The proposed approach holds several advantages for LN targeted tumor therapy: high uptake in the TDLN, ability to present the therapeutic substance to cancer cells in its natural form, and low toxicity to local LN tissue. Supplementary Material supplementFig. antibodies against DR4, DR5 and their corresponding isotype controls. (B) Percent viability of SW620 cells at different concentrations of TRAIL treatment as measured using MTT assay. Bars symbolize imply and standard deviation from 3 wells for each treatment condition. (C) Representative bright field micrographs of SW620 cells at 0 ng/mL and 100 ng/mL TRAIL treatment after 24 hr in culture. The brightfield micrograph of TRAIL treated cells shows lifeless cells (reddish arrows) characterized by a rounded morphology when compared to live cells (green arrows) that are spread and show a spindle-shaped morphology. Level bar=100 m. Fig. S3: Growth of main tumor in treatment groups: (A) Sequential noninvasive bioluminescence imaging shows the growth of main tumor in mice under different treatment conditions. Three mice per group are shown at week 2, 4 and 6 for each treatment ITK inhibitor 2 condition. The color scale indicates bioluminescence signal intensity. (B) Quantification of total flux indicating the primary tumor growth with time from a circular ROI around the primary tumor. Each point represents the imply standard deviation for 5 mice at each time point (4 mice at week 6 for buffer and soluble TRAIL treatment groups). (C) Box and whisker plot comparing total flux from the primary tumor in mice from different treatment groups at week 2. NS, Not Significant (Wilcoxon rank-sum test). (D) Box and whisker plot comparing the total flux from the primary tumor in mice from different treatment groups at week 6. *p 0.05, **p 0.01 (Wilcoxon rank-sum test) and n=5 mice per group (4 mice for buffer and soluble TRAIL treatment groups). Fig. S4: TRAIL liposome toxicity to liver: (A) Following subcutaneous injection of TRAIL/IgG liposomes or TRAIL/Anti-NK1.1 liposomes (every 3 days for 6 weeks), C57BL/6 mice were euthanized and blood harvested by cardiac puncture. Serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) liver enzymes in control mice and TRAIL/IgG and TRAIL/Anti-NK1.1 treated mice showed no significant difference RGS18 in enzyme levels. Bars represent imply standard deviation for enzyme levels. n=3 mice per group. NS, not significant (student t-test). (B) Representative H&E stained section of liver from control and treatment mice showing no evidence of cytotoxicity in in liver owing to TRAIL/Anti-NK1.1 treatment. Level bar=100 m. Fig. S5: Body weight of animals in different treatment groups: Body weight from week 1 to week 6 of mice in control group and treatment groups. Each point represents the imply standard deviation excess weight (grams) from 5 mice in each group. Control group represents age-matched and strain-matched mice that received no tumor cells or drug. Fig. S6: Percentage of NK cells infiltrating the primary tumor: Circulation cytometry scatter plots showing the percentage of NK cells infiltrating the primary tumor isolated from a representative mouse in each treatment group at the end of the study. NIHMS735889-product.docx (45M) GUID:?ABB9A382-A65F-43A6-ABFD-2712D216285C Abstract Tumor draining lymph nodes are the first site of metastasis in most types of cancer. The extent of metastasis in the lymph nodes is usually often used in staging malignancy progression. We previously showed that nanoscale TRAIL liposomes conjugated to human natural killer cells enhance their endogenous therapeutic potential in killing malignancy cells cultured in designed lymph node microenvironments. In this work, it is shown that liposomes decorated with apoptosis-inducing ligand TRAIL and an antibody against a mouse natural killer cell marker are carried to the tumor draining inguinal lymph nodes and prevent the lymphatic spread of a subcutaneous tumor in ITK inhibitor 2 mice. It is shown that targeting natural killer cells with TRAIL liposomes enhances their retention time within the tumor draining lymph nodes to induce apoptosis in malignancy cells. It is concluded that this approach can be used to kill cancer cells within the tumor draining lymph nodes to prevent the lymphatic spread of malignancy. LN microenvironments . When cocultured with human malignancy cell lines that are known to metastasize to LN in experimental animal models, engineered super natural killer cells were able to induce ITK inhibitor 2 apoptosis in malignancy cells to a significantly higher degree compared to unmodified NK cells. The goal of the present study was to determine if TRAIL liposomes targeted to NK cells that traffic to the tumor draining inguinal LN of mice bearing a subcutaneous human xenograft tumor could effectively prevent the metastasis of a primary tumor to the TDLN. Orthotopic models are also used for studying malignancy metastasis.