![]() MDSCs and TAMs were isolated from all tumor models by generating a single-cell suspension from the tumor by using collagenase I (1 mg/mL) and DNase (100 μg/mL) for 1 hour with shaking at 37☌. Tumors were utilized for studies starting at 100 mm 3 for the start-of-treatment studies and harvested at a size of 600–800 mm 3 for nontreatment studies, unless otherwise specified Cell lines Outgrowth of the tumor was monitored by length versus width measurements for solid flank tumors. RM-1 tumors were additionally implanted as an intraperitoneal tumor by injecting 1 × 10 6 cells into the intraperitoneal space and allowing to grow for 7 days. Myc-Cap tumors were implanted orthotopically in the prostate of male FVB mice at a dose of 1 × 10 5 cells per mouse. EMT6, MB49, RM-1, TRAMP-C2, and CT26 were implanted on the flank of the animals at a dose of 1 × 10 6 cells per mouse. 4T1 tumors were implanted in the mammary fat pad of female BALB/cJ mice at a dose of 1 × 10 6 cells per mouse. Unless otherwise specified, mice were housed on corn cob bedding and fed complete chow. Mice were used at 8–12 weeks of age for tumor implantation studies. Materials and MethodsīALB/cJ, FVB/NJ, and C57BL/6J mice were purchased from The Jackson Laboratory and housed in accordance with Purdue Animal Use and Care Committee guidelines. We further show that a potent folate-targeted TLR7 agonist (TLR7a) that is too toxic to administer in nontargeted form can be safely and specifically targeted to myeloid cells within the TME by conjugation to folate, resulting in reprogramming of tumor myeloid cells, abrogation of MDSC/TAM immunosuppressive activities, enhancement of T-cell infiltration, induction of antitumor activity, and significant improvement in overall survival. Herein, we demonstrate that FRβ + MDSCs and TAMs freshly isolated from the TME are the dominant immunosuppressive myeloid populations and that highly specific delivery of both imaging and therapeutic agents to these cells in the TME can be achieved with folate targeting. Given that FRβ is also localized to inflammatory sites and the TME, we initiated studies to define the functional properties of FRβ + TME-derived myeloid cells. Ongoing research by our group has shown the immediate suppressive function of MDSCs to be located within inflammatory sites and the TME ( 14). Previous studies have shown that folate receptor beta (FRβ) constitutes a marker for TAMs ( 9) and other forms of activated macrophages ( 10), however, use of this marker has focused to date on imaging of autoimmune diseases ( 11, 12), with little effort devoted to exploiting it for therapeutic applications or evaluating its association with function ( 13). In fact, to date, no diagnostic marker has been identified that distinguishes immunosuppressive from nonimmunosuppressive MDSCs or TAMs. Because all solid tumors accumulate MDSCs and TAMs, a general strategy to both identify and reprogram these cells should be broadly applied in the characterization and treatment of multiple tumors.ĭespite significant advances in MDSC and TAM biology ( 1, 7, 8), both populations are still primarily defined functionally, because antigenic markers that segregate with MDSCs and TAMs are also expressed on nonimmunosuppressive myeloid populations ( 8). The data also establish FRβ as the first marker that distinguishes immunosuppressive from nonimmunosuppressive subsets of MDSCs and TAMs. These data reveal a broadly applicable strategy across tumor types for reprogramming MDSCs and TAMs into antitumorigenic immune cells using a drug that would otherwise be too toxic to administer systemically. ![]() Delivery of a folate-targeted TLR7 agonist to these cells (i) reduced their immunosuppressive function, (ii) increased CD8 + T-cell infiltration, (iii) enhanced M1/M2 macrophage ratios, (iv) inhibited tumor growth, (v) blocked tumor metastasis, and (vi) improved overall survival without demonstrable toxicity. This FRβ + subpopulation could be selectively targeted with folate-linked drugs. Here, we explore the properties of MDSCs and TAMs from freshly isolated mouse and human tumors and find that an immunosuppressive subset of these cells can be distinguished from the nonimmunosuppressive population by its upregulation of folate receptor beta (FRβ) within the TME and its restriction to the TME. Prominent among immunosuppressive cells are myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM) that inhibit T cells via release of immunosuppressive cytokines and engagement of checkpoint receptors. Although immunotherapies of tumors have demonstrated promise for altering the progression of malignancies, immunotherapies have been limited by an immunosuppressive tumor microenvironment (TME) that prevents infiltrating immune cells from performing their anticancer functions.
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