To investigate the possible effects that CIN may have in mediating macrophage immune response, we chose to work with the poorly immunogenic B16F10 mouse melanoma model. To induce CIN in B16F10, we treated cells with the MPS1 inhibitor reversine (Hong et al., 2022; Kitajiam et al., 2022; Santaguida et al., 2010; Santaguida et al., 2017). Treatment occurred for 24 h, after which the drug was washed out, and cells were allowed to recover for an additional 48 h (Fig. 1A). After the recovery period, we quantified CIN in B16F10 cells, characterized its effects in early-stage tumors, and studied potential effects on macrophages in vitro (Fig. 1A). We first confirmed that MPS1i treatment increased the frequency of micronuclei formation in interphase cells (Fig. 1B), as micronuclei are often used as a surrogate for CIN and genome instability (Cohen-Sharier et al., 2020; Crasta et al., 2012; Harding et al., 2017; Mackenzie et al., 2017). Regardless of the MPS1i concentration used, we saw substantial increases over the cell line’s baseline level of micronuclei formation. We further verified these results with other MPS1i inhibitors AZ3146 and BAY 12-17389 (Fig. S1A) to confirm the effect was not somehow limited to just reversine. Upon confirmation of increased micronuclei induction, we then proceeded to quantitatively confirm and assess copy number variations that resulted from MPS1i treatment using single-cell RNA-sequencing. For DMSO-treated B16F10 cells, approximately 10% of the population were considered aneuploid outliers (Fig. 1C-top; S1B-C) – cells with copy number profiles that are 2.5 standard deviations away from the distribution peak (see Methods). However, 34% of the MPS1i-treated B16F10 cells were aneuploid (Fig. 1C-bottom; S1B-C).

After confirming that MPS1i indeed induces CIN and quantifiable aneuploidy in B16F10 cells, we next sought to understand how CIN may affect early-stage immune response and tumor development. We proceeded to establish tumors in mice with either MPS1i-treated or DMSO-treated B16F10 (following the same schema outlined in Fig. 1A). We isolated tumors from mice at two timepoints, five and ten days after initial challenge, and then we processed whole-tumors for bulk RNA-sequencing (Fig. 1D). Comparison of tumors comprised of MPS1i-treated and DMSO-treated cells by differential gene expression showed distinct downregulated transcripts for numerous genes encoding M2-like (pro-cancer) macrophage polarization markers (Fig. 1E). At day 5 post-challenge, we see that classical M2-like, pro-cancer markers Arg1, Marco, and Cd274 are downregulated. However, several M2 markers are upregulated at this timepoint: Mrc1, Cd163, and genes related to the complement component 1a (C1q). By day 10, Marco, Mrc1, Cd163, Cd274, and the C1q complex genes are now all downregulated. Arg1, however, returns to either basal or increased expression on day 10. Overall, though, we generally see globally decreased expression for most markers by day 10. Furthermore, cytokines Ccl2, Ccl4, Ccl7, Ccl22, and Il21r, which associated with an M2-like, pro-cancer macrophage phenotype (Cerezo-Wallis et al., 2020), were found to be consistently downregulated at both days 5 and 10 post-challenge. Gene set enrichment analysis further revealed that numerous pathways related cell cycle and proliferation were also downregulated (Fig. 1F), consistent with previous studies (Cohen-Sharir et al., 2020; Wang et al., 2021). These downregulated pathways suggest longer tumor doubling time since cells have yet to adapt to these CIN-associated proliferation deficits.

The identification of downregulated M2-like macrophage markers in CIN-afflicted tumors next led us to better assess how early-stage CIN in cancer may influence macrophages specifically. Previous studies (Cerezo-Wallis et al., 2020; Xian et al., 2021) have found that the secretome can have profound and rapid polarization effects on bone marrow-derived macrophages (BMDMs) in vitro. We proceeded to perform a similar secretome study, in which conditioned media from MPS1i-treated or DMSO-treated B16F10 cells was collected and added to previously differentiated BMDMs. BMDMs were treated with conditioned media for 24 h, after which they were collected and processed for single-cell RNA-sequencing (Fig. 1G). Unsupervised clustering analysis using uniform manifold approximation and projection (UMAP) nonlinear dimensionality reduction identified four distinct macrophage population clusters: two clusters (0 and 2) that consisted of approximately 75% of macrophages treated with MPS1i-treated B16F10 secretome and two clusters (1 and 3) that consisted of approximately 75% macrophages treated with DMSO-treated B16F10 secretome (Fig. 1H).

