Pancreatic Cancer Cell Exosome-Mediated Macrophage Reprogramming and the Role of MicroRNAs 155 and 125b2 Transfection using Nanoparticle Delivery Systems

Exosomes are nano-sized endosome-derived small intraluminal vesicles, which are important facilitators of intercellular communication by transporting contents, such as protein, mRNA, and microRNAs, between neighboring cells, such as in the tumor microenvironment. The purpose of this study was to understand the mechanisms of exosomes-mediated cellular communication between human pancreatic cancer (Panc-1) cells and macrophages (J771.A1) using a Transwell co-culture system. Following characterization of exosome-mediated cellular communication and pro-tumoral baseline M2 macrophage polarization, the Panc-1 cells were transfected with microRNA-155 (miR-155) and microRNA-125b-2 (miR-125b2) expressing plasmid DNA using hyaluronic acid-poly(ethylene imine)/hyaluronic acid-poly(ethylene glycol) (HA-PEI/HA-PEG) self-assembling nanoparticle-based non-viral vectors. Our results show that upon successful transfection of Panc-1 cells, the exosome content was altered leading to differential communication and reprogramming of the J774.A1 cells to an M1 phenotype. Based on these results, genetic therapies targeted towards selective manipulation of tumor cell-derived exosome content may be very promising for cancer therapy.


Supplementary Information
Methods and Results 1. Evaluation of Baseline Macrophage Polarity and Establishment of Panc-1 and J774.A1 Cells in Co-culture System

Evaluation of Baseline Macrophage Polarity
To induce the pro-inflammatory subtype (M1), J774A.1 macrophages were cultured for 6 hours in the presence of 100 ng/mL IFN-γ and LPS. To induce the antiinflammatory phenotype (M2), J774A.1 macrophages were culture for 6 hours in the presence of 100 ng/mL IL-4 for M2a phenotype, or 100 ng/mL IL-10/TGF-b for M2c phenotype.

Total RNA Extraction
The total RNA extraction was isolated from the cell lysates using the High Pure RNA Isolation kit. RNA extraction kit from all the cell pellets that were obtained from untreated J774A1, M1 polarized J774A1 for 6 hours, M1 polarized J774A1 for 72 hours, M1 polarized J774A1 cells co-culture with Panc-1for 72 hours, M2 polarized J774A1 for 6 hours conditions and quantified by using Nano Drop 2000. The extracted total RNA was stored at -80°C for further use.

RT-PCR Analysis of Macrophage Phenotype Expression Markers
The amplification of M1 macrophage marker, IL-1 beta gene and M2 macrophage markers, arginase-1 gene were performed on the extracted total RNA samples by using the Verso ® cDNA Synthesis Kit for first cDNA strand synthesis and Platinum TaqDNA ® Polymerase for DNA amplification. Beta-actin was used as a housekeeping gene. The following are primer sequences, which were used for the amplification. Reverse transcription PCR was run on the thermo cycler (BioRad T100, Hercules, CA) for 2 min at 94 °C and 40 cycles of 94 °C for 30 sec, 55 °C for 30 sec, and 72 °C for 60 sec for all the primers. The amplified reaction products were visualized by running them on 2 % E Gels with SYBR Safe for 30 minutes. The gels were imaged by using ChemiDocTM XRS imaging system and the intensity or the density of the bands on the gels was quantified on the using ImageLab TM software on the system. Densitometric analysis of the bands was done to evaluate the expression levels of total RNA of the genes for specific M1/M2 macrophage endogenous markers.

Indirect Co-culture of Pancreatic Cancer Cells and Macrophage
Murine J774A.1 macrophages (2 x 10 5 cells/dish) were seeded in 100 mm polystyrene cell culture dishes in DMEM, supplemented with 10% FBS and 1% penicillin/streptomycin. Pancreatic cancer cells Panc-1 (3 x 10 5 cells/insert) were seeded into the upper chamber of a 75 mm Polycarbonate Transwell ® Insert of 3.0 µm pore size in DMEM with 10% FBS and 1% penicillin/streptomycin. The following day, the J774A.1 macrophages were treated with IFN-γ and LPS at a concentration of 100 ng/ml for 6 hours, with the aim of promoting the classical or M1 phenotype of macrophages.
After 6 hours, the media were replaced by new DMEM into the 100 mm cell culture dishes, and the culture inserts with Panc-1 cells were placed into the culture dishes containing J774A.1 macrophages, and incubated up to 72 hours in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. Cells were cultured in a standard humidified incubator at 37 o C. After co-culture with Panc-1cell, the phenotype of J774A.1 cells had been analyzed by RT-PCR.

Dose-dependent Studies of J774A.1 Re-programming with Panc-1 Exosomes
The aim of the studies is to reprogram the J774A.1 macrophages via the effect of Panc-1 exosomes and MiR-155 modified Panc-1 exosomes. Murine J774A.1 macrophages (2 x 10 5 cells/dish) were seeded in 100 mm polystyrene cell culture dishes in DMEM. Cells were cultured in a standard humidified incubator at 37 o C overnight. J774A.1 macrophages were cultured for a period of 6 hours in the presence of IFN-γ and LPS at a concentration of 100ng/ml, with the aim of promoting the classical or M1 phenotype of macrophages. After 6 hours, the media were replaced by new DMEM and different protein concentrations of exosomes, 40 µg, 80 µg, 120 µg, 160 µg, were added into the media for 72 hours. The J774A.1 macrophages were also treated with IL-4 and LPS/IFN-ϒ, respectively as positive control. The expression of M1 and M2 specific marker genes of J77A.1 macrophages at the mRNA level was then ascertained using two-step RT-PCR to determine phenotypic gene expression.

Macrophage Re-programming via MicroRNA-Mediated Re-programming Tumor
Exosomes Similar to dose-dependent studies of J774A.1 re-programming with Panc-1 derived exosomes, the aim of this study is to evaluate the effect of miR155/miR125b-2 modified exosomes on macrophage re-programming. Untreated J774A.1 macrophages were initially stimulated with 100ng/ml IL-4 to induce their polarization to M2 phenotype in each group. The miR155 and miR125b-2 modified exosomes were collected from plasmid DNA transfected panc-1 cells via HA-PEI/HA-PEG nanoparticles using same transfection protocol. The M2 states J774A.1 macrophages were further co-cultured with 160 µg of miR155/miR125b-2 modified exosomes in a standard humidified incubator at 37 o C for 48 hours. After 48 hours, the J774A.1 macrophages were collected for evaluating the polarization states via specific M1/M2 endogenous markers. The expression of IL-1β, iNOS, and Arginase were then determined by qPCR using the ΔΔCt Method. The LightCycler® 480 SYBR Green I Master Mix was used for preparing various reactions and β -Actin was used as an endogenous control. The Roche Light Cycle 480 Instrument was used for running the reactions.