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The tumor microenvironment is deeply involved in the process of tumor growth and development. In this study, we focused on cancer-associated fibroblasts (CAFs) and their derived exosomes on the lymphoma microenvironment to uncover their clinical significance. CAFs were established from primary lymphoma samples, and exosomes secreted from CAFs were obtained by standard procedures. We then investigated the roles of CAFs and their derived exosomes in the survival and drug resistance of lymphoma cells. CAFs supported the survival of lymphoma cells through increased glycolysis, and the extent differed among CAFs. Exosomes were identified as a major component of the extracellular vesicles from CAFs, and they also supported the survival of lymphoma cells. The suppression of RAB27B, which is involved in the secretion of exosomes, using a specific siRNA resulted in reduced exosome secretion and decreased survival of lymphoma cells. Moreover, anti-pyrimidine drug resistance was induced in the presence of exosomes through the suppression of the pyrimidine transporter, equilibrative nucleoside transporter 2 (ENT2), and the suppression of ENT2 was significant in in vivo experiments and clinical samples. RNA sequencing analysis of miRNAs in exosomes identified miR-4717-5p as one of the most abundant miRNAs in the exosome, which suppressed the expression of ENT2 and induced anti-pyrimidine drug resistance in vitro. Our results suggest that exosomes including miR-4717-5p secreted from CAFs play a pivotal role in the lymphoma microenvironment, indicating that they are a promising therapeutic target.

Summary Extracellular vesicles (EVs) are classified as large EVs (l-EVs, or microvesicles) and small EVs (s-EVs, or exosomes). S-EVs are thought to be generated from endosomes through a process that mainly depends on the ESCRT protein complex, including ALG-2 interacting protein X (ALIX). However, the mechanisms of l-EV generation from the plasma membrane have not been identified. Membrane curvatures are generated by the bin-amphiphysin-rvs (BAR) family proteins, among which the inverse BAR (I-BAR) proteins are involved in filopodial protrusions. Here, we show that the I-BAR proteins, including missing in metastasis (MIM), generate l-EVs by scission of filopodia. Interestingly, MIM-containing l-EV production was promoted by in vivo equivalent external forces and by the suppression of ALIX, suggesting an alternative mechanism of vesicle formation to s-EVs. The MIM-dependent l-EVs contained lysophospholipids and proteins, including IRS4 and Rac1, which stimulated the migration of recipient cells through lamellipodia formation. Thus, these filopodia-dependent l-EVs, which we named as filopodia-derived vesicles (FDVs), modify cellular behavior.

Chondroitin sulfate E (CSE) is a polysaccharide containing mainly disaccharide units of D-glucuronic acid (GlcA) and 4,6-O-disulfated N-acetyl-D-galactosamine (GalNAc) residues (E-unit) in the amount of ∼ 60%. CSE is involved in many biological and pathological processes. In this study, we established new monoclonal antibodies, termed E-12C and E-18H, by using CSE that contained more than 70% of E-units as an immunogen. These antibodies recognized CSE but not other CSs isomers or dermatan sulfate (DS). We evaluated the reactivities of the antibodies to 6-O-sulfated CSA (6S-CSA) and DS (6S-DS) that possessed ∼ 60% of GalNAc (4S, 6S) moieties in their structures. Neither of the antibodies reacted with 6S-DS. The antibodies strictly distinguished the structural difference of GlcA and L-iduronic acid in the polysaccharide. Binding affinities of the antibodies were determined by a surface plasmon resonance assay using CSE and 6S-CSA. The binding affinities were strongly associated with the molecular weight of CSE and the E-unit content of 6S-CSA. Moreover, we demonstrated that the antibodies are applicable to histochemical analysis. In conclusion, the new anti-CSE monoclonal antibodies specifically recognize the E-unit of CSE. The antibodies will become useful tools for the investigation of the biological and pathological significance of CSE.

Sialic acid bound to glycans in glycolipids and glycoproteins is essential for synaptic plasticity and memory. Sialidase (EC 3.2.1.18), which has 4 isozymes including Neu1, Neu2, Neu3 and Neu4, regulates the sialylation level of glycans by removing sialic acid from sialylglycoconjugate. In the present study, we investigated the distribution of sialidase activity in rat hippocampus and the role of sialidase in hippocampal memory processing. We previously developed a highly sensitive histochemical imaging probe for sialidase activity, BTP3-Neu5Ac. BTP3-Neu5Ac was cleaved efficiently by rat Neu2 and Neu4 at pH 7.3 and by Neu1 and Neu3 at pH 4.6. When a rat hippocampal acute slice was stained with BTP3-Neu5Ac at pH 7.3, mossy fiber terminal fields showed relatively intense sialidase activity. Thus, the role of sialidase in the synaptic plasticity was investigated at mossy fiber terminal fields. The long-term potentiation (LTP) at mossy fiber-CA3 pyramidal cell synapses was impaired by 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA), a sialidase inhibitor. DANA also failed to decrease paired-pulse facilitation after LTP induction. We also investigated the role of sialidase in hippocampus-dependent spatial memory by using the Morris water maze. The escape latency time to reach the platform was prolonged by DANA injection into the hippocampal CA3 region or by knockdown of Neu4 without affecting motility. The results show that the regulation of sialyl signaling by Neu4 is involved in hippocampal memory processing.