The gene expression profiles of these clusters showed that the two clusters (1 and 3) that consisted mostly of macrophages treated with DMSO-treated B16F10 secretome had increased expression of anti-inflammatory, M2-like pro-cancer macrophage polarization markers (Fig. 1I). Consistent with the whole-tumor bulk RNA-seq, we were successfully able to capture increased expression of Mrc1 in both clusters 1 and 3 and observed increased expression of Ccl2, Ccl4, and Ccl7 in cluster 3. More importantly though, gene expression revealed that clusters 1 and 3 tended to downregulate many genes associated with a pro-inflammatory, M1-like anti-cancer response (Fig. 1J). Clusters 0 and 2, on the other hand, which consisted mostly of MPS1i-treated B16F10 secretome, showed little-to-no expression for most M2-like, pro-cancer markers (Fig. 1I) and increased expression of pro-inflammatory, anti-cancer markers (Fig. 1J). These results suggest that early-stage CIN in cancer cells engages pro-inflammatory macrophage activity.

Next, we sought to experimentally confirm the polarization effects. Here, we use B16F10 CD47 knockout (KO) cells (Fig. S2A) to remove the inhibitory effects of CD47 on phagocytosis and more easily study macrophage engulfment ability (Fig. S2B-C). Moving forward, we chose to use a B16F10 tumoroid model to better simulate the biophysical properties of the 3D tumor microenvironment and proliferative capacity of tumor masses (Fig. 2A) (Dooling et al., 2022). We chose this model primarily because 2D phagocytosis assays fail to consider the mechanical cohesive forces between tumor cells and other biophysical aspects that can affect immune response (Nia et al., 2020). We have previously found that macrophages need to phagocytose cooperatively—large numbers of macrophages working together to overcome the mechanical strength of cohesion between solid tumor cells—to achieve durables responses (Dooling et al., 2022). CIN can also often induce additional effects on cells in its early stages, such as increases in cell size due to cytokinesis defects and poly-aneuploidy (Chunduri & Storchová, 2019; Mallin et al., 2022). Single macrophages may struggle to phagocytose these large targets in a conventional 2D assay (Champion et al., 2006), even with IgG opsonization, which we experimentally confirmed with MPS1i-treated cells (Fig. S2B). MPS1i treatment, in fact, increases cell size, as measured by flow cytometry (Fig. S2C), although size effects are confounded by decreased Tyrp1 expression, the antigen targetable for opsonization using anti-Tyrp1 (Fig. S2D). CD47 levels were relatively consistent across treatment conditions and similar to non-perturbed levels (Fig. S2E) for B16F10 sgCtrl. We thus hypothesized that cooperative phagocytosis (Dooling et al., 2022) may be able to overcome the CIN-induced size increases and reduced binding of anti-Tyrp1. BMDMs were added to pre-assembled tumoroids at a 3:1 ratio, and this “immuno-tumoroid” co-culture was maintained for five days. We first assessed surface protein expression of macrophage polarization markers. Consistent with the polarization trends in our whole-tumor bulk RNA-sequencing and single-cell RNA-sequencing of BMDMs (Fig. 1E, 1I-J), we find that BMDMs co-cultured in tumoroids of MPS1i-treated B16F10 showed increased expression of M1-like, anti-cancer macrophage markers MHCII and CD86 while having decreased expression of M2-like, pro-cancer markers CD163 and CD206 (Fig. 2B-C).

We next wondered if these polarization studies translated to functional effects on macrophage-mediated phagocytosis and clearance of tumoroids. We again prepared tumoroids, later adding BMDMs to these pre-assembled tumoroids at a 3:1 ratio with either anti-Tyrp1 or mouse IgG2a isotype control for IgG opsonization. In our 3D tumoroid in vitro assays, we found that macrophages can suppress the growth of chromosomally unstable tumoroids and clear them, surprisingly both with and without anti-Tyrp1 (Fig. 2D-E), regardless of MPS1i concentration used for treatment. In contrast, DMSO-treated B16F10 cells (chromosomally stable with regular proliferative capacity) require anti-Tyrp1 for macrophages to clear them (Fig. 2D, S3B). These results link the phenotypic studies with functional effects: CIN can induce anti-cancer macrophage phenotypes to favor macrophage-mediated clearance.

