Mast Cells As a Therapeutic Target for Hodgkin Lymphoma: Bortezomib Inhibits Mast Cell-Induced Modification of the Tumor Microenvironment
Authors: Mizuno Hiroki , Nakayama Takayuki , Miyata Yasuhiko , Saito Shigeki , Satoshi Nishiwaki , Nakao Norihiko , Takeshita Kyosuke , Tomita Akihiro , Naoe Tomoki 
Published: October 2019

Abstract
Abstract 3634

Background:
A variety of inflammatory cells are present the microenvironment of Hodgkin lymphoma (HL); these cells enhance the survival of lymphoma cells and suppress tumor immunity. HL is frequently associated with the mast cell infiltration that correlates directly with disease severity, but the mechanisms underlying this relationship remain unclear.

Aims:
To examine whether mast cells can promote the growth of HL by modifying the tumor microenvironment and to determine whether mast cells can be a therapeutic target for HL.

Methods:
The human HL cell lines, L428, HDLM2, and KMH2, bone marrow-derived mast cells (BMMCs), and spleen-derived mast cells (SPMCs) from C57BL/6 mice were used in our analyses. The proliferative effect of in vitro co-culture was assessed by a colorimetric assay. HL transplantation assays were performed in NOD/SCID mice using HL cells with or without BMMCs. To study the effects of anti-cancer drugs on mast cell functions, BMMCs were treated with or without bortezomib or lenalidomide. Tumor size was measured and histopathological analyses were carried out to determine the effectiveness of the drugs. The expression profile of angiogenesis-related proteins was confirmed using the Angiogenesis Array Kit (R&D Systems, Minneapolis). To analyze the in vitro effects of bortezomib on the BMMCs, VEGF-A, CCL2, and b-hexosaminidase expressions were measured by ELISA and a b-hexosaminidase assay. The statistical significance of inter-group differences was evaluated by Student's t-test.

Results:
On in vitro co-culture assays, BMMCs weakly induced the proliferation of only KMH2 cells, and SPMCs did not induce the proliferation of any HL cell lines. On the in vivo transplantation assays, HL cells gave rise to tumors in NOD/SCID mice more rapidly when inoculated subcutaneously together with BMMCs than when inoculated HL cells alone. The mean size of tumors derived from inoculated HL cells with BMMCs was significantly greater than that of tumors derived from inoculated HL cells alone (e.g., L428 vs. L428 + BMMC, mean size: 108.39 mm3 vs. 225.19 mm3, respectively, at day 5; p = 0.0026). Microscopically, tumors derived from inoculated HL cells with BMMCs showed increased vasculature and fibrosis, whereas tumors derived from inoculated HL cells alone were generally hypovascularized with less fibrosis and were necrotic in most areas. An antibody array using cell lysates to determine the source of proangiogenic factors showed that HL cells minimally produced proangiogenic factors, but that mast cells produced them abundantly. Next, we examined whether bortezomib can target mast cell functions by inhibiting the secretion of mast cell products. Bortezomib inhibited degranulation of b-hexosaminidase, PGE2-induced rapid release of CCL2, and continuous release of vascular endothelial growth factor-A from mast cells, even at concentrations that did not induce cell death, and profoundly decreased expressions of angiopoietin-1, endoglin, HB-EGF and VEGF-B. On an in vivo transplantation assay in the presence or absence of bortezomib, the mean size of tumors derived from inoculated HL cells plus untreated BMMCs were significantly greater than those of tumors derived from inoculated HL cells plus bortezomib-treated BMMCs (e.g., L428 + intact BMMC vs. L428 + bortezomib-treated BMMC, mean size: 105.6 mm3 vs. 57.7 mm3, respectively, at day 6; p = 0.0255). Microscopically, tumors derived from inoculated HL cells together with intact BMMCs were highly vascularized and fibrotic, whereas tumors derived from inoculated HL cells plus bortezomib-treated BMMCs were generally not. Results from a similar analysis using lenalidomide showed that its effect on BMMCs was much lower than that of bortezomib.

Discussion:
Mast cells had the ability to promote the growth of HL on in vivo transplantation assay, but not on in vitro co-culture assay, indicating that there may be an indirect event via the promotion of angiogenesis that acts on the tumor microenvironment. Bortezomib effectively inhibited the mast cell-induced growth of Hodgkin's cell tumors in vivo by blocking the release of secretory granules from mast cells, but suppress of mast cells could not have a complete remission. As a treatment strategy for the future, it may be necessary to combine bortezomib with other drugs or irradiation.

Conclusions:
Mast cells have the ability to promote the growth of HL, and may be a promising target for the treatment of HL.

Disclosures:
No relevant conflicts of interest to declare.