Exosomes Derived from Cancer Associated Fibroblasts Elicit Survival and Drug Resistance of Primary Lymphoma Cells
Authors: Kunou Shunsuke , Shimada Kazuyuki , Hikita Tomoya , Aoki Tomohiro , Sakamoto Akihiko , Hayakawa Fumihiko , Oneyama Chitose , Kiyoi Hitoshi 
Published: October 2019

Abstract
Background
Cancer associated fibroblasts (CAFs) are one of the major components constituting the tumor microenvironment and known to be deeply involved in the growth and metastasis of solid tumors. We previously reported that CAFs supported survival of primary lymphoma cells via the increased glycolytic metabolism (Aoki et al. Oncotarget 2017). Recent findings indicate that exosomes secreted from tumor cells play an important role of the survival and metastasis. Although the information about exosomes secreted from tumor cells has been accumulated, the role of them secreted from cells constituting the tumor microenvironment has been largely unexplored.
Purpose
To uncover the role of exosomes in lymphoma microenvironment, we investigated the function of them derived from CAFs those were isolated from primary lymphoma samples.
Methods
CAFs were successfully isolated from primary lymph node samples of various types of non-Hodgkin lymphoma including follicular lymphoma, diffuse large B-cell lymphoma, angioimmunoblastic T-cell lymphoma, mantle cell lymphoma, and T lymphoblastic lymphoma (N=20), and subsequently exosomes secreted from the representative 4 CAFs were obtained by a standard procedure using ultracentrifugation. Exosomes were confirmed by immunoblotting, electron microscope, and the nano tracking analysis. The functional role of CAFs and exosomes in an interaction between lymphoma cells and its microenvironment were investigated. Written informed consent for the experimental use of patient lymph node samples was obtained, and all experimental procedures were approved by the institutional review board of Nagoya University Hospital.
Results
We isolated CAFs from different types of non-Hodgkin lymphoma samples, and studied whether the survival of patient lymphoma cells could be supported in co-culture with CAFs. The survival of cells differed depending on CAFs indicating the diversity of the ability of CAFs to support lymphoma cells. Then we analyzed cellular glycolysis and ATP production of lymphoma cells in co-culture with CAFs. As expected, the increase of glycolysis and production of ATP differed among 4 CAFs probably due to the extent of the Warburg effect. Next, we investigated whether CAFs secreted extracellular vesicles into culture supernatant. We found that vesicles with CD9 positive and matched exosomes in size were accumulated in culture media (Figure A, B), and the amount of released exosomes differed among CAFs in line with the ability to support the survival of lymphoma cells. Exosomes displayed survival support of lymphoma cells in a dose-dependent manner(Figure C), and cellular glycolysis and ATP production were increased in the presence of exosomes as well as CAFs, which indicated that the ability of CAFs to support lymphoma cells was, at least in part, elicited by exosomes. While exosomes secreted from CAFs with the strong ability to support lymphoma cells displayed the support of the survival, the survival of lymphoma cells was not observed in the presence of exosomes derived from CAFs with the weak support of lymphoma cells even in higher dose of exosomes, indicating that not only the quantity but also the quality of exosomes could be a determinant of the survival effect. Next, we focused on exosomes secretion-related proteins of nSMase2 and Rab27b to uncover the underlying mechanism of secretion of exosomes. NSMase2 required for exosomes formation and Rab27b involved in the migration of multivesicular body from the pericule to the membrane were strongly expressed in CAFs with higher ability to support lymphoma cells. Using nSMase2- and Rab27b- specific siRNA, the amount of exosomes secretion was reduced resulting in the decreased survival support. Finally, we studied the presence of CAFs or exosomes associated with the drug resistance. As expected, primary lymphoma cells demonstrated resistance to gemcitabine and cytarabine in the presence of CAFs or exosomes, and the resistance was restored in the presence of CAFs transduced with Rab27b-specific siRNA.
Conclusion
Our results suggest that CAFs and exosomes secreted from them are involved in the survival and drug resistance of patient lymphoma cells and play a pivotal role in the microenvironment of non-Hodgkin lymphoma. Exosomes would be a novel attractive therapeutic target.

Disclosures
Kiyoi: Sanofi K.K.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Celgene Corporation: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; FUJIFILM Corporation: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Phizer Japan Inc.: Research Funding; Novartis Pharma K.K.: Research Funding; Astellas Pharma Inc.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Bristol-Myers Squibb: Honoraria.