The data sets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.. to both solid and hematologic tumor cells in vitro, and inhibited tumor growth in immune-competent tumor-bearing mice. Furthermore, pep-20 promoted macrophages to mobilize the antitumor T-cell response with minimal toxicity. Furthermore, systemic administration of the derivate pep-20-D12 showed robust synergistic antitumor efficacy in combination with IR. Conclusion In summary, these results demonstrated that CD47/SIRP blocking peptides, pep-20 and its derivate, could serve as promising candidates to promote macrophages-mediated phagocytosis and immune response in cancer immunotherapy. proposed that CD47 blockade with a low dose at the beginning, SH3RF1 followed by a higher grade maintenance dose, minimized the side effects of anemia. 32 Our work was partly consistent with this strategy. Compared with antibodies and fusion proteins, the T1/2 of Coumarin 30 peptides is relatively short, which may weaken the therapeutic effect to some extent. In return, adverse effects could be managed timely and effectively for clinical treatments. Furthermore, administering a large dose of inhibitors in a short period may break the balance of pro-phagocytic and anti-phagocytic signals which are partly regulated by cellular homeostasis, even normal cells are not subjected to phagocytosis because of lack of pro-phagocytic signaling.38 In response to erythrocyte decline, peptide treatment may be a relatively milder strategy that enables Coumarin 30 maintaining relative homeostasis or returning to a stable state after a short period of disequilibrium via a compensatory effect. While macrophages are the main effector cells of CD47/SIRP pathway from current studies, it is undeniable that the presence of Fc receptor (FcR)-mediated ADCC and antibody-dependent cellular phagocytosis (ADCP) effectively synergize antitumor therapy by phagocytes, even the contradictory relationship between antitumor effects and potential side effects can be balanced.39 Therefore, agents of CD47/SIRP blockade for enhancement of phagocytosis function by blocking dont eat me signals combined with cancer-targeting therapeutic antibodies, FcR-mediated ADCC and ADCP, may be a more effective approach to potently eliminate tumors at the current stage.12 34 40 Previous studies have provided evidence that macrophages could effectively phagocytose tumor cells, and dendritic cells (DCs) could elicit increased activation of T cells in the tumor microenvironment after anti-CD47 therapy. Since DCs and macrophages are important components of innate immune system for phagocytosis and antigen presentation which can bridge innate and adaptive immunity, it is possible that they are synergistic to the removal of tumor cells, and to stimulate CD8+ T cells and initiate antitumor T-cell immune response.41 42 In the present study, the pep-20-mediated phagocytosis of tumors by macrophages initiated antitumor T-cell immune response with a marked increase of CD8+ T cells in tumor tissues, and the IFN–secreting CD8+ T cells were significantly amplified.43 Macrophages engulfed OVA-expressing tumor cells and presented antigen, thus effectively activating OT-IT cell proliferation and secretion of IFN- cytokines in the presence of pep-20. In addition, removal of macrophages invalidated the therapeutic effects of pep-20 and impaired the activation of CD8+ T cells in tumor-bearing mice. Collectively, these results suggested that pep-20 depended on macrophages to exert antitumor effects and participated in motivating antitumor immune response. A variety of therapeutic peptides and derivative drugs have been approved by the Food and Drug Administration for diverse disease diagnosis and treatments.44 Despite the limitations in wide applications of peptide drugs, such as low solubility, relatively short circulating T1/2, poor stability and limited oral bioavailability, many strategies including cyclization, PEGylation modifications, adjunction of D-amino acids and conjugation with serum albumin have been utilized Coumarin 30 to refine the peptide drugs.45C47Retro-inverso peptide ensures the stability of proteases which possess similar side-chain topologies and biological activities to interact with its receptor. However, pep-20 containing -helices structure may not maintain the same biological activity as parent L-peptides to the retro-inverso isomerization Coumarin 30 due to the change at the secondary structure.48 D-amino acid substitutions at the N-terminal and C-terminal of the peptide which showed little effect on the -helical structure were adopted to circumvent these problems.49 Pep-20-D12 containing D-amino acid substitutions at the N-terminal and Coumarin 30 C-terminal proved to be highly stable to enzymatic degradation and effectively maintain blocking activity. RT results in inducing tumor cell death and triggers antitumor immune response, but in most cases, the response is insufficient to maintain, and relapses always occur.28 The combination of pep-20-D12 with IR treatment exerted more effectiveness.