## 3 Discussion  
Here, we show that CD47 ablation is insufficient on its own to affect tumor progression in a poorly immunogenic, metastatic model that is widely used as a pre-clinical model. Our KO and WT control tumors across various B16F10 lung metastases indicate a consistent median survival of ~20-25 days that reflects exponential-like growth (Figure 4A,B for CD47 KO and Figures 5C and 7B for WT B16). Clinical trials also show that CD47 monotherapy is rarely, if ever, effective against human cancer [4; 5]. Anti-CD47 blockade in combination with a second opsonizing mAb can show efficacy in the clinic against liquid cancer, but  
Figure 7: Pre-loading APB with microparticle cargo hinders tumor suppression. (**A**) Antibody-engineered macrophages/monocytes pre-loaded with cargo prior to treatment. (i) Schematic of in vivo marrow pre-load therapy to assess off-target effects on phagocytosis. Freshly harvested marrow cells phagocytose IgG-opsonized polystyrene microparticles for 1 h prior to tail-vein injections. (ii) Representative images of marrow cells with polystyrene microparticles, with excess particles removed by centrifugation prior to injection. (iii) Quantification of microparticles engulfed per marrow cell in a field of view (FOV). About 40% of marrow cells engulfed up to ~10 particles. (**B**) In vivo phagocytosis of WT B16F10 cells in APB marrow treatments. (i) Lung tumor burdens in control (untreated) and 2\(\times\) APB + microparticle pre-loaded groups. Mice were treated with antibody-engineered marrow on days 4 and 6 in addition to tail-vein injections of 250 \(\upmu\)g anti-Tyrp1 per Figure 2B through day 13. Mean and s.e.m. (n.s. not significant, Ordinary one-way ANOVA with [PERSON]’s multiple comparisons, \(n=5\) per three groups, including 2\(\times\) APB in Figure 5B). (ii) Projected survival curves. solid tumors likely present larger barriers and limits to anti-CD47-based blockade even in combination treatments. Similarly, subcutaneous B16F10 tumors treated with anti-Tyrp1 show little to no effect of anti-CD47 [24; 25]. Our CD47 knockout is 100% (rather than incomplete per earlier studies), and a combination with the tumor-opsonizing IgG TA99 proves effective against established lung metastases of B16F10s. The effect is consistent and significant, with survival extended 2-3-fold across several different studies (Figure 8). Our anti-SIRP\(\alpha\) blocked macrophages also proved to be effective in combination with tumor opsonization. Heterogeneity of lung tumor nodules and explosive growth can be difficult to suppress. Anti-Tyrp1 therapy of mice bearing CD47-deleted metastases nonetheless resulted in lower overall tumor burden even though mice succumbed with similar clinical symptoms as untreated controls. These observations point to a small number of escaping tumors causing moribundity, but otherwise exceptional tumor control by tissue-resident macrophages [36]. The model of phagocytic suppression of rapidly growing B16F10 metastases is extended to slow-growing A549 liver metastases that are effectively repressed by treatment. The A549/NSG model lacks other effector immune cells, consistent with a key role for macrophages and phagocytic function. Overall, our analyses of tumor growth, variability, and phagocytosis provide insight into immune cell-tumor relationships that might translate to a patient's response to such therapy. Our results with a standard pre-clinical cancer model contrast with many early reports that anti-CD47 monotherapy can suppress tumor growth in various mouse models (often xenogeneic in immunodeficient mice). The only \"monotherapy\" to succeed to date against patient tumors is an intratlumally administered SIRP\(\alpha\)-IgG1 chimera in fungoides myosin (a type of lymphoma), with evidence that the IgG1 serves to opsonize while SIRP\(\alpha\) binds CD47 [37; 38].