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.

The nonreceptor tyrosine kinase c-Src is frequently over-expressed or hyperactivated in various human cancers and contributes to cancer progression in cooperation with up-regulated growth factor receptors. However, Src-selective anticancer drugs are still in clinical trials. To identify more effective inhibitors of c-Src-mediated cancer progression, we developed a new screening platform using Csk-deficient cells that can be transformed by c-Src. We found that purvalanol A, developed as a CDK inhibitor, potently suppressed the anchorage-independent growth of c-Src-transformed cells, indicating that the activation of CDKs contributes to the c-Src transformation. We also found that purvalanol A suppressed the c-Src activity as effectively as the Src-selective inhibitor PP2, and that it reverted the transformed morphology to a nearly normal shape with less cytotoxicity than PP2. Purvalanol A induced a strong G2-M arrest, whereas PP2 weakly acted on the G1-S transition. Furthermore, when compared with PP2, purvalanol A more effectively suppressed the growth of human colon cancer HT29 and SW480 cells, in which Src family kinases and CDKs are activated. These findings demonstrate that the coordinated inhibition of cell cycle progression and tyrosine kinase signaling by the multi-selective purvalanol A is effective in suppressing cancer progression associated with c-Src up-regulation.

The tyrosine kinase c-Src is upregulated in various human cancers irrespective of its negative regulator Csk, but the regulatory mechanisms remain unclear. Here, we show that a lipid raft-anchored Csk adaptor, Cbp/PAG, is directly involved in controlling the oncogenicity of c-Src. Using Csk-deficient cells that can be transformed by c-Src overexpression, we found that Cbp expression is markedly downregulated by c-Src activation and re-expression of Cbp efficiently suppresses c-Src transformation as well as tumorigenesis. Cbp-deficient cells are more susceptible to v-Src transformation than their parental cells. Upon phosphorylation, Cbp specifically binds to activated c-Src and sequesters it in lipid rafts, resulting in an efficient suppression of c-Src function independent of Csk. In some human cancer cells and tumors, Cbp is downregulated and the introduction of Cbp significantly suppresses tumorigenesis. These findings indicate a potential role for Cbp as a suppressor of c-Src-mediated tumor progression.

The c-src proto-oncogene product, c-Src, is frequently over-expressed and activated in various human malignant cancers, implicating a role for c-Src in cancer progression. To verify the role of c-Src, we analyzed the transforming ability of c-Src in mouse embryonic fibroblasts that lack Csk, a negative regulator of Src family kinases. Although Csk deficiency is not sufficient for cell transformation, c-Src over-expression induced characteristic transformed phenotypes including anchorage-independent growth and tumorigenecity. These phenotypes were dose-dependently inhibited by the re-expression of Csk, indicating that there is a certain threshold for c-Src transformation, which is determined by the c-Src : Csk ratio. In contrast to v-Src, c-Src induced the phosphorylation of a limited number of cellular proteins and elicited a restricted change in gene expression profiles. The activation of some critical targets for v-Src transformation, such as STAT3, was not significantly induced by c-Src transformation. Several genes that are involved in cancer progression, that is, cyclin D1 and HIF-1α, were induced by v-Src, but not by c-Src. Furthermore, v-Src tumors exhibited aggressive growth and extensive angiogenesis, while c-Src tumors grew more slowly accompanied by the induction of hematomas. These findings demonstrate that c-Src has the potential to induce cell transformation, but it requires coordination with an additional pathway(s) to promote tumor progression in vivo.

The junctional adhesion molecule (JAM) family is a key molecule in a process called transendothelial migration or diapedesis. Here, we report implications of JAM-C in cancer metastasis. We first determined the mRNA expression of JAMs in 19 kinds of cancer cell lines. JAM-C was expressed in most of tumors having potent metastatic properties. Especially in murine K-1735 melanoma cell lines, the highly metastatic sublines (M2 and X21) strongly expressed JAM-C when compared with the poorly metastatic ones (C-10 and C23). Next, we investigated the role of JAM-C in cancer metastasis by using human JAM-C (hJAM-C) gene-transfected HT1080 fibrosarcoma cells. In comparison with mock-transfected HT1080 cells, these cells showed a significant increase in the adhesion to various extracellular substrates and the invasion across a Matrigel™-coated membrane. The knockdown of hJAM-C using small interfering RNA resulted in the suppression of both the adhesion and the invasion of HT1080 cells, suggesting that endogenous hJAM-C might be involved in tumor metastasis. Finally, we studied the role of hJAM-C in an in vivo experimental metastatic model. The results showed that the overexpression of hJAM-C in HT1080 cells significantly decreased the life spans of the tumorbearing mice. In contrast, the knockdown of hJAM-C in HT1080 cells suppressed the weight gain of the lungs with metastatic colonies. We conclude that the expression of JAM-C promotes metastasis by enhancing both the adhesion of cancer cells to extracellular matrices and the subsequent invasion.