Replication Study: The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors

Abstract

In 2015, as part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Chroscinski et al., 2015) that described how we intended to replicate selected experiments from the paper "The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors "(Willingham et al., 2012). Here we report the results of those experiments. We found that treatment of immune competent mice bearing orthotopic breast tumors with anti-mouse CD47 antibodies resulted in short-term anemia compared to controls, consistent with the previously described function of CD47 in normal phagocytosis of aging red blood cells and results reported in the original study (Table S4; Willingham et al., 2012). The weight of tumors after 30 days administration of anti-CD47 antibodies or IgG isotype control were not found to be statistically different, whereas the original study reported inhibition of tumor growth with anti-CD47 treatment (Figure 6A,B; Willingham et al., 2012). However, our efforts to replicate this experiment were confounded because spontaneous regression of tumors occurred in several of the mice. Additionally, the excised tumors were scored for inflammatory cell infiltrates. We found IgG and anti-CD47 treated tumors resulted in minimal to moderate lymphocytic infiltrate, while the original study observed sparse lymphocytic infiltrate in IgG-treated tumors and increased inflammatory cell infiltrates in anti-CD47 treated tumors (Figure 6C; Willingham et al., 2012). Furthermore, we observed neutrophilic infiltration was slightly increased in anti-CD47 treated tumors compared to IgG control. Finally, we report a meta-analysis of the result.

Keywords: CD47; Reproducibility Project: Cancer Biology; cancer biology; metascience; mouse; replication; reproducibility; targeted therapy.

Figure 1.. Blood toxicity analysis.

Female FVB mice bearing orthotopic MT1A2 breast tumors were randomized to receive IgG isotype control (IgG) (n = 7) or anti-mouse CD47 (CD47) (n = 7) antibodies. Mice bearing small or undetectable tumors were designated for baseline reading (NT) (n = 6). Five days after the beginning of treatment blood samples collected via retro-orbital bleed were analyzed on a hematology analyzer. Dot plots with means reported as crossbars for each hematological parameter. For each parameter a one-way ANOVA was performed and the alpha level or p-value was adjusted using the Bonferroni correction. (A) White blood cells (WBC), one-way ANOVA; F(2,17) = 15.09, uncorrected p=0.00017 with alpha level = 0.0033; (Bonferroni corrected p=0.0026). (B) Neutrophils (NE), one-way ANOVA; F(2,17) = 1.02, uncorrected p=0.381 with alpha level = 0.0033; (Bonferroni corrected p>0.99). (C) Lymphocytes (LY), one-way ANOVA; F(2,17) = 20.84, uncorrected p=2.67×10⁻⁵ with alpha level = 0.0033; (Bonferroni corrected p=0.00040). (D) Monocytes (MO), Welch's one-way ANOVA; F(2,8.52) = 9.98, uncorrected p=0.0058 with alpha level = 0.0033; (Bonferroni corrected p=0.0877). (E) Eosinophils (EO), one-way ANOVA; F(2,17) = 0.06, uncorrected p=0.942 with alpha level = 0.0033; (Bonferroni corrected p>0.99). (F) Basophils (BA), one-way ANOVA; F(2,17) = 4.20, uncorrected p=0.0330 with alpha level = 0.0033; (Bonferroni corrected p=0.495). (G) Red blood cells (RBC), one-way ANOVA; F(2,17) = 424.9, uncorrected p=3.07×10⁻¹⁵ with alpha level = 0.0033; (Bonferroni corrected p=4.60×10⁻¹⁴). (H) Hemoglobin (Hb), one-way ANOVA; F(2,17) = 502.1, uncorrected p=7.61×10⁻¹⁶ with alpha level = 0.0033; (Bonferroni corrected p=1.14×10⁻¹⁴). (I) Hematocrit (HCT), one-way ANOVA; F(2,17) = 283.0, uncorrected p=8.93×10⁻¹⁴ with alpha level = 0.0033; (Bonferroni corrected p=1.34×10⁻¹²). (J) Mean corpuscular volume (MCV), one-way ANOVA; F(2,17) = 11.81, uncorrected p=0.00061 with alpha level = 0.0033; (Bonferroni corrected p=0.0091). (K) Mean corpuscular hemoglobin (MCH), one-way ANOVA; F(2,17) = 10.64, uncorrected p=0.00101 with alpha level = 0.0033; (Bonferroni corrected p=0.0151). (L) Mean corpuscular hemoglobin concentration (MCHC), one-way ANOVA; F(2,17) = 0.61, uncorrected p=0.552 with alpha level = 0.0033; (Bonferroni corrected p>0.99). (M) Red blood cell distribution width (RDW), Welch's one-way ANOVA; F(2,10.46) = 30.62, uncorrected p=4.25×10⁻⁵ with alpha level = 0.0033; (Bonferroni corrected p=0.00064). (N) Platelets (PLT), one-way ANOVA; F(2,17) = 0.62, uncorrected p=0.548 with alpha level = 0.0033; (Bonferroni corrected p>0.99). (O) Mean platelet volume (MPV), one-way ANOVA; F(2,17) = 6.98, uncorrected p=0.0061 with alpha level = 0.0033; (Bonferroni corrected p=0.092). Additional details for this experiment can be found at https://osf.io/g57ch/. DOI: http://dx.doi.org/10.7554/eLife.18173.002

