Search

Macrophages often pervade solid tumors, but their nearest neighbor organization is understudied and potentially enables key functions such as phagocytosis. Here, we observe dynamic macrophage clusters in tumors under conditions that maximize cancer cell phagocytosis and use reductionist approaches to uncover pathways to cluster formation and roles for tumor-intrusive pseudopodia, which we term 'intrudopodia'. Macrophage clusters form over hours on low- adhesion substrates after M1 polarization with interferons, including T cell-derived cytokines, and yet clusters prove fluid on timescales of minutes. Clusters also sort from M2 macrophages that disperse on the same substrates. M1 macrophages upregulate specific cell-cell adhesion receptors but suppress actomyosin contractility, and while both pathways contribute to cluster formation, decreased cortical tension was predicted to unleash pseudopodia. Macrophage neighbors in tumor spheroids indeed extend intrudopodia between adjacent cancer cell junctions - at least when phagocytosis conditions are maximized, and coordinated intrudopodia help detach and individualize cancer cells for rapid engulfment. Macrophage clusters thereby provide a cooperative advantage for phagocytosis to overcome solid tumor cohesion.

Solid tumors generally exhibit chromosome copy number variation, which is typically caused by chromosomal instability (CIN) in mitosis. The resulting aneuploidy can drive evolution and associates with poor prognosis in various cancer types as well as poor response to T-cell checkpoint blockade in melanoma. Macrophages and the SIRPα-CD47 checkpoint are understudied in such contexts. Here, CIN is induced in poorly immunogenic B16F10 mouse melanoma cells using spindle assembly checkpoint MPS1 inhibitors that generate persistent micronuclei and diverse aneuploidy while skewing macrophages toward a tumoricidal 'M1-like' phenotype based on markers and short-term anti-tumor studies. Mice bearing CIN-afflicted tumors with wild-type CD47 levels succumb similar to controls, but long-term survival is maximized by SIRPα blockade on adoptively transferred myeloid cells plus anti-tumor monoclonal IgG. Such cells are the initiating effector cells, and survivors make de novo anti-cancer IgG that not only promote phagocytosis of CD47-null cells but also suppress tumor growth. CIN does not affect the IgG response, but pairing CIN with maximal macrophage anti-cancer activity increases durable cures that possess a vaccination-like response against recurrence., Competing Interests: BH, MW, HZ, SP, LD, JA, AC, MT, NO, TM, DD No competing interests declared, (© 2023, Hayes et al.)

Chromosomal instability (CIN), a state in which cells undergo mitotic aberrations that generate chromosome copy number variations, generates aneuploidy and is thought to drive cancer evolution. Although associated with poor prognosis and reduced immune response, CIN generates aneuploidy-induced stresses that could be exploited for immunotherapies. In such contexts, macrophages and the CD47-SIRPα checkpoint are understudied. Here, CIN is induced pharmacologically induced in poorly immunogenic B16F10 mouse melanoma cells, generating persistent micronuclei and diverse aneuploidy while skewing macrophages towards an anti-cancer M1-like phenotype, based on RNA-sequencing profiling, surface marker expression and short-term antitumor studies. These results further translate to in vivo efficacy: Mice bearing CIN-afflicted tumors with wild-type CD47 levels survive only slightly longer relative to chromosomally stable controls, but long-term survival is maximized when combining macrophage-stimulating anti-tumor IgG opsonization and some form of disruption of the CD47-SIRPα checkpoint. Survivors make multi-epitope, de novo anti-cancer IgG that promote macrophage-mediated phagocytosis of CD47 knockout B16F10 cells and suppress tumoroids in vitro and growth of tumors in vivo . CIN does not greatly affect the level of the IgG response compared to previous studies but does significantly increase survival. These results highlight an unexpected therapeutic benefit from CIN when paired with maximal macrophage anti-cancer activity: an anti-cancer vaccination-like antibody response that can lead to more durable cures and further potentiate cell-mediated acquired immunity.

