Page 159 - 2019_03-Haematologica-web

Maraviroc inhibits cHL crosstalk and xenograft growth
reactive lymphoid hyperplasia.7,8 Both CCL5 and its recep- tor CCR5 are constitutively expressed by cHL-derived cell lines7 by tumor cells from cHL lymph node tissues and by bystander cells including stromal cells and lymphocytes.7 The CCR5 receptor expressed by cHL cells is fully func- tional and its ligands function as both paracrine and autocrine growth factors.7
The interactions of cHL tumor cells with a variety of non-tumor reactive cells accumulating in cHL tissues mediate tumor cell growth, formation of an immunosup- pressive, protective tumor microenvironment (TME), neo- angiogenesis,9 and drug resistance.10,11 Increasing evidence suggests that not only T cells,12 but also mesenchymal stro- mal cells (MSCs)13 and monocytes,14,15 contribute to the TME in cHL.11,16 MSCs, by modulating NKG2D expression in T cells and its ligand in tumor cells, reduce the immune response against cHL cells.13 A high number of infiltrating macrophages,17,18 predominantly derived from circulating monocytes,19 and a high absolute monocyte count in peripheral blood both correlate with poor cHL progno- sis.20,21 These observations likely reflect the ability of cHL cells to reprogram macrophages towards immunosuppres- sive tumor-associated macrophages (TAMs).20,21
Given current knowledge about cell-cell interactions in cHL, there is interest in drugs that can interfere with this crosstalk.22-25 But since drug discovery is expensive and time-consuming, drug repurposing is an attractive approach for finding new cancer treatments.26 One such repurposed drug is the CCR5 antagonist maraviroc.27 Approved by the US Food and Drug Administration for the treatment of HIV infection, maraviroc causes few side effects in humans, even during long-term therapy.28,29 As an anticancer drug, maraviroc has different effects: it blocks metastasis of basal breast cancer cells;30 it decreases the migration of regulatory T cells; it reduces metastatic breast cancer growth in the lungs;31 and it inhibits the accumula- tion of fibroblasts in human colorectal cancer.32 Maraviroc reprograms immunosuppressive myeloid cells and reinvig- orates antitumor immunity by targeting the autocrine CCL5-CCR5 axis in bone marrow.6 It also polarizes macrophages towards an M1-like functional state.27
Our working hypothesis is that cHL cancer cells, by secreting CCL5, recruit both MSCs and monocytes to the TME, and then reprogram these cell types to make them pro-tumorigenic. Thus, blocking the CCR5 receptor should inhibit not only tumor growth, as we previously observed,7 but also the recruitment of cells to form the protective, immunosuppressive TME.
Here, we investigated the role of CCL5-CCR5 signaling in the interactions of monocytes and MSCs with cHL cells, using, in particular, three-dimensional multicellular heterospheroids33 formed by tumor cells, monocytes and MSCs, as well as an in vivo cHL model and tissues from cHL patients.
Methods
Maraviroc (Sigma-Aldrich) was dissolved in DMSO at 51.8 mM. Other reagents are detailed in Online Supplementary Methods, together with protocols for cell migration, proliferation, clonogenic growth and senes- cence assays, immunosuppression, synergy, flow cytome- try, ELISAs and other cell-based assays, immunofluores- cence, survival of tumor xenografts and statistical analysis.
Cell culture and conditioned media
Authenticated cHL-derived cell lines L-1236, L-428, KM- H2, HDLM-2, and L-540 (DSMZ, Germany) were cultured in RPMI-1640 medium containing 10% fetal calf serum (FCS). To prepare conditioned medium (CM) from cHL cell lines, cells were seeded at 2.0×105/ml in RPMI-1640 plus 10% FCS, and the medium was collected after 72 h. Human bone marrow-derived and adipose tissue-derived mesenchymal stromal cells (MSCs) (BM-MSCs and AT- MSC, respectively) were purchased from Lonza (Verviers, Belgium). cHL-MSCs from frozen lymph nodes were gen- erated as described in Online Supplementary Methods. BM- MSCs, AT-MSC and cHL-MSCs were maintained in Mesenchymal Stem Cell Growth Medium Bulletkit (Lonza) to avoid differentiation.
Monocytes were isolated from peripheral blood mononuclear cells (PBMCs) from healthy donor blood using CD14 Microbeads, Human (Miltenyi Biotec). To generate tumor-educated MSCs (E-MSCs) and monocytes (E-monocytes), MSCs and monocytes were cultured sepa- rately for 6 days in complete culture medium (RPMI-1640, 10% FCS) containing 20% CM from cHL cell lines; half the volume of medium was replaced every other day. To prepare CM from these tumor-educated cells, they were washed and cultured in fresh medium for 72 h.
3D culture of heterospheroids
Heterospheroids were generated by co-culturing various combinations of cHL cells, HL-MSCs and monocytes (1.0 × 104/mL of each cell type) in RPMI-1640 medium con- taining 1% FCS, using plates coated with 20 mg/mL poly- HEMA (Sigma) to prevent adhesion. After 4 days, heteros- pheroids were tested for CCL5 secretion into the medium by ELISA. In some experiments, heterospheroids were treated with maraviroc, alone or with doxorubicin, for 6 days. Growth was evaluated using the PrestoBlue Cell Viability Reagent (Invitrogen).
Tumor xenograft experiments
Animal experiments were approved by the Italian Ministry of Health (no. 671/2015/PR). We used ten 4- week-old female athymic nude/nude mice (Harlan Laboratories) and ten 4-week-old male NOD/SCID gamma chain deficient (NSG) mice (Charles River). L-540 (200×106 cells/animal) and L-428 cells (10×106 cells/animal) were suspended in Matrigel (diluted 1:3 in PBS) and inoc- ulated into the flank of nude mice (L-540) or NSG mice (L-428). When tumors were palpable, animals were divid- ed into two equal groups and treated every day (L-540) or every other day (L-428) with maraviroc (intraperitoneal injection, 10 mg/kg)34 or vehicle (PBS). Body weight and tumor volume were measured daily.
Immunohistochemistry tissue microarray analysis of cHL patients
The study protocol was approved by the institutional review board of the University Medical Center Groningen. We recruited 65 patients with cHL (Online Supplementary Table S1). All study subjects provided writ- ten informed consent. Immunohistochemistry was per- formed for CCL5 (C-19 antibody, 1:200 dilution, Santa Cruz Biotechnology) (antigen retrieval in 10 mM Tris-HCl pH 9, 1 mM EDTA). CD68 was detected with KP1 anti- body (1:4000 dilution, Dako) (antigen retrieval in 10 mM citrate buffer, pH 6).
haematologica | 2019; 104(3)
565