords: chemoresistance; nanoparticles; obesity; tumor.

Figure 1. Effect of adipocyte conditioned medium (CM) on tumor cell proliferation, invasiveness and response to gemcitabine. (A) The effect of adipocyte-CM (0.1%), in comparison to fibroblast-CM, on the proliferation of M-Wnt, E-Wnt and MCF-7 tumor cells. (B) The effect of adipocyte-CM on the cytotoxicity of gemcitabine HCl and GemC18-NPs on M-Wnt, E-Wnt and MCF-7 cells. (C) Migration of M-Wnt and MCF-7 cells in an in vitro assay. Each experiment was performed in triplicate (or more) on different days and repeated at least twice. Different letters (a, b, c) indicate significant differences in each cell line (p < 0.05).

Figure 2. Mouse body weight and tumor growth in ovariectomized C57BL/6 mice. OVX C57BL/6 mice were fed a control diet (10% Kcal fat, n = 39) or a DIO diet (60% Kcal fat, n = 39) for the duration of the study. Obese mice had significantly increased body weight, as compared with control mice (A) (* p < 0.05, Control vs. Obese in week 17). (B) M-Wnt mammary tumor size at endpoint (i.e., week 24) (n = 12−13). M-Wnt tumor cells were injected in the 9th fat pad in week 17. In weeks 20, 21, 22 and 23, mice were injected via the tail vein with GemC18-NPs, a molar equivalent of gemcitabine HCl or mannitol as a vehicle control. The doses of the gemcitabine and GemC18-NPs were adjusted based on the weight of individual mice in the week of every injection. Values with different letters are significantly different (p < 0.05). (C) Mouse body weight after M-Wnt tumor cell injection and during gemcitabine treatments (n = 12−13).

Figure 3. The effect of diet and gemcitabine treatment on the histopathology of M-Wnt tumors in mice. (A) H&E staining and IHC staining for pHH3, Ki-67, p-S6, CD31 and perilipin expressions in tumors. Scale bars for H&E are 400 μm, 100 μm for others. (B) Quantification of pHH3-, Ki-67-, p-S6-, CD31- and perilipin-positive cells and the mean vessel area determined by CD31 staining, from representative images. Data are expressed as mean ± SEM (n = 3−5 slides/group). The numbers of +1, +2 and +3 in the pHH3 graph indicate the density of pHH3 staining. Different letters (a, b, c or a’, b’, c’) indicate significant differences for each biomarker.