Figure 1—figure supplement 1.. Blood toxicity analysis.

Female FVB mice bearing orthotopic MT1A2 breast tumors were randomized to receive IgG isotype control (IgG) (n = 7) or anti-mouse CD47 (CD47) (n = 7) antibodies. Mice bearing small or undetectable tumors were designated for baseline reading (NT) (n = 6). Five days after the beginning of treatment blood samples collected via retro-orbital bleed were analyzed on a hematology analyzer. Normal reference ranges (Range) for each parameter are from Drew Scientific Hemavet 950FS. WBC = White blood cells; NE = Neutrophils; LY = Lymphocytes; MO = Monocytes; EO = Eosinophils; BA = Basophils; RBC = Red blood cells; Hb = Hemoglobin; HCT = Hematocrit; MCV = Mean corpuscular volume; MCH = Mean corpuscular hemoglobin; MCHC = Mean corpuscular hemoglobin concentration; RDW = Red blood cell distribution width; PLT = Platelets; MPV = Mean platelet volume. Additional details for this experiment can be found at https://osf.io/g57ch/. DOI: http://dx.doi.org/10.7554/eLife.18173.003

Figure 2.. Final tumor weights of immune competent hosts treated with control or CD47 targeted antibodies.

At the end of the predefined study period (Day 31), tumors from mice bearing orthotopic MT1A2 breast tumors treated every other day with IgG isotype control (IgG) (n = 7) or anti-mouse CD47 (anti-CD47) (n = 6) antibodies were excised and weighed. Dot plot with means reported as crossbars and error bars represent s.e.m. Two-tailed Welch’s t-test between IgG and anti-CD47 treated tumors; t(9.66) = 1.796, p=0.104. Additional details for this experiment can be found at https://osf.io/g57ch/. DOI: http://dx.doi.org/10.7554/eLife.18173.004

Figure 2—figure supplement 1.. Tumor volumes of immune competent hosts treated with control or CD47 targeted antibodies.

This is the same experiment as in Figure 2. Following orthotopic injection of MT1A2 cells mice were monitored for development of tumors. Caliper measurements were taken at 9, 11, 18, and 25 days after cell injection to calculate tumor volume. Fourteen mice with detectable tumors were randomly assigned to treatment at 11 days post cell inoculation (dashed line). At the end of the predefined study period, tumors from mice treated with either IgG isotype control (IgG) (n = 7) or anti-mouse CD47 (anti-CD47) (n = 6) antibodies were excised and weighed. Tumor volume at day 42 post cell injection was calculated from weight and density (1.05 g/ml). Exploratory analysis on tumor weights excluding three tumors that regressed during the course of the study (indicated by asterisk): two-tailed Welch’s t-test between IgG and anti-CD47 treated tumors; t(7.94) = 0.745, p=0.478, Glass' =$\Delta$ −0.58, 95% CI [−1.88, 0.80]. Additional details for this experiment can be found at https://osf.io/g57ch/. DOI: http://dx.doi.org/10.7554/eLife.18173.005

Figure 3.. Meta-analysis of effect.

Effect size (Glass' $\Delta$) and 95% confidence interval are presented for Willingham et al. (2012), this replication attempt (RP:CB), and a meta-analysis to combine the two effects of tumor weight comparisons. Sample sizes used in Willingham et al. (2012) and this replication attempt are reported under the study name. Random effects meta-analysis of tumors treated with IgG compared to anti-CD47 (meta-analysis p=0.745). Additional details for this meta-analysis can be found at https://osf.io/ha2bx/. DOI: http://dx.doi.org/10.7554/eLife.18173.007