We also attempted to use two other MPS1i inhibitors to see if we achieved similar results: tumoroids made of AZ3146-treated cells, which BMDMs could only suppress at very high concentrations (Fig. S3C-i), and tumoroids made of BAY 12-17389-treated cells, which were heavily suppressed and/or cleared similarly to tumoroids comprised of reversine-treated cells (Fig. S3C-ii). Given that AZ3146-treated cells tended to proliferate well, we next sought to verify if tumoroids made of cells treated with either BAY 12-17389 or reversine would eventually grow. We observed that tumoroids decreased in size 24 h after plating, most likely due to some cell death (Fig. S3D). Afterwards though, tumoroids generally showed some proliferation, albeit with deficits. This suggests that the anti-cancer macrophage activity observed may require CIN be severe enough to induce some proliferation deficits. Otherwise, low-level CIN may be tolerable for cancer cells, eventually favor their evolution (Vasudevan et al., 2020), and allow cancer cells to still proliferate more rapidly than phagocytosis kinetics can manage.

Lastly, we sought to confirm both our transcriptomic analyses and tumoroid surface marker expression before proceeding to long-term in vivo experimentation. We proceeded to establish tumors in mice with either MPS1i-treated or DMSO-treated B16F10 cells (the drug treatment followed the same schema outlined in Fig. 1A). We then isolated tumors five days post-challenge for immune infiltrate analyses by flow cytometry (Fig. 2F, S5). We found that CIN-afflicted tumors had approximately 3-fold more CD45+ immune cells (Fig. 2G-i), 2.5-fold more CD45+ CD11b+ (myeloid) cells (Fig. 2G-ii), and a near 6-fold increase in F4/80-positive macrophage (Fig. 2G-1. iii) compared to their DMSO counterparts. Furthermore, we found that CIN-afflicted tumors showed approximately 6-fold more CD86^hi CD206^lo (M1-like, anti-cancer) macrophages compared to their DMSO counterparts. We should note that these tumors also saw an increase in CD86^lo CD206^hi (M2-like, pro-cancer) macrophages compared to DMSO control (Fig. 2G-iv)., but this increase is expected given that there are generally more macrophages infiltrating these tumors. Despite the increase in M2-like macrophages, these tumors made of MPS1i-treated cells still have roughly 2-fold more M1-like macrophages (Fig. 2G-iv). These results suggest that although other immune cell types infiltrate tumors, which is consistent with upregulated surface expression of H2-K^b (Fig. S4), macrophage functional activity is indeed positively affected by CIN-afflicted cancer cells.

While previous studies show that complete CD47 ablation can favor suppression and even complete rejection of IgG-opsonized B16F10 tumors (Andrechak et al., 2022; Dooling et al., 2022; Hayes et al., 2022; Kamber et al., 2021), inter-experimental variation is high, particularly regarding complete tumor rejection and clearance. Furthermore, how CIN could influence these results is unknown. Therefore, we sought to assess if CIN in B16F10, which thus far has shown to skew macrophages toward an anti-cancer phenotype and away from pro-cancer one, can translate to improved efficacy and consistency in macrophage-oriented therapies. For these subsequent in vivo experiments, we first pre-treated B16F10 cells with either MPS1i or DMSO for 24 h, washed the drug out, and then allowed the cells to recover for an additional 48 h (Fig. 3A). After the recovery period elapsed, we established tumors in mice, with either MPS1i-treated or DMSO-treated B16F10 cells. To further test for CD47-mediated effects, we used either B16F10 CD47 KO or B16F10 sgCtrl, expressing wild-type CD47 (WT) levels. Starting four days post-challenge, mice received either anti-Tyrp1 or mIgG2a isotype control for opsonization.

