Gastroenterology

Gastroenterology

Volume 128, Issue 7, June 2005, Pages 1796-1804
Gastroenterology

Rapid communication
Human Colorectal Cancer Cells Induce T-Cell Death Through Release of Proapoptotic Microvesicles: Role in Immune Escape

https://doi.org/10.1053/j.gastro.2005.03.045Get rights and content

Background & Aims: Normal and neoplastic cells release microvesicles, whose effects on the immune system still need to be elucidated. Because human colorectal cancer cells are hypothesized to escape immune recognition by expressing proapoptotic molecules, we investigated whether microvesicles bearing Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand and inducing apoptosis of activated T cells are secreted by colorectal cancer cells both in vitro and in affected patients. Methods: Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand expression were analyzed in colorectal cancer cells and purified microvesicles by flow cytometry, Western blotting, and immunoelectron microscopy. Microvesicle tumor origin was assessed through simultaneous detection of lysosomal (CD63) and adenocarcinoma (carcinoembryonic antigen) markers. Proapoptotic activity of microvesicles was evaluated by annexin V/propidium iodide staining and caspase activation in T cells, including CD8+ T lymphocytes from colorectal cancer patients. Results: Colorectal cancer cells showed a granular pattern of tumor necrosis factor-related apoptosis-inducing ligand and Fas ligand expression, suggesting a secretory behavior. These proapoptotic molecules were detected on isolated microvesicles, together with class I HLA, CD63, and carcinoembryonic antigen. Microvesicles induced Fas ligand-mediated and tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis of activated CD8+ T cells generated from colorectal cancer patients. Microvesicles with comparable phenotypes and functions were found in plasma from patients with advanced disease, whereas vesicular structures expressing Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand were also detected in colorectal cancer specimens. Conclusions: These data show that colorectal cancer induces T-cell apoptosis through the release of Fas ligand-bearing and tumor necrosis factor-related apoptosis-inducing ligand-bearing microvesicles both in vitro and in vivo. This mechanism of immune escape has potential implications as a prognostic factor and could be targeted for the development of new antitumor therapies in colorectal cancer patients.

Section snippets

Colorectal Cancer Lines, Colorectal Cancer Patients, and Purification of Released Microvesicles

The CRC line SW403 was purchased from the American Type Culture Collection (Manassas, VA), and 1869 col (provided by Dr C. Maccalli, Istituto Superiore di Sanità, Rome, Italy) 17 and CRC28462 were short-term lines generated from liver metastases of CRC patients. All the lines stained positively for pan-epithelial (BerEP4), cytokeratin (CD18, LP34, and MNF116), and class I HLA markers and were negative for the fibroblast marker 5B5. These lines formed colonies in soft agar, were tumorigenic in

Expression of FasL and TRAIL in Human Colorectal Cancer Cells and Their Released Microvesicles

Expression of both FasL (Figure 1A) and TRAIL (Figure 1B and C), as detected by immunocytochemistry on CRC cell lines, displayed a granular distribution that was apparently confined to the intracellular compartments. TRAIL staining seemed to vectorially accumulate either at the cell-to-cell contact sites or at the center of pseudocrypts formed in culture (Figure 1C). IEM showed FasL and TRAIL localization in defined intracellular MVs (approximately 50–100 nm), primarily located in proximity to (

Discussion

Here we report that human CRC cells induce apoptosis of T lymphocytes through the release of FasL- and TRAIL-bearing MVs. These lysosome-derived organelles, characterized by a 50–100-nm diameter and a high content of cytosolic and membrane proteins derived from the producing cell, are released by CRC in vitro and are found in plasma and tumor specimens from patients with advanced CRC. These data shed new light on the role of FasL in immune evasion of CRC. The acquired expression of this

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    Supported by grants from the Italian Association for Cancer Research (Milan) and Fondo pergli Investimenti della Ricerca di Base, Ministero dell’Instruzione, dell’Università e della Ricerca (FIRB-MIUR) RBNE017B4C (Rome).

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