Preclinical evaluation of macrophage reprogramming in treatment of CTCL

Abstract Introduction: Reprogramming tumor-supportive macrophages may have therapeutic utility in cutaneous T-cell lymphoma (CTCL) (Wu et al., J Invest Dermatol 2014). Targetable pathways in tumor-associated macrophages include signaling through mannose receptor, CD206 (Jaynes et al., Sci Transl Med 2020), and pattern-recognition receptor, toll-like receptor 4 (TLR4) (Oblak et al., Clin Dev Immunol 2011). Here, we evaluate TLR4 and CD206 in CTCL-associated macrophages to explore their therapeutic potential. Methods: We utilized scRNA-seq to assess macrophages in CTCL lesional skin biopsies. Additionally, we assessed TLR4 and CD206 activity in an immunocompetent CTCL mouse model and in cancer cell-macrophage co-culture systems. In the co-culture systems, human or murine monocytic cells were differentiated into macrophages and subsequently polarized to anti- or pro-inflammatory phenotypes in the presence or absence of CTCL cell lines in transwells. TLR4 deficient mice and the small molecule TLR4 inhibitor, TAK-242, were used to abrogate TLR4 function. To stimulate CD206, we used a novel CD206 binding peptide, RP-832c. Results: Human CTCL lesional skin biopsies (scRNA-seq N CTCL = 7, healthy control = 4; immunofluorescence N = 5) and mouse CTCL tumors (N = 5) displayed co-localized expression of CD206 and TLR4 on macrophages. CTCL-associated macrophages in skin lesions and in co-cultures exhibited a tumor-promoting phenotype with a 2-fold decrease in TNF-α expression (p <0.005), a 1.33-fold increase in CD206 expression (p <0.005). In addition, CTCL-associated macrophages exhibited a 4-fold increase in TLR4 expression in co-cultures (p <0.005). Macrophage phenotype shifted from tumor-promoting (IL-10 expressing) to tumor-suppressive (CD86 expressing) in macrophage co-cultures treated with RP-832c (1.25-fold increase in CD86 expression, p <0.05) or TAK-242 (5-fold increase in CD86 expression, p <0.05). Similarly, MBL2 mouse tumors treated with RP-832c (daily treatment with 10mg/kg body weight, provided by Riptide Biosciences) or TAK-242 (daily intraperitoneal injections 3mg/kg body weight) displayed a shift from immunosuppressive to inflammatory macrophage phenotype and attenuated tumor growth. In mice lacking TLR4 in the microenvironment or in CTCL-associated macrophages, CTCL tumor growth, measured by tumor volumes over time, was diminished compared to controls (N = 5 per group, p <0.0001). Conclusions: Our data suggest that TLR4 inhibition and CD206 activation in the CTCL tumor microenvironment inhibit tumor growth by inducing a shift from tumor-promoting to tumor-suppressive macrophages. Therefore, CD206 binding peptide, RP-832c, and small molecule TLR4 inhibitor, TAK-242, present promising therapeutic options in CTCL.