Multistep, effective drug distribution within solid tumors.

The distribution of drugs within solid tumors presents a long-standing barrier for efficient cancer therapies. Tumors are highly resistant to diffusion, and the lack of blood and lymphatic flows suppresses convection. Prolonged, continuous intratumoral drug delivery from a miniature drug source offers an alternative to both systemic delivery and intratumoral injection. Presented here is a model of drug distribution from such a source, in a multistep process. At delivery onset the drug mainly affects the closest surroundings. Such ‘priming’ enables drug penetration to successive cell layers. Tumor ‘void volume’ (volume not occupied by cells) increases, facilitating lymphatic perfusion. The drug is then transported by hydraulic convection downstream along interstitial fluid pressure (IFP) gradients, away from the tumor core. After a week tumor cell death occurs throughout the entire tumor and IFP gradients are flattened. Then, the drug is transported mainly by ‘mixing’, powered by physiological bulk body movements. Steady state is achieved and the drug covers the entire tumor over several months. Supporting measurements are provided from the LODER™ system, releasing siRNA against mutated KRAS over months in pancreatic cancer in-vivo models. LODER™ was also successfully employed in a recent Phase 1/2 clinical trial with pancreatic cancer patients.


Animals
Female C57B/6 5-week-old mice were purchased from Harlan, Israel. All mice were kept in a specific pathogen-free facility. Mice were handled according to the criteria outlined in the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health. All experiments were approved by the Animal Care Committee of the Hebrew University.

Tumor models
The mice were allowed to acclimate to the facility for at least one week prior to manipulation. Mice had free access to water and chow at all times. All animal procedures were performed under general anesthesia with intraperitoneally (i.p.) administered xylazine, 10 mg/g body weight (Chanelle Pharmaceuticals Manufacturing, Loughrea, Galway, Ireland) and ketamine, 450 mg/g body weight (Fort Dodge Animal Health, Fort Dodge, IA). After surgery, mice were allowed food and water ad libitum.

Subcutaneous tumors
Tumor xenografts were established by subcutaneous injection of log-phase growth viable cells, 10 7 (in 150 μL PBS) in case of Panc1 cells or 10 6 (in 100 μL PBS) in case of Panc-02 cells; the cells were injected into the flanks of the mice. When tumors reached an average volume of 80 mm 3 , mice were divided into equal groups. LODERs were implanted into tumors under anesthesia. The tumor volume was calculated according to the following formula: V = [largest diameter * small diameter 2 ]/2

Intra-pancreatic orthotopic tumors
The mice were anesthetized; their abdomens were sterilized with alcohol (70%) and were positioned laterally. A small, left abdominal flank incision was made, and the pancreas tail with the spleen was carefully exposed under aseptic conditions. The tumor cells (10 6 cells/30μL PBS) were injected into the tail of pancreas using a 27G tuberculin syringe. After replacement of the pancreas into the abdominal cavity, the incision was closed in two layers using an absorbable surgical 6-0 vicryl suture for the peritoneum and a 4-0 vicryl suture for the skin. After surgery, mice were inspected daily. Tumor growth was followed by measurement of Luciferase levels. When the tumors were detected, mice were stratified and divided into treatment groups according to the Luciferase levels and treated as noted. For LODER™insertion, mice were anesthetized, the pancreas was exposed as described, and LODERs were attached to the tumor using a 7-0 vicryl suture. The abdominal cavities were closed as described. Pancreatic tumor growth was followed by Luciferase measurement twice a week.

Immunohistochemical staining
Immunohistochemistry was conductedon 5 μm thick formalin-fixed, paraffin-embedded tissue sections by standard procedures. Deparaffinization and rehydration SUPPLEMENTARY DATA www.impactjournals.com/oncotarget/ Oncotarget, Supplementary Materials 2015 were followed by antigen retrieval using a pressure cooker with Glycine buffer (pH9) and CDC47. CDC47 primary antibodywas diluted 1:50 (Biocare Medical #CM137b from Pharmatrade). Secondary antibodies were from DAKO. Staining was developed with diamonobenzine using a kit from Zymed for H&E staining. TUNEL staining was performed using the In Situ Cell Death Detection Kit (Riche, cat# 11684795910). Slides were visualized using a Nikon microscope and analyzed using the Nis elements computer program (Nikon Instruments Inc.)

Priming
Priming (~first 1-2 days) -onset of drug release leads to apoptosis at the closest cell layers surrounding the drug delivery system, enabling more drug to penetrate further outwards. The tumor is still impervious to diffusion far from the closest layers.
To accelerate priming, LODER™ was designed to release a burst of 20% of its contents on the first day, which for aLODER™ containing a total drug load of 5 μg translates into 4 × 10 13 molecules. Substituting a 1 mm 3 layer of volume into which the drug penetrates on the first day, which would contain 7.1 × 10 5 cells (as obtained from the [cell density] 3/2 ) in 'untreated' tumor, in Table  1), an average of 0.53 × 10 8 siG12D molecules per cell is expected. Therefore, even if the cell relative uptake is much smaller than the 0.21%, the lowest value in Table 2, the number of released molecules during that step will be sufficient to support immediate drug entrance into the cell layer at a depth of ~165 μm, allowing the priming step.

Statistical analysis
All data were subjected to statistical analysis using the Excel software package (Microsoft, Courtaboeuf, France). A two-tailed Student's t-test was used to determine the difference between the groups. Differences were considered significant at P < 0.05. Data are given as the mean ± SEM