Soluble factors in the AML secretome drive macrophage polarization toward an M2-like state promoting an immunosuppressive microenvironment Free

Tumor-secreted proteins are critical mediators through which cancer cells dynamically reshape and modulate their surrounding microenvironment. In acute myeloid leukemia (AML), this strategy is particularly evident in the bone marrow (BM), where leukemic blasts hijack the niche to sustain their growth, evade immune surveillance, and resist therapy. Among the non-hematopoietic components of the BM, mesenchymal stromal cells (MSCs) emerged as key orchestrators toward the creation of a tumor-supportive niche. We previously demonstrated that MSCs isolated from AML pediatric patients at diagnosis (AML-MSCs) are transcriptionally and functionally distinct from those of healthy age-matched donors (h-MSCs), showing enhanced support for leukemic cell proliferation and impaired regulation of normal hematopoiesis (Borella G. et al Blood 2021).

To better recapitulate the BM niche, we developed a biomimetic 3D model using a scaffold composed of hydroxyapatite and collagen I. To dissect secretome dynamics during leukemia onset, we co-cultured AML-MSCs or h-MSCs with primary AML blasts for 7, 14 and 21 days and performed proteomic analysis by mass spectrometry coupled with stable isotope labeling by amino acids labeling (n=3). This approach enabled the identification of 15 newly differentially secreted proteins between the AML- and h-MSCs conditions. Notably, 9 proteins were specifically upregulated in the AML niche and functionally associated with cancer progression, inflammation and angiogenic pathways. Among them, PAI-1, CHI3L1 and PTX3 emerged as the most strongly secreted and were collectively termed “PCP”. We measured PCP levels of AML- or h-MSCs alone or in co-cultures with AML blasts (n=15) by ELISA assay. Interestingly, while co-culture with blasts increased CHI3L1 and PTX3 levels, which are also directly secreted by the leukemic cells themselves, PAI-1 was produced only by MSCs and was markedly up secreted in AML-MSCs, suggesting their intrinsically higher capacity for PAI-1 production compared to h-MSCs. Moreover, preliminary quantification of PCP levels in plasma samples collected from AML patients at diagnosis (n=16) confirmed their presence, further highlighting the predictive relevance of the 3D model.

To explore their functional role, we exposed Hematopoietic Stem Cells (HSCs) derived from induced pluripotent stem cells (iPSCs) and healthy PBMCs-derived monocytes to PCP (as recombinant proteins). iPSCs-derived HSCs cultured with PCP exhibited a skewed differentiation toward a macrophage-like phenotype, confirmed by cell morphology and flow cytometry. Likewise, healthy monocytes differentiated for 7 days in the presence of PCP (AML-Ms) acquired a M2-like phenotype, as evidenced by a 2.7-fold increase in CD206 expression and a characteristic spindle-shaped morphology. Cytokine analysis revealed that AML-Ms secrete significantly higher levels of IL-6 compared to control macrophages, a key cytokine typical of tumor-associated macrophages (TAMs).

Functionally, AML-Ms were investigated for immunosuppressive properties: AML-Ms reduced autologous T-cell proliferation (<10% CFSElow) and activation (<50% CD25+) after 72h co-culture. Importantly, AML-Ms derived from different donors (n=4) decreased T cell-mediated cytotoxicity against primary AML blasts compared to macrophage not stimulated with PCP (p<0.05). To mimic the leukemic niche, we cultured either h- or AML-MSCs for 7 days in the 3D scaffolds, then seeded AML blasts (n=4) and then PBMC-derived monocytes in the presence or absence of PCP, and after 21 days we added autologous T cells. At day +24 we observed that AML blasts proliferated significantly more (by luciferase monitoring) when PCP were present (p=0.0064), highlighting the reconstituted leukemic niche able to induce AML-Ms which dampened T-cell cytotoxic activity. Thus, we argued whether AML-Ms could affect CAR-T cells function targeting an AML specific antigen. We showed that CAR-T cells co-cultured for 3 days with AML-Ms (induced by PCP) had a reduced lytic potency against AML blasts (<20%, E:T=1:1, n=4; p=0.0021) vs. CAR-T cells co-cultured with control macrophages.

These findings deconvolute the role of PCPs in supporting immune evasion within the AML niche. Transcriptomic analyses are ongoing to identify PCP driven TAMs properties. Notably, PCPs are under evaluation as promising therapeutic targets to restore immune surveillance and to improve efficacy of immunotherapy in AML.