Abstract 1558: The critical role of lymph node stromal cell-derived microvesicles in colorectal cancer metastasis
Authors: Zhang Xin , Sullivan Ryan , Hite Nathan , Maresh Grace , Hellmers Linh , Lin Zhen , Flemington Erik , Salomon Carlos , Green Heather , Margolin David , Li Li 
Published: July 2016

Introduction: Metastatic disease is responsible for 90% of colorectal cancer (CRC) deaths. Studies suggest that metastasis is closely associated with the presence of CRC tumor-initiating cells (Co-TIC) and their interaction with the lymph node (LN) stromal microenvironment. Prior to developing extra-nodal metastasis, these cells acquire a chemotherapy-resistant phenotype developing genetic alterations making them resistant to conventional treatments. In addition to cell-cell contact and secreted molecules, a recently discovered means of intercellular signaling is the exchange of extra-cellular vesicles. These microvesicles (MVs) carry complex biological information, including mRNA, miRNA, as well as soluble and transmembrane proteins that can affect the behavior of target cells. MVs have been detected in patient specimens with diverse malignancies and may play a role in communication between the LN stromal microenvironment and Co-TIC. We hypothesize that MVs are involved in intracellular trafficking between LN stromal cells and CRC cells promoting tumor formation and distant organ metastasis.

Methods: MVs released by human mesenteric LN stromal cells (LNSC) derived from surgical specimens and the established LN stromal cell line (HK cell) were isolated using differential centrifugation and gradient purification. The MVs were visualized using GFP-HK cell and RFP-HT-29 cell (CRC cell line) and florescence microscopy. The functional properties of LN stromal MVs and their effect on CRC proliferation and metastasis was analyzed using established in vitro co-culture models and a humanized orthotopic intra-rectal (IR) injection mouse model, tracked by bioluminescent imaging (BLI).

Results: A 100,000 g pellet containing MVs derived from LNSC and HK cells have a similar size profile when analyzed by NanoSight. Budding CD63-RFP tagged MVs were released by LNSC and HK cells and uptake by GFP tagged CRC cells was confirmed through time-lapse experiments using deconvoluting microscopy. When HK cell or LN stromal cell-derived MVs were co-cultured with HT-29 cells in vitro, they supported HT-29 cell growth at a similar level as that of HK cell or LN stromal cell conditioned media, respectively. By adding LNSC- or HK-derived MVs to HT-29-Luc cells or patient derived CRC cells (CRC-Pt-Luc cells) in our IR model, we demonstrated that MVs enhanced CRC tumor growth as well as distant organ metastasis in vivo.

Conclusion: MVs isolated from LNSCs traffic between the stromal cells and CRC cells. These MVs promote tumor formation and distant organ metastasis in vivo suggesting that they play a crucial role in the communication between the LN stromal microenvironment and CRC cells. Further analyzing the functional properties of effector MV RNAs may help identify novel targetable candidates for therapeutic strategies that target CRC metastasis using our unique patient derived orthotopic mouse model.

Citation Format: Xin Zhang, Ryan Sullivan, Nathan Hite, Grace Maresh, Linh Hellmers, Zhen Lin, Erik Flemington, Carlos Salomon, Heather Green, David Margolin, Li Li. The critical role of lymph node stromal cell-derived microvesicles in colorectal cancer metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1558.