## 2 Results
### Tumor Clearance Induced by Opsonization and CD47 Deletion Prolongs Survival, but Some Tumors Escape  
To directly assess survival, we followed small cohorts of untreated and treated mice over time. All untreated mice with CD47 KO lung metastases exhibited numerous clinical symptoms that required euthanasia (hunched posture, slow movement, labored breathing), whereas mice treated with anti-Tyrp1 showed a median survival \(\sim\)30% longer (Figure 4A). Furthermore, untreated controls all required sacrifice within a narrow window of 2-3 days (at \(\sim\)23 days), whereas all mice in the treatment group survived longer, with a large variation and 20% surviving up to 2-fold longer. Figure 4: Tumor clearance induced by opsonization AND CD47 deletion prolongs survival, but a few large tumor masses still cause death. (**A**) Survival of anti-Tyrp1-treated mice induced with 2 \(\times\) 10\({}^{5}\) CD47 KO B16F10 cells intravenously. 250 ug anti-Tyrp1 doses beginning on Day 4 post inoculation prolong the survival of mice by about 30%, but mice are unable to be cured completely (\(n\) = 5 per group, *** \(p\) < 0.01, Log-rank test). Right: mice are healthy and exploratory, then rapidly deteriorate in health in less than 24 h, showing clinical symptoms of hunched posture, labored breathing, slow reactivity, and weight loss. (**B**) Projected survival of anti-Tyrp1-treated mice induced with 2 \(\times\) 10\({}^{5}\) CD47 KO B16F10 intravenously modeled with a power law fit based on single timepoint estimates of growth. With 75 ug anti-Tyrp1 on the same dosing timeline, the estimated projected survival advantage is \(\sim\)6–7 days (\(n\) = 10 per group, **** \(p\) < 0.0001, Log-rank test). Inset: exponential-type growth projections of six representative mice. (**C**) CD47 KO B16F10 tumor burden on lung lobes at sacrifice. Anti-Tyrp1-treated mice have lungs with markedly reduced tumor burden relative to untreated mice, yet demonstrate similar clinical symptoms, requiring sacrifice (\(n\) = 5 per group, * \(p\) < 0.05, unpaired t-test). (**D**) Survival of mice in days versus lung surface tumor area at sacrifice. An exponential-type model shows that survival is inhibited by lung tumor burden. Applying our nonlinear analyses (per Figures 2B and 3B) to single snapshot measurements on sacrificed mice (e.g., day 16), we estimated survival curves of cohorts (Figure 4B) that align very well with our direct measurements of survival (Figure 4A). More specifically, we fit an exponential-type curve to the datapoint for each sacrificed mouse, and then we identify when the curve crosses a critical tumor burden (see Methods). The Kaplan-Meier curves show that the projected long-term survival is nearly the same as the measured survival, with 20% again living twice as long or longer than the controls (\(\sim\)20 days). Although these experiments used different anti-Tyrp1 doses in the treated cohorts (3 mg/kg vs. 10 mg/kg), the overall agreement suggests that the higher anti-Tyrp1 dose in the symptoms-based cohort is in therapeutic excess. Strikingly, despite the similar clinical symptoms between treated and untreated mice, lower lung tumor burden was present on the long-surviving, treated mice (Figure 4C,D). Mice with the lowest lung tumor burden measured at sacrifice survived the longest. Clinical symptoms leading to rapid decompensation were consistent with compression of airways, blood vessels, and surrounding lung tissues leading to hypoxia, and post-mortem dissection indeed showed a few large tumor masses (Figure S7). We confirmed the identity of these melanized masses as B16F10 tumors by disaggregating and growing the cells in culture. Thus, a small number of metastases can outpace clearance by macrophages and cause death in this fast-growing model. Regardless, complete CD47 disruption combined with a tumor-opsonizing antibody significantly suppresses metastatic-type tumor growth and prolongs survival.