In solid tumours, the abundance of macrophages is typically associated with a poor prognosis. However, macrophage clusters in tumour-cell nests have been associated with survival in some tumour types. Here, by using tumour organoids comprising macrophages and cancer cells opsonized via a monoclonal antibody, we show that highly ordered clusters of macrophages cooperatively phagocytose cancer cells to suppress tumour growth. In mice with poorly immunogenic tumours, the systemic delivery of macrophages with signal-regulatory protein alpha (SIRPα) genetically knocked out or else with blockade of the CD47-SIRPα macrophage checkpoint was combined with the monoclonal antibody and subsequently triggered the production of endogenous tumour-opsonizing immunoglobulin G, substantially increased the survival of the animals and helped confer durable protection from tumour re-challenge and metastasis. Maximizing phagocytic potency by increasing macrophage numbers, by tumour-cell opsonization and by disrupting the phagocytic checkpoint CD47-SIRPα may lead to durable anti-tumour responses in solid cancers., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Phagocytic elimination of solid tumors by innate immune cells seems attractive for immunotherapy, particularly because of the possibilities for acquired immunity. However, the approach remains challenging, with blockade of the macrophage checkpoint CD47 working in immunodeficient mice and against highly immunogenic tumors but not in the clinic where tumors are poorly immunogenic. Even when mouse tumors of poorly immunogenic B16F10 melanoma are opsonized to drive engulfment with a suitable monoclonal antibody (mAb), anti-CD47 blockade remains insufficient. Using both in vitro immuno-tumoroids and in vivo mouse models, we show with CRISPR interference (CRISPRi) that a relatively uniform minimum repression of CD47 by 80% is needed for phagocytosis to dominate net growth when combined with an otherwise ineffective mAb (anti-Tyrp1). Heterogeneity enriches for CD47-high cells, but mice that eliminate tumors generate prophagocytic IgGs that increase in titer with CD47 repression and with tumor accumulation of macrophages, although deeper repression does not improve survival. Given well-known limitations of antibody permeation into solid tumors, our studies clarify benchmarks for CD47 disruption that should be more clinically feasible and safer but just as effective as complete ablation. Additionally, safe but ineffective opsonization in human melanoma trials suggests that combinations with deep repression of CD47 could prove effective and initiate durable immunity., (© The Author(s) 2023. Published by Oxford University Press on behalf of National Academy of Sciences.)

The mechanical environment of a cell can have many effects, but whether it impacts the DNA sequence of a cell has remained unexamined. To investigate this, we developed a live-cell method to measure changes in chromosome numbers. We edited constitutive genes with GFP or RFP tags on single alleles and discovered that cells that lose Chromosome reporters (ChReporters) become non-fluorescent. We applied our new tools to confined mitosis and to inhibition of the putative tumor suppressor myosin-II. We quantified compression of mitotic chromatin in vivo and demonstrated that similar compression in vitro resulted in cell death, but also rare and heritable ChReptorter loss. Myosin-II suppression rescued lethal multipolar divisions and maximized ChReporter loss during three-dimensional (3D) compression and two-dimensional (2D) lateral confinement, but not in standard 2D culture. ChReporter loss was associated with chromosome mis-segregation, rather than just the number of divisions, and loss in vitro and in mice was selected against in subsequent 2D cultures. Inhibition of the spindle assembly checkpoint (SAC) caused ChReporter loss in 2D culture, as expected, but not during 3D compression, suggesting a SAC perturbation. Thus, ChReporters enable diverse studies of viable genetic changes, and show that confinement and myosin-II affect DNA sequence and mechano-evolution., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

CD47 on healthy cells, cancer cells, and even engineered particles can inhibit phagocytic clearance by binding SIRPα on macrophages. To mimic and modulate this interaction with peptides that could be used as soluble antagonists or potentially as bioconjugates to surfaces, we made cyclic "nano-Self" peptides based on the key interaction loop of human CD47. Melanoma cells were studied as a standard preclinical cancer model and were antibody-opsonized to adhere to and activate engulfment by primary mouse macrophages. Phagocytosis in the presence of soluble peptides showed cyclic > wildtype > scrambled activity, with the same trend observed with human cells. Opsonized cells that were not engulfed adhered tightly to macrophages, with opposite trends to phagocytosis. Peptide activity is nonetheless higher in human versus mouse assays, consistent with species differences in CD47-SIRPα. Small peptides thus function as soluble antagonists of a major macrophage checkpoint.

The macrophage checkpoint interaction CD47-SIRPα is an emerging target for cancer therapy, but clinical trials of monoclonal anti-CD47 show efficacy only in liquid tumors when combined with tumor-opsonizing IgG. Here, in challenging metastatic solid tumors, CD47 deletion shows no effect on tumor growth unless combined with otherwise ineffective tumor-opsonization, and we likewise show wild-type metastases are suppressed by SIRPα-blocked macrophages plus tumor-opsonization. Lung tumor nodules of syngeneic B16F10 melanoma cells with CD47 deletion show opsonization drives macrophage phagocytosis of B16F10s, consistent with growth versus phagocytosis calculus for exponential suppression of cancer. Wild-type CD47 levels on metastases in lungs of immunocompetent mice and on human metastases in livers of immunodeficient mice show that systemic injection of antibody-engineered macrophages also suppresses growth. Such in vivo functionality can be modulated by particle pre-loading of the macrophages. Thus, even though CD47-SIRPα disruption and tumor-opsonizing IgG are separately ineffective against established metastatic solid tumors, their combination in molecular and cellular therapies prolongs survival.