## 2 Results  
### Tumor-Opsonizing Antibodies Drive Regression of Metastases Only When CD47 Is Disrupted  
We used CRISPR/Cas9 to eliminate CD47 from the surface of B16F10 cells without the affecting surface levels of another relevant protein, tyrosinase-related protein 1 (Tyrp1; Figure S1) [19]. Tyrp1 is targetable in vivo with a mouse monoclonal IgG2a antibody (anti-Tyrp1 clone TA99) that activates Fc receptors (FcR) [20; 21] and can suppress wild-type (WT) B16F10 tumors if injected within ~1 day of B16F10 inoculation [20; 22]. TA99 monotherapy is generally ineffective in established WT B16F10 tumors (Figure 2 Aii and Figure S2A,B), which concurs with recent clinical trial results of anti-TYRP1 monotherapy against human melanoma that demonstrated safety but no anti-tumor efficacy [23]. Our dosage range for TA99 is similar to that in the clinical trial: 1-10 mg/kg was here compared to safety dose testing of 5, 10, 20, and 30 mg/kg in the trial.  
For all in vivo B16F10 experiments, metastases are allowed to establish in the lungs prior to tail-vein injections of anti-Tyrp1 (Figure 2 Bi: treatment from days 4 to 15). Tumor burden was quantified from melanized area and/or nodules on the surfaces of mouse lung lobes post-sacrifice (Figure S2C,D); we avoid live-animal imaging methods such as those using luciferase because they add immunogenicity. For WT B16F10 metastases expressing wild-type levels of CD47, high TA99 doses (250 \(\upmu\)g: \(\sim\)10 mg/kg) show no significant effect on lung tumors compared to untreated or isotype IgG2a-treated controls 14 days after tumor inoculation (Figure 2 Aii and Figure S2B). Yet, when CD47 is deleted from the tumors, even moderate doses (75 \(\upmu\)g, \(\sim\)3 mg/kg) suppress growth even at days 16 and 20 (Figure 2 Bi-iii). Whereas untreated lungs show melanoma covering nearly the entire lung surface, anti-Tyrp1-treated lung lobes exhibit relatively few, smaller nodules.  
We show that the anti-Tyrp1 doses are effective against established CD47 KO tumors, whereas the WT tumors show no significant change (Figure S2A), even when systemic injections of murine anti-CD47 are combined with the anti-Tyrp1 (Figure S3). The latter result might be explained in part by previous observations that systemic anti-CD47 can only access and bind a fraction of CD47 on the surface of these tumor cells [24; 25]. Thus, in contrast to WT tumors, complete disruption of CD47's inhibitory \"don't eat me\" signal enables FcR-mediated phagocytosis to suppress growth of CD47 KO metastatic tumors. Most past studies of WT that show efficacy of TA99 began treatment on days 0 to 1 after tumor inoculation [22; 24; 25], affecting initial cell engraftment rather than treating the more clinically relevant, established tumors. Even in one early study where TA99 was injected later [22], mAb doses were \(\sim\)8-fold higher than here.  
CD47-deleted lung metastases across a cohort measured at different time points exhibit exponential (or power law) growth, and the growth is identical to WT tumors (Figure 2 Biii,iv). These results align with current clinical results that generally show that anti-CD47 monotherapy does not affect tumor growth. Our studies show that tumor variance increases with time, which is consistent with the exponential-type growth that is typical of unhindered proliferation, including B16 tumors at subcutaneous sites [26]. Longitudinal measurements of B16 lung metastasis growth are challenging without modified cells (e.g., without immunogenic reporters), but one study that showed similar exponential-type B16 growth used computed tomography (CT) [27]. Our own CT imaging of tumor margins (Figure S4) suggested higher resolution and higher confidence in the mean kinetics when measured by optical imaging of a cohort at sacrifice (Figure S2). Overall, the doubling of CD47 KO metastases is suppressed \(\sim\)4-5-fold by anti-Tyrp1 (Figure 2 Biii), suggesting that anti-Tyrp1 drives tumor cell phagocytosis.