Gemcitabine enhances the transport of nanovector-albumin-bound paclitaxel in gemcitabine-resistant pancreatic ductal adenocarcinoma

Abstract

The mechanism for improved therapeutic efficacy of the combination therapy with nanoparticle albumin-bound paclitaxel (nAb-PTX) and gemcitabine (gem) for pancreatic ductal adenocarcinoma (PDAC) has been ascribed to enhanced gem transport by nAb-PTX. Here, we used an orthotopic mouse model of gem-resistant human PDAC in which increasing gem transport would not improve the efficacy, thus revealing the importance of nAb-PTX transport. We aimed to evaluate therapeutic outcomes and transport of nAb-PTX to PDAC as a result of (1) encapsulating nAb-PTX in multistage nanovectors (MSV); (2) effect of gem on caveolin-1 expression. Treatment with MSV/nAb-PTX + gem was highly efficient in prolonging animal survival in comparison to other therapeutic regimens. MSV/nAb-PTX + gem also caused a substantial increase in tumor PTX accumulation, significantly reduced tumor growth and tumor cell proliferation, and increased apoptosis. Moreover, gem enhanced caveolin-1 expression in vitro and in vivo, thereby improving transport of nAb-PTX to PDAC. This data was confirmed by analysis of PDACs from patients who received gem-based neo-adjuvant chemotherapy. In conclusion, we found that nAb-PTX treatment of gem-resistant PDAC can be enhanced by (1) gem through up-regulation of caveolin-1 and (2) MSV through increasing accumulation of nAb-PTX in the tumor.

Keywords: Drug resistance; Gemcitabine; Multistage nanovectors; Pancreatic cancer; Transport; nAb-paclitaxel.

Figure 1

Survival of nude mice bearing L3.6pl orthotopic PDAC (n = 9–10) in response to therapy with control (PBS), gem, nAb-PTX, MSV, MSV/nAb-PTX, nAb-PTX + gem, and MSV/nAb-PTX + gem. Gem was given at a concentration of 50 mg/kg, nAb-PTX or MSV/nAb-PTX at a concentration of 75 mg/kg equivalent to 7.5 mg/kg PTX, and MSVs at a dose of 10⁹ particles/mouse.

Figure 2

Suppression of tumor growth in response to nAb-PTX + gem or MSV/nAb-PTX + gem vs. no treatment control (PBS) in nude mice bearing L3.6pl orthotopic PDAC (n = 8): A) PDAC weight. B) Quantification of proliferating Ki67 positive cells. C) Quantification of apoptotic cells measured with the TUNEL assay. Field of view: 430 μm2. (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Figure 3

Accumulation of PTX in pancreatic tumor and liver of nude mice bearing L3.6pl orthotopic PDAC determined by LC-MS/MS (n = 5). (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Figure 4

Increased expression of albumin transporter cav-1 in vitro (A), and in vivo (B) in response to gem treatment: (A1) Cav-1 expression obtained through ELISA of mouse endothelial cells lysates treated with gem 5, 10, and 20 nM in vitro (*P < 0.05); (A2) Fluorescent images of untreated mouse endothelial cells stained for cav-1 (green) and cell nuclei (DAPI, blue); (A3) Fluorescent images of mouse endothelial cells treated with gem 20 nM and stained for cav-1 (green) and cell nuclei (DAPI, blue). Scalebar: 100 μm; (B1) Quantification of mean fluorescent intensity of cav-1 immunofluorescence in tumor sections from nude mice bearing L3.6pl orthotopic PDAC in vivo. (**** P < 0.0001); (B2) Fluorescent images of PDAC from untreated mice (PBS group) stained for cav-1 (green) and cell nuclei (DAPI, blue); (B3) Fluorescent images of PDAC from mice treated with gem 50 mg/kg stained for cav-1 (green) and cell nuclei (DAPI, blue). Scalebar: 100 μm.

Figure 5

Increased expression of albumin transporter cav-1 in PDAC samples from patients (see Supplementary Table 1) in response to gem treatment: (A) image processing workflow applied to analyze 30 patient specimens stained with cav-1. Tissue segmentation was employed in order visualize tumoral and stromal components, while a marker area detection algorithm was used to identify the cav-1 positive tissue. (B) Immunohistological quantification of cav-1 in the PDAC tumors of the patients pre-treated with gem-based neoadjuvant chemotherapy compared to the patients without neoadjuvant chemotherapy, ANOVA (P = 0.0032)

Figure 6

Proposed mechanism for gem and MSV-induced preferential accumulation of nAb-PTX in tumor tissue. (A) normal tumor vasculature, (B) upon intraperitoneal injection of gem the expression of gp60 and caveolin-1 increases, (C) after intravenous injection, nAb-PTX accumulates in tumor, (D) MSV delivery further improves preferential localization of nAb-PTX in the tumor due to the formation of vascular depots in tumor blood vessels, (E) Mechanism for nAb-PTX transport across the endothelial barrier through transcytosis mediated by gp60 receptor and caveolin-1.