Inhibiting Monocyte Recruitment to Prevent the Pro-Tumoral Activity of Tumor-Associated Macrophages in Chondrosarcoma

Abstract

Chondrosarcomas (CHS) are malignant cartilaginous neoplasms with diverse morphological features, characterized by resistance to chemo- and radiation therapies. In this study, we investigated the role of tumor-associated macrophages (TAM)s in tumor tissues from CHS patients by immunohistochemistry. Three-dimensional organotypic co-cultures were set up in order to evaluate the contribution of primary human CHS cells in driving an M2-like phenotype in monocyte-derived primary macrophages, and the capability of macrophages to promote growth and/or invasiveness of CHS cells. Finally, with an in vivo model of primary CHS cells engrafted in nude mice, we tested the ability of a potent peptide inhibitor of cell migration (Ac-d-Tyr-d-Arg-Aib-d-Arg-NH2, denoted RI-3) to reduce recruitment and infiltration of monocytes into CHS neoplastic lesions. We found a significant correlation between alternatively activated M2 macrophages and intratumor microvessel density in both conventional and dedifferentiated CHS human tissues, suggesting a link between TAM abundance and vascularization in CHS. In 3D and non-contact cu-culture models, soluble factors produced by CHS induced a M2-like phenotype in macrophages that, in turn, increased motility, invasion and matrix spreading of CHS cells. Finally, we present evidence that RI-3 successfully prevent both recruitment and infiltration of monocytes into CHS tissues, in nude mice.

Crosstalk between Macrophages and Myxoid Liposarcoma Cells Increases Spreading and Invasiveness of Tumor Cells

“…Immunohistochemistry (IHC) was performed on 4 µm thin FFPE tissue sections as previously described [29], using automated slide stainers BenchMark (Ventana Medical System-Roche) and Leica Bond-III, (Leica Biosystems,), according to manufacturer's instructions. To characterize lymphocytic infiltrate, CD3 (clone 2GV6, ready to use), CD4 (clone SP35, ready to use), and CD8 (clone SP57, ready to use) monoclonal antibodies (Roche) were applied on tissue sections for 15 min at 25 • C. Monoclonal antibody directed to nuclear transcription factor forkhead box P3 (FOXP3) (clone D2W8E, 1:250, Cell Signaling Technology) was utilized to identify regulatory T cells.…”

“…Peripheral blood mononuclear cells (PBMC)s, and serum were individually collected. PBMCs were harvested by density gradient centrifugation as previously described [29], using the Lympholyte-poly Cell Separation Media (Cedarlane Laboratories, #CL5015, Cedarlane, Burlington, ON, Canada) according to the manufacturer's instructions. Monocytes were isolated from PBMCs by positive selection of CD14+ cells using the Monocyte Isolation Kit II purchased by Miltenyi Biotec, #130-091-153 (79% pure by visual and cytofluorimetric analysis) and transferred to tissue culture plates in RPMI-1640 medium (Cytiva HyClone™ #SH30096.01, Cytiva, Marlborough, MA, USA), supplemented with 10% autologous human serum, penicillin (100 U/mL), and streptomycin (100 µg/mL).…”

“…Non-Contact co-cultures were carried out as described [29], using 24-multiwell plates and transwell polyethylene terephthalate permeable supports that allow the exchange of soluble factors purchased by Corning (Falcon ® , #353095, NY USA). Briefly, human monocytes (1 × 10 6 cells/well) were seeded in the lower compartment in RPMI-1640 medium supplemented with 10% heat inactivated human serum.…”

“…Monocytes recovered from co-cultures with/without MLPS cells were analyzed by flow cytometry as described [29], using PE-conjugated anti-CD68 REAfinity™ (Miltenyi Biotec #130-114-460) and APC-conjugated anti-CD163 REAfinity™ (Miltenyi Biotec, #130-112-129, Bergisch Gladbach, Germany) antibodies. Samples were acquired with the BD FACSCanto II (BD Biosciences, Franklin Lakes, NJ, USA), and data analyzed by the FlowJo v10.0.7 software (Tree Star, Inc, Ashland, OR, USA), after gating on the myeloid population in the FSC/SSC plot.…”

“…More recently, molecular profiling studies allowed to identify a number of immune therapeutic targets in bone sarcomas [27]. Otherwise, most of soft tissue sarcomas are considered non-immunogenic [1], few reports investigating the composition of TME in soft tissue sarcomas have been published, and clinical responses in trials with checkpoint inhibitors still remain unsatisfactory [28][29][30][31][32].…”