MPS1i-induced chromosomal instability favors induction of pro-phagocytic de novo IgG and can lead to durable acquired immunity.
(A) Schematic illustrating protocol for sera collection from surviving mice from Fig. 3C-D and follow-up experiments to characterize potential de novo anti-cancer IgG antibodies and their functionality both in vitro and in vivo. Serum from all mice was collected at least 100 days after initial tumor challenge.
(B) (i) Representative flow cytometry histograms showing that convalescent sera from survivors in Fig. 3C-D contain IgG2a/c (top) and IgG2b (bottom) that bind to both WT and Tyrp1 KO B16F10 cells.
(ii) Median fluorescence intensity quantification of IgG2a/c and IgG2b binding from sera from surviving mice. Convalescent sera show statistically significant increase in both IgG2a/c and IgG2b titers. Binding to even Tyrp1 KO cells suggests broader recognition of antigens unique to B16F10. Statistical significance was calculated by an unpaired two-sample Kolmogorov-Smirnov test (**** p < 0.0001). For IgG2a/c quantification: for binding against Tyrp1+ cells, n = 23 distinct naïve serum samples and n = 28 distinct convalescent serum samples from surviving mice; for binding against Tyrp1 KO cells, n = 23 distinct naïve serum samples and n = 27 distinct convalescent serum samples. For IgG2b quantification: for binding against Tyrp1+ cells, n = 14 distinct naïve serum samples and n = 16 distinct convalescent serum samples from surviving mice; for binding against Tyrp1 KO cells, n = 13 distinct naïve serum samples and n = 17 distinct convalescent serum samples.
(iii) Western blotting of B16F10 lysate with either naïve sera or first challenge survivor sera as primary probe followed by anti-mouse IgG [H+L] secondary staining. Numerous bands appear when immunoblotting with convalescent survivor sera (and more than when immunoblotting with naïve sera), qualitatively confirming binding to numerous antigens and suggesting acquired immunity beyond Tyrp1.
(C) (i) Phagocytosis of serum-opsonized CD47 KO or CD47/Tyrp1 double KO B16F10 cells by BMDMs on 2D tissue culture plastic. Additionally, B16F10 cells opsonized with either anti-Tyrp1 or mouse IgG2a were included as controls for comparisons. Serum IgG derived from survivors has both opsonization and pro-phagocytic functional ability against B16F10. Furthermore, convalescent sera IgG from survivors is still able to drive engulfment of Tyrp1 KO cells, further suggesting targeting of antigens beyond Tyrp1. Statistical significance was calculated by two-way ANOVA and Tukey’s multiple comparison test (mean ± SD, n = 21-23 distinct sera samples collected from survivors for B16F10 CD47 KO phagocytosis per condition and n = 14-23 for B16F10 CD47/Tyrp1 double KO phagocytosis per condition).
(ii) Convalescent sera from first challenge survivors can repress growth of B16F10 CD47 KO immuno-tumoroids (with macrophages). Tumoroid growth was measured by calculating the GFP+ area at the indicated timepoints (mean ± SD, n = 16 total tumoroids from two independent experiments for each condition, except n = 8 for opsonization with sera from naïve mice). Statistical significance was calculated by Brown-Forsythe and Welch ANOVA tests with Dunnett T3 corrections for multiple comparisons (ns, not significant; ** p < 0.01; **** p < 0.0001). Scale bars = 0.5 mm.
(D) B16F10 CD47 KO cells were pre-opsonized with either convalescent sera from first challenge survivors, anti-Tyrp1 or mouse IgG2a isotype control. All mice were subcutaneously injected with 2×105 pre-opsonized B16F10 CD47 KO cells.
(i) Tumor growth curves shown are at early timepoints where growth is still in the linear regime. Linear fits highlight growth suppression of tumors comprised of B16F10 CD47 KO cells pre-opsonized with convalescent sera and anti-Tyrp1, compared to the mouse IgG2a isotype counterparts. Mean ± SEM for all timepoints, with n = 16 mice with tumors pre-opsonized with convalescent sera (each from a distinct survivor), n = 9 mice with tumors pre-opsonized with anti-Tyrp1, and n = 14 mice with tumors pre-opsonized with mouse IgG2a isotype control. Statistical significance was calculated by ordinary one-way ANOVA and Tukey’s multiple comparison test at days 9, 11, and 13 (* p < 0.05; **** p < 0.0001). Significance represented in plot legend is representative of all three timepoints.
