Having established that lymphoma: macrophage ratios differ within the BM and spleen at late timepoints, we next asked whether the altered ratio could be due to BM lymphoma cells being less prone to macrophage phagocytosis. Paeonol (Peonol) a super-phagocytic subset that expressed CD36/FcgRIV. Together, these findings define a novel mechanism through which high-dose alkylating brokers promote macrophage-dependent lymphoma clearance. Introduction The alkylating agent cyclophosphamide (CTX) first became available in 1959 (1,2). Soon thereafter, CTX was noted to have remarkable single-agent activity in the treatment of endemic Burkitt lymphoma. In fact, a fraction of high-grade lymphomas could be cured with a single dose of CTX (3,4), a response that is wholly unique among aggressive cancers. The mechanisms through which CTX exerts this profound efficacy have Paeonol (Peonol) remained largely unclear. Cell lines developed from high-grade lymphomas like Burkitt lymphoma have comparable sensitivities to alkylating brokers as they do to topoisomerase II poisons (e.g. doxorubicin, etoposide), vinca alkaloids and other chemotherapies (5-7). Thus, there does not appear to be a lymphoma cell-autonomous sensitivity specific to alkylating brokers. This leaves the remarkable activity of high-dose CTX unexplained, but one Paeonol (Peonol) possibility is usually that it involves the lymphoma microenvironment. High doses of CTX Paeonol (Peonol) are extremely lymphodepleting (8,9), so it is usually unlikely that adaptive immunity plays a large role in its activity. In contrast, macrophages are largely resistant to chemotherapy, including high-doses of alkylating Thbd brokers like CTX. Chemotherapies such as doxorubicin and cyclophosphamide can be immunogenic and increase macrophage-mediated clearance of tumor cells (10,11). Of note, BL and other high-grade lymphomas with rearrangements commonly have a starry sky appearance under the microscope due to infiltration of the microenvironment by lymphoma-associated macrophages (12,13). Monoclonal antibodies like rituximab and alemtuzumab (Alem), which bind to CD20 and CD52, respectively, are widely utilized in the treatment of lymphomas. These antibodies function through various mechanisms, including antibody-dependent cellular phagocytosis (ADCP) by macrophages, antibody-dependent cellular cytotoxicity (ADCC) by NK cells and complement-dependent cytotoxicity (CDC) (14-16). Both rituximab and Alem have reduced activity at sites of bulky Paeonol (Peonol) disease (17,18), suggesting at least two possibilities: (1) the antibodies have poor penetration into sites of bulky disease and/or (2) bulky disease represents a later stage of disease progression, in which the lymphoma microenvironment is usually less amenable to antibody-dependent lymphoma killing. We previously treated NOD.SCID.human lymphomas? Second, do other alkylating brokers recapitulate the effects observed with high-dose CTX? Third, what components of bone marrow remodeling that occur during disease progression drive therapeutic resistance? Fourth, are macrophages required for CTX-mediated killing in the BM microenvironment? Fifth, how does CTX induce crosstalk between lymphoma cells and macrophages? Finally, does the crosstalk change the transcriptional and phenotypic says of macrophages to promote phagocytosis? Here we utilize models of human DHL to specifically address mechanisms underlying the notable activity of high-dose cyclophosphamide described in patients with aggressive lymphomas. Results Alkylating brokers overcome therapeutic resistance of human lymphoma cells in the BM. DFBL-20954 and DFBL-69487 are DHL PDXs that harbor translocations of both and (Supplementary Physique 1A) (26). Both DFBL-20954 and DFBL-69487 are CD52high/CD20low/unfavorable (Physique 1A, Supplementary Physique 1B), consistent with a subset of DHLs (27,28) and observed with acquired resistance to rituximab-based therapy (29). In fact, both PDXs were established from biopsies obtained after treatment failure with R-CHOP, which includes rituximab and a lower dose of CTX (750mg/m2). Open in a separate window Physique 1: Alkylating Brokers Overcome Bone Marrow Antibody Resistance(A) Flow cytometric analysis of surface CD20 and CD52 expression on DFBL-20954 and DFBL-69487. (B) On day 8 of treatment, spleen was harvested and a single femur was flushed from mice treated with PBS, Cyclophosphamide (CTX), Doxorubicin (Dox) Alem (Alem) or combinations, as indicated. Total cells were counted and.