As expected, all tumors comprised of DMSO-treated B16F10 sgCtrl showed exponential growth and no survivors (Fig. 3B), consistent with previous studies (Dooling et al., 2022; Hayes et al., 2022). These results re-confirm the inhibitory effect that CD47 has on macrophage-mediated immunity (Ingram et al., 2017; Sockolosky et al., 2016; Willingham et al., 2012), suppressing macrophage immune response even with anti-Tyrp1 IgG opsonization. Mice with CIN-afflicted B16F10 sgCtrl tumors ultimately showed exponential growth and no survivors as well, but they did show increased median survival. Furthermore, median survival increased even further when mice were treated with anti-Tyrp1, such that all mice were considered partial responders (survival of 20+ days, one week longer than median survival of tumors comprised of DMSO-treated B16F10 sgCtrl without anti-Tyrp1 treatment). All tumors comprised of DMSO-treated B16F10 CD47 KO showed exponential growth and no survivors (Fig. 3C), regardless of anti-Tyrp1 treatment or not. However, CIN-afflicted B16F10 CD47 KO tumors showed more positive outcomes. Even without anti-Tyrp1, all challenged mice with CIN-afflicted CD47 KO tumors were either cured completely (28%) or considered partial responders. When paired with anti-Tyrp1 treatment, 97% of mice challenged with CIN-afflicted CD47 KO tumors survive.

Long-term survival results show that challenging mice with both MPS1i-treated and DMSO-treated B16F10 sgCtrl failed to yield any survivors, regardless of anti-Tyrp1 opsonization or not (Fig. 3D-i). Similarly, we also failed to generate any survivors among mice challenged with DMSO-treated B16F10 CD47 KO cells, regardless of anti-Tyrp1 opsonization or not (Fig. 3D-ii). This again highlights a challenge in optimizing macrophage-mediated therapies: achieving consistency in long-term therapeutic outcomes. Mice challenged with MPS1i-treated B16F10 CD47 KO cells, however, were able to survive, both without anti-Tyrp1 (28% survival) and with anti-Tyrp1 (97% survival) (Fig. 3D-ii). To determine if the degree of CIN affected survival, we also challenged mice with B16F10 CD47 KO cells treated with varying concentrations of reversine. Regardless of MPS1i concentration, >80% of mice survive when also treated with anti-Tyrp1 (Fig. S6). These results show that, in physiologically relevant microenvironments, early-stage CIN can favor survival when paired with IgG opsonization and CD47 disruption. This further suggests that macrophages are key effector cells in achieving survival against CIN-afflicted tumors, since 97% survival was achieved under conditions of maximal phagocytosis.

Although no mice challenged with CIN-afflicted B16F10 sgCtrl survived, MPS1i increase median survival significantly when paired with anti-Tyrp1 opsonization (Fig. 3D-i). This suggests that macrophages still display some anti-cancer activity, despite the inhibitory effects of CD47, and are important effector cells in final therapeutic outcome. To better support the hypothesis that macrophages are indeed key effector cells in rejecting CIN-afflicted tumors, we established tumors comprised of wild-type (WT) B16F10 in mice. Although WT tumors are generally unaffected by anti-CD47 and anti-Tyrp1 (Dooling et al., 2022; Hayes et al., 2022), we hypothesized that we could eliminate CIN-afflicted WT B16F10 tumors by providing adoptive transfer of marrow to increase macrophage numbers to compensate for the CD47-mediated inhibition of endogenous macrophages. Furthermore, we engineered marrow by priming Fcγ receptors with anti-Tyrp1, initially inhibiting CD47-SIRPα interaction via anti-SIRPα antibody blockade or combining both (Fig. 3E-i). As expected, mice challenged with CIN-afflicted WT tumors and treated with regular, unprimed marrow only had ∼14% survival (Fig. 3E-ii & iii). This result, however, was identical to chromosomally stable WT tumors treated with marrow primed with both anti-SIRPα and anti-Tyrp1. These results suggest increased macrophages numbers do provide some minor benefit. As the marrow engineering becomes more rigorous to maximize phagocytosis, survival improves. 37% of mice challenged with CIN-afflicted WT tumors survived when treated with marrow that was initially engineered with anti-SIRPα. 50% survived with marrow primed with anti-Tyrp1. Lastly, 80% survived with anti-Tyrp1-primed and anti-SIRPα-blocked monocytes (Fig. 3E-ii & iii). Altogether, these results support three conclusions: (1) the increase in survival attributed to anti-SIRPα supports the idea that CD47 modulates therapeutic outcome even during CIN; (2) the increase in survival attributed to anti-Tyrp1 highlights the importance of IgG opsonization; (3) the high success from the combination emphasizes macrophages playing a key role in achieving complete rejection and clearance.

