Malignant mesothelioma (MM) is an aggressive tumor associated to asbestos exposure We aim to understand how macrophages support MM progression and whether their phenotype affects the cell crosstalk in the tumor microenvironment (TME), which comprises a large number of macrophages (MOs) High Mobility Group Box 1 (HMGB1) is a chromatin protein crucial in MM onset and development, as it contributes to MO recruitment and sustains chronic inflammation, promoting a proinflammatory TME. BoxA, an 89-amino acid fragment corresponding to the first domain of HMGB1, antagonizes HMGB1 activity and shows therapeutic effects in MM. BoxA binds to the CXCR4 receptor and induces CD47 internalization, leading to the phagocytosis of tumor cells by MOs. This mechanism of cancer-immune surveillance, termed immunogenic surrender (IGS), identifies MOs as fundamental cells in immune responses mediated by BoxA. The colony-stimulating factor 1 receptor (CSF-1R) pathway modulates MO proliferation and polarization; our goal is to modulate the CSF-1R pathway in TAMs to enhance the therapeutic effects of BoxA.

We generated 3D spheroids of MM cells co-cultured with mouse bone marrow-derived macrophages (BMDMs) or human monocytes. To assess the effects of MOs polarization states, we compared spheroid growth in co-cultures with M0 (unstimulated), M1-like, or M2-like polarized MOs. Spheroid growth was measured via imaging, and MO polarization was assessed by qPCR and flow cytometry. To validate MOs contribution to MM progression in vivo, we blocked CSF-1R using a monoclonal antibody in a syngeneic MM mouse model, applying two experimental settings: before and after tumor engraftment. TME composition was analyzed by IHC, flow cytometry, and RT-PCR.

BMDMs initially polarized to M0 acquired a pro-tumoral M2-like phenotype when co-cultured with 3D MM spheroids. M2-like MOs promoted spheroid growth. Treatment with anti–CSF-1R monoclonal antibody (CSF-1R) shifted MO polarization from an M2- to an M1-like phenotype in co-culture settings. Moreover, conditioned medium from MM cells polarized MOs towards M2-like phenotype, an effect that was reversed upon CSF-1R treatment. CSF-1R blocking in a syngeneic mouse model of MM delayed tumor growth and extended mice survival. Analysis of TME revealed a reduction in the total number of MOs and an increase in M1-like MOs. Interestingly, treatment with CSF-1R also influenced HMGB1 localization in tumor cells, increasing its cytosolic levels relative to nuclear levels; this suggests that TAMs may induce tumor cells to secrete more HMGB1 in an attempt to sustain their own growth.

MOs sustain MM growth and aCSF-1R treatment reduces it by skewing TAM polarization toward an M1-like state. This may synergize with BoxA, making MM and other inflammation-driven tumors more responsive to immunotherapy.