Phagocytic elimination of solid tumors is an attractive mechanism for immunotherapy- particularly because of the possible induction of anti-cancer immunity. The phagocytic potential of macrophages is limited, however, by the CD47-SIRPa; checkpoint, and how much CD47 disruption is needed for efficacy remains unclear, even when tumors are opsonized by a pro-phagocytic antibody. Here, CRISPR-interference (CRISPRi) is applied with a large set of sgRNAs to produce a broad range of CD47 knockdowns in B16F10 melanoma, which is generally found to be resistant to the heavily studied PD-1 blockade. Guided by 3D immuno-tumoroid results, we identify a critical CD47 density below which macrophage-mediated phagocytosis dominates proliferation in the presence of an otherwise ineffective pro-phagocytic antibody (anti-Tyrp1). Growing tumors and immuno-tumoroids generally show selection for CD47-positive cells, but some mice reject tumors having >97% mean repression of CD47 or even having 80% repression-unless mixed with 50% repressed cells. Interestingly, long-term survivors have de novo pro-phagocytic IgG antibodies that increase in titer with depth of repression and also with early accumulation of tumor macrophages. Given well-known limitations of antibody permeation into solid tumors, our studies set a benchmark for anti-CD47 blockade and suggest deep disruption favors acquired immunity.

中文翻译：

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通过错配 CRISPRi 滴定 CD47 揭示不完全抑制可以消除 IgG 调理的肿瘤，但 CD47 异质性限制了抗肿瘤 IgG 的诱导

实体瘤的吞噬消除是免疫治疗的一种有吸引力的机制，特别是因为可能诱导抗癌免疫。然而，巨噬细胞的吞噬潜能受到 CD47-SIRPa 的限制；检查点，以及需要多少 CD47 破坏才能达到疗效仍不清楚，即使肿瘤被前吞噬抗体调理。在这里，CRISPR 干扰 (CRISPRi) 与大量 sgRNA 一起应用，以在 B16F10 黑色素瘤中产生广泛的 CD47 敲低，这通常被发现对大量研究的 PD-1 阻断具有抗性。在 3D 免疫肿瘤样结果的指导下，我们确定了一个关键的 CD47 密度，在该密度以下，巨噬细胞介导的吞噬作用在存在其他无效的促吞噬抗体（抗 Tyrp1）的情况下主导增殖。生长的肿瘤和免疫类肿瘤通常显示对 CD47 阳性细胞的选择，但一些小鼠排斥 CD47 平均抑制 >97% 甚至具有 80% 抑制的肿瘤 - 除非与 50% 抑制的细胞混合。有趣的是，长期幸存者具有从头产生的促吞噬 IgG 抗体，其效价随着抑制深度的增加以及肿瘤巨噬细胞的早期积累而增加。鉴于众所周知的抗体渗透到实体瘤中的局限性，我们的研究为抗 CD47 阻断设定了基准，并表明深度破坏有利于获得性免疫。长期幸存者具有从头产生的促吞噬 IgG 抗体，其滴度随着抑制深度的增加以及肿瘤巨噬细胞的早期积累而增加。鉴于众所周知的抗体渗透到实体瘤中的局限性，我们的研究为抗 CD47 阻断设定了基准，并表明深度破坏有利于获得性免疫。长期幸存者具有从头产生的促吞噬 IgG 抗体，其滴度随着抑制深度的增加以及肿瘤巨噬细胞的早期积累而增加。鉴于众所周知的抗体渗透到实体瘤中的局限性，我们的研究为抗 CD47 阻断设定了基准，并表明深度破坏有利于获得性免疫。