Macrophages and related phagocytic cells make up innate immunity, a first line of defense against pathogens and disease, but they are also responsible for initiating acquired immunity. The activation of the acquired immune response involves two branches of immunity: humoral (mediated by macromolecules such as antibodies) and cell-mediated immunity (involving T cells, for example). We hypothesized that mice that survived challenges with CIN-afflicted tumors would show signs of an acquired immune response due to their high survival rate.

We collected convalescent serum from survivors to quantify de novo anti-cancer IgG antibodies that may have resulted from successful rejection and clearance of CIN-afflicted tumors (Fig. 4A). Sera was collected and then subsequently used in antibody binding testing and Western blotting to confirm emergence of anti-B16F10 antibodies. We first quantified IgG2a and IgG2b titers in convalescent sera, both of which have been previously found to engage mouse macrophage Fcγ receptors (Bruhns, 2012; Nimmerjahn et al., 2010) that are typically required for macrophage-mediated phagocytosis. B16F10 cells, either Tyrp1-expresing or Tyrp1 KO, were incubated with sera (convalescent from survivors or naïve from unchallenged mice) and then counterstained with conjugated antibodies against IgG2a/c and IgG2b (Fig. 4B-i & ii). All mice that survived challenges from CIN-afflicted tumors yielded sera that showed significantly large increases in IgG2a/c binding against both Tyrp1-positive and Tyrp1 KO B16F10 cells. We similarly saw a statistically significant increases in IgG2b binding against both Tyrp1-positive and Tyrp1 KO B16F10 cells using the same sera, although the increases in binding were more variable. Altogether, we see that surviving CIN-afflicted tumors generates IgG-rich sera, like previous studies (Dooling et al., 2022; Hayes et al., 2022). The increases in binding observed against Tyrp1 KO cells also suggest that IgG antibodies target a repertoire of antigens beyond Tyrp1, which we further qualitatively confirmed via Western blotting (Fig. 4B-iii). Convalescent sera were used to immunoblot against B16F10 lysates, revealing many bands at multiple molecular weights and more bands than when immunoblotting with naïve sera, supporting our hypothesis of antigen broadening beyond Tyrp1. Lastly, we also tested IgG2a and IgG2b titers from additional in vivo experiments: survivors from both titrated CIN-afflicted tumors from Fig. S6 and adoptive marrow transfers in Fig. 3E. Similarly, convalescent sera from all these mice show increases in IgG2a/c and IgG2b in both Tyrp1-expressing and Tyrp1 KO B16F10 cells (Fig. S7). These results confirm that regardless of the method used to exploit CIN, induction of anti-cancer IgG can be expected.

To test where these de novo serum antibodies functionally promote macrophage-mediated phagocytosis, we performed conventional 2D phagocytosis assays in which cancer cell suspensions were opsonized with sera (or anti-Tyrp1 or mouse IgG2a isotype as controls) (Fig. 4C-i). Under conditions of CD47 KO, we see that nearly all unpurified convalescent sera increased phagocytosis relative to naïve serum and mIgG2a isotype control. Furthermore, this increase in phagocytosis is identical to that provided by anti-Tyrp1 (both ∼5-fold higher than baseline). We also find that sera continue to promote phagocytosis even in B16F10 CD47/Tyrp1 double KO cells (∼3-fold higher than baseline). Convalescent sera are still able to provide increases in phagocytosis against double KO cells, whereas anti-Tyrp1 expectedly does not drive phagocytosis due to lack of antigen. This again supports the hypothesis of acquired immunity with de novo IgG antibodies that target B16F10 antigens beyond Tyrp1. Upon confirming the functional effect of de novo IgG in the convalescent sera, we then wondered if convalescent serum IgG would be able to suppress tumoroid growth, given that this model better captures both the biophysical microenvironment and proliferative capacity of tumors (Dooling et al., 2022). Indeed, we found that convalescent serum IgG added simultaneously with macrophages to B16F10 CD47 KO tumoroids led to either tumoroid elimination or significantly suppressed growth (Fig. 4C-ii), although the efficacy was less potent as anti-Tyrp1. This suggests that perhaps the polyclonal de novo IgGs here still lack the specificity benefits that accompany a monoclonal antibody such as anti-Tyrp1. Nonetheless, these results demonstrate induction of a generally potent anti-cancer antibody response B16F10 in a CD47 KO context.

