Abstract Introduction: Intercellular communication is critical for cancer growth and progression. Research to date has focused on gap junctions, micro-vesicles/exosomes, and diffusible factors. There is limited understanding of how efficient cell-to-cell communication occurs between distant cells in the complex tumor microenvironment. Tunneling nanotubes (TNTs) are thin, non-adherent actin filament-based, open conduits that facilitate direct sharing of various cellular cargos between cells. TNTs can be 50-800 nanometers in diameter and up to several hundred micrometers in length in vitro. Our lab was the first to provide in vivo evidence of TNTs in human solid tumors. We hypothesized that TNT formation is significantly upregulated in human cancers compared to their non-malignant counterparts. In this study, we examined the rate of TNT formation in cell lines from multiple human cancers. Methods: Cell lines from four cancers (colon, mesothelioma, pancreatic, and ovarian cancer) were plated at similar density in 6-well tissue culture plates. The number of TNTs and cells in 10 randomly chosen fields of each well were quantified every 24 hrs for up to 96 hrs and representative images were taken. Experiments were performed in duplicate for each cell line. Results: The number of TNTs per cell (TNTs/cell), which we have titled “TNT index,” was calculated in order to control for cell proliferation. The ratio of TNTs formed between malignant colorectal cells compared benign adenoma ranged from 20:1 to 100:1 by 96 hrs. For mesothelioma, malignant:mesothelial TNTs/cell ratio ranged from 8:1 to 78:1 over 72 hrs. The mesothelioma cells were too confluent at 96 hrs to quantify TNTs. The pancreatic malignant:stellate human pancreatic ductal epithelial TNTs/cell ratio ranged between 2:1 to 63:1. Interestingly, we found that non-malignant ovarian epithelial (IOSE) cells had a significantly higher rate of TNT formation than malignant ovarian cells. Conclusions: We have demonstrated that malignant cells have significantly higher rates of TNT formation compared to stromal or premalignant cells of their corresponding tumor. TNTs potentially provide malignant cells a direct route for transmitting influential messages across long distances in the complex tumor microenvironment. We believe that upregulation of TNTs in cancer may amplify cellular cross-talk and thus increase rates of tumor recurrence and invasion. Citation Format: Snider Desir, Subbaya Subramanian, Clifford Steer, Emil Lou. Tunneling nanotube formation is significantly upregulated in invasive cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3189. doi:10.1158/1538-7445.AM2015-3189