Our preclinical tumor models as a result appear to recapitulate, at least in part, the protumorigenic part of TAMs in B cell lymphoma (38). Blockade of the CD47-SIRP connection is a promising TAM-directed therapeutic approach, with the aim of promoting ADCP of tumor cells by macrophages (39, 40). support tumor progression while limiting antitumor immune reactions (1, 2). Tumor-associated macrophages (TAMs) are the most abundant infiltrating immune cell type in the TME of many human being cancers (3). TAMs are associated with poor prognosis and manifest an anti-inflammatory phenotype that promotes angiogenesis, matrix redesigning, tumor growth, and metastasis (4, 5). However, TAMs also possess antitumor potential dependent on cytotoxicity for and phagocytosis of tumor cells (6). Reprogramming of TAMs from a protumoral state toward an antitumoral state might therefore provide the basis for fresh malignancy therapeutics (7). Transmission regulatory protein (SIRP) is an immunoglobulin (Ig) superfamily protein that is highly indicated on macrophages and dendritic cells (8C11). Connection of the extracellular region of SIRP with its ligand CD47 elicits signaling from the tyrosine phosphatase SHP-1 that inhibits Fc receptorCmediated antibody-dependent cellular phagocytosis (ADCP). Connection of CD47 on tumor cells with SIRP on TAMs therefore constitutes an innate immune checkpoint that interferes with the detection and phagocytosis of the former cells from the second option (10, 12). We previously showed that an antibody to SIRP that blocks its connection with CD47 markedly enhanced the inhibitory effect of rituximab, an antibody to human being FLT3-IN-2 (h) CD20, within the growth of human being B cell lymphoma FLT3-IN-2 in immunodeficient mice (13, 14). Given that these studies targeted endogenous mouse macrophages in the immunodeficient animals, however, a preclinical model that recapitulates the status of FLT3-IN-2 human being TAMs in the TME is required to evaluate further the therapeutic effect of antibodies to hSIRP (anti-hSIRP) before their potential medical use. Humanized immune system (HIS) mice are immunodeficient mice reconstituted having a human being immune system by transfer of human being CD34+ hematopoietic stem and progenitor cells (HSPCs) (15C18). Recent advances in the development of HIS mice have allowed studies of the connection between human being immune components and human being tumors (19, 20). In particular, HIS mouse models are essential for evaluation of the antitumor effects of therapeutics that take action only on human being immune cells, such as antibodies to the immune checkpoint molecule PD-1 (21). However, evaluation of the effects of tumor immunotherapeutic providers that target human being macrophages (such as anti-hSIRP) has not been possible FLT3-IN-2 with such models because the differentiation of human myeloid cells is not supported in immunodeficient mouse strains such as NOD experiments. 2.7. Development of PDX models A renal subcapsular patient-derived xenograft (PDX) model FLT3-IN-2 of DLBCL was generated as previously described (27), with minor modifications. In brief, tumor tissue was obtained from DLBCL patients during surgery and was processed within 24h. Each fresh tumor specimen was cut into pieces of 2 by 2 by 2 mm and maintained in sterile RPMI 1640 medium until xenotransplantation. Mouse surgery was performed in a laminar-flow hood under sterile conditions. Introduction of an ~1-cm incision into FGF20 the left flank of an anesthetized MITRG mouse was followed by incision of the peritoneum and exteriorization of the left kidney. A small pocket was created between the kidney capsule and parenchyma with the use of sharp tweezers and a stereomicroscope. A piece of tumor tissue was inserted into the pocket, the kidney was gently eased back into the peritoneal cavity, the peritoneal incision was aligned with 5-0 surgical threads, and the skin incision was fixed with a surgical stapler. Mice were maintained for 4 to 8 weeks until the tumor was palpable, after which they were killed, the tumor (P0) was excised and digested as described above for flow cytometry, and the isolated cells were stored at C80C until use. 2.8. Tumor cell engraftment and treatment Cell lineCderived xenograft (CDX) models of B cell lymphoma were established as described previously (13, 14), with minor modifications. In brief, Raji or RajiGFP/Luc cells (5 105 in 100 l of PBS) were injected subcutaneously into the right flank of HIS-MITRG or MITRG mice at 6 to 8 8 weeks of age. The mice were injected intraperitoneally with control mIgG (200 g), rituximab (200 g), SE12C3 (200.