We then proceeded to test the function of convalescent serum in vivo by opsonizing B16F10 CD47 KO cells just prior to subcutaneous implantation in naïve mice. For comparison, we also opsonized B16F10 CD47 KO cells with either anti-Tyrp1 (positive control) or mIgG2a control (negative control). We found that both convalescent sera and anti-Tyrp1 suppressed tumor growth by days 11 and 13 relative to mIgG2a control (Fig. 4D-i). Interestingly, we found that convalescent sera showed a trend of suppressing growth more than anti-Tyrp1, although this was not statistically significant. We continued to monitor all mice for long-term survival, and we found that this pre-opsonization with both convalescent sera and anti-Tyrp1 eliminated tumors in challenged mice with near identical cure rates (25% and 22%, respectively) (Fig. 4D-ii). Altogether, we found that the convalescent sera from mice originally challenged with chromosomally unstable tumors has potent anti-cancer effects in vitro and in vivo.

The anti-cancer IgG antibody development in survivors led us to further hypothesize that we should see improved median survival and/or survival rate if surviving mice were re-challenged. We therefore challenged surviving mice with a second injection of either DMSO-treated or B16F10 CD47 KO cells (Fig. 4E). If additional survivors resulted from this experiment, we also intended to undergo a third challenge, akin to a prime-boost strategy for anti-cancer vaccination. It should be highlighted that starting from this second challenge, no mice received anti-Tyrp1. This was done to maximally challenge acquired immunity and to better simulate the possibility of recurrence post-therapy. Age-matched naïve mice receiving their first challenge responded similarly to the younger cohorts (Fig. 3B). Of the previously cured mice, only a single mouse (of 11 total) survived a challenge with DMSO-treated B16F10 cells (Fig. 4F). However, median survival increased (21 days) compared to their naïve counterparts (14 days), supporting the initial hypothesis of prolonged survival. Survivors that were re-challenged again with MPS1i-treated cells, however, showed 100% survival, even in the absence of anti-Tyrp1 (Fig. 4F). Age-matched naïve mice receiving their first challenge of MPS1i-treated cells responded relatively similarly (a single survivor out of 6 total, 17% survival) to the younger cohort (28% survival). This complete success rate against a second challenge of CIN-afflicted B16F10 CD47 KO further supports an acquired immune response, at least against ongoing chromosomally unstable cells.

Mice that failed to reject re-challenge tumors comprised of DMSO-treated B16F10 CD47 KO in Fig. 4F were euthanized and had their tumors harvested to measure their immune cell infiltrate by flow cytometry (Fig. S8). Although these mice did not survive, we found that these previous survivors showed roughly a 2-fold increase in the number of immune cells in their tumors (Fig. 4G-i), a 2-fold increase in the number of CD3+ CD8+ T cells (Fig. 4G-ii), and a near 3-fold increase in the number of F4/80+ macrophages (Fig. 4G-iii) compared to their naïve counterparts. We further found that of the F4/80+ macrophage infiltrate, previous survivors had roughly four times as many MHCII-high macrophages (M1-like, anti-cancer). These immune infiltrate analyses suggest that although the acquired immune response in these previous survivors was still not potent enough to clear chromosomally stable and regularly proliferating B16F10 CD47 KO tumors, it did enhance cell-mediated immunity.

Lastly, we performed a third tumor challenge on second challenge survivors with untreated B16F10 CD47 KO cells (chromosomally stable and regular proliferating). Mice were left untreated post-challenge (no anti-Tyrp1). ∼56% of these mice completely resisted tumor growth. Of the four mice that developed tumors, two had to be euthanized prematurely due to tumor rupture but were indeed showing signs of growth suppression. The last two mice consisted of two long-term partial responders: one whose tumor did not reach terminal burden until 82 days post-challenge and another who experienced stable tumor regression with almost no regrowth. Overall, the 56% survival rate in the third challenge, in the absence of anti-Tyrp1, and the partial responses observed in mice that grew tumors suggest a durable immunological response that results from facing and clearing ongoing chromosomally unstable tumors, at least in the context of CD47 disruption.