(ii) Survival curves up to 100 days of mice from (D-i) with pre-opsonized tumors. Both convalescent sera and anti-Tyrp1 provide similar survival benefits, suggesting potent de novo IgG opsonization and anti-cancer function. Statistical significance was determined by the Log-rank (Mantel-Cox) test (ns, not significant; ** p < 0.01; **** p < 0.0001).
(E) Schematic illustrating the series of experimental tumor challenges to assess acquired immunity. Survivors from the first challenge (Fig. 3C-D) were again challenged with either DMSO or MPS1i-treated B16F10 CD47 KO cells. Survivors from this second tumor challenge were once again challenged, this time with untreated B16F10 CD47 KO.
(F) Survival curves of survivors from Fig. 3C-D for a second tumor challenge experiment. Prior to tumor inoculation, B16F10 CD47 KO cells were treated with 2.5 μM MPS1i (reversine) or the equivalent volume of DMSO vehicle control. Cells were treated for 24 h, after which they were washed twice with PBS and allowed to recover for an additional 48 h. After the recovery period elapsed, all mice were subcutaneously injected with 2×105 B16F10 CD47 KO cells. Experimental conditions are as follows: n = 7 age-matched naïve mice (never tumor-challenged) injected with DMSO-treated B16F10 CD47 KO cells, n = 11 surviving mice (from Fig. 3C-D) injected DMSO-treated B16F10 CD47 KO cells, n = 6 age-matched naïve mice injected with MPS1i-treated B16F10 CD47 KO cells, and n = 8 surviving mice (from Fig. 3C-D) injected with MPS1i-treated B16F10 CD47 KO cells. Previous survivors challenged with DMSO-treated B16F10 CD47 KO cells show increased median survival (21 days) compared to their naïve counterpart (14 days). All previous survivors that were again challenged with MPS1i-treated B16F10 CD47 KO cells survive. All mice challenged were from three independent experiments. Statistical significance was determined by the Log-rank (Mantel-Cox) test (** p < 0.01; *** p < 0.001; **** p < 0.0001).
(G) Non-survivors from the second tumor challenge in (F) were euthanized after tumor size was >150 mm2, and their tumors were excised and disaggregated for immune infiltrate analysis by flow cytometry. (i) Quantification of CD45+ (immune) cells in the excised tumors, showing that first challenge survivors still show ∼2.5-fold increased tumor immune cell infiltrate despite reaching terminal burden. n = 4 mice for age-matched naïve control, n = 5 mice that survived the first tumor challenge. (ii) Quantification of tumor infiltrating CD8a+ cytotoxic T cell relative to the total number of tumor cells. First challenge survivors show ∼2-fold increase in CD8a+ T cells. n = 4 mice for age-matched naïve control, n = 3 mice that survived the first tumor challenge. (iii) Quantification of tumor infiltrating F4/80+ macrophages relative to the total number of tumor cells. First challenge survivors show ∼3-fold increase in macrophages. n = 4 mice for age-matched naïve control, n = 5 mice that survived the first tumor challenge. (iv) Quantification of MHCII+ tumor infiltrating F4/80+ macrophages relative to the total number of F4/80 macrophages. First challenge survivors show ∼3-fold increase in MHCII+ macrophages. n = 4 mice for age-matched naïve control, n = 5 mice that survived the first tumor challenge. For all experiments, mean ± SEM shown, and statistical significance was calculated by an unpaired two-tailed t-test with Welch’s correction (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).
(H) (i) Third tumor challenge survival curves of long-term survivors from (G). All mice were challenged with 2×105 B16F10 CD47 KO cells (n = 9 mice from three independent experiments), delivered subcutaneously. For benchmarking and statistical comparison, survival curves from Fig. 3F for naïve mice (n = 7 mice from three independent experiments) and second challenge (n = 11 mice from three independent experiments) are included. Long-term survivors challenged a third time show ∼70% survival without any additional therapeutic modality, suggesting significantly improved acquired immune response. Statistical significance was determined by the Log-rank (Mantel-Cox) test (** p < 0.01; *** p < 0.001; **** p < 0.0001).
(ii) Individual tumor growth curves for third challenge in long-term survivors (n = 9 mice from three independent experiments) shown in (H-i). In total, 4 mice developed tumors, two of which had to be euthanized prematurely due to tumor rupture despite not reach a terminal burden of 125 mm2. These mice are still considered and included as casualties in the survival curve analysis. The two remaining mice show significantly slower tumor growth than naïve mice challenged with regular B16F10 CD47 KO (median survival of 14 days) and can be considered durable partial responders.