Chromosomal instability induced in cancer can enhance macrophage-initiated immune responses that include anti-tumor IgG.

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.

Chromosomal instability can favor macrophage-mediated immune response and induce a broad, vaccination-like anti-tumor IgG response.

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.

Titrating CD47 by mismatch CRISPR-interference reveals incomplete repression can eliminate IgG-opsonized tumors but limits induction of antitumor IgG.

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.

Confinement plus myosin-II suppression maximizes heritable loss of chromosomes, as revealed by live-cell ChReporters.

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.

Cooperative phagocytosis of solid tumours by macrophages triggers durable anti-tumour responses.

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.

CD47-SIRPα Checkpoint Disruption in Metastases Requires Tumor-Targeting Antibody for Molecular and Engineered Macrophage Therapies.

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.

Human CD47-Derived Cyclic Peptides Enhance Engulfment of mAb-Targeted Melanoma by Primary Macrophages.

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.

Multivalent, Soluble Nano-Self Peptides Increase Phagocytosis of Antibody-Opsonized Targets while Suppressing "Self" Signaling.

Macrophages engulf "foreign" cells and particles, but phagocytosis of healthy cells and cancer cells is inhibited by expression of the ubiquitous membrane protein CD47 which binds SIRPα on macrophages to signal "self". Motivated by some clinical efficacy of anti-CD47 against liquid tumors and based on past studies of CD47-derived polypeptides on particles that inhibited phagocytosis of the particles, here we design soluble, multivalent peptides to bind and block SIRPα. Bivalent and tetravalent nano-Self peptides prove more potent (Keff ∼ 10 nM) than monovalent 8-mers as agonists for phagocytosis of antibody opsonized cells, including cancer cells. Multivalent peptides also outcompete soluble CD47 binding to human macrophages, consistent with SIRPα binding, and the peptides suppress phosphotyrosine in macrophages, consistent with inhibition of SIRPα's "self" signaling. Peptides exhibit minimal folding, but functionality suggests an induced fit into SIRPα's binding pocket. Pre-clinical studies in mice indicate safety, with no anemia that typifies clinical infusions of anti-CD47. Multivalent nano-Self peptides thus constitute an alternative approach to promoting phagocytosis of "self", including cancer cells targeted clinically.

Macrophage checkpoint blockade: results from initial clinical trials, binding analyses, and CD47-SIRPα structure-function.

The macrophage checkpoint is an anti-phagocytic interaction between signal regulatory protein alpha (SIRPα) on a macrophage and CD47 on all types of cells - ranging from blood cells to cancer cells. This interaction has emerged over the last decade as a potential co-target in cancer when combined with other anti-cancer agents, with antibodies against CD47 and SIRPα currently in preclinical and clinical development for a variety of hematological and solid malignancies. Monotherapy with CD47 blockade is ineffective in human clinical trials against many tumor types tested to date, except for rare cutaneous and peripheral lymphomas. In contrast, pre-clinical results show efficacy in multiple syngeneic mouse models of cancer, suggesting that many of these tumor models are more immunogenic and likely artificial compared to human tumors. However, combination therapies in humans of anti-CD47 with agents such as the anti-tumor antibody rituximab do show efficacy against liquid tumors (lymphoma) and are promising. Here, we review such trials as well as key interaction and structural features of CD47-SIRPα.

The macrophage checkpoint CD47 : SIRPα for recognition of 'self' cells: from clinical trials of blocking antibodies to mechanobiological fundamentals.

Immunotherapies against some solid tumour types have recently shown unprecedented, durable cures in the clinic, and the most successful thus far involves blocking inhibitory receptor 'checkpoints' on T cells. A similar approach with macrophages is emerging by blocking the ubiquitously expressed 'marker of self' CD47 from binding the inhibitory receptor SIRPα on macrophages. Here, we first summarize available information on the safety and efficacy of CD47 blockade, which raises some safety concerns with the clearance of 'self' cells but also suggests some success against haematological (liquid) and solid cancers. Checkpoint blockade generally benefits from parallel activation of the immune cell, which can occur for macrophages in multiple ways, such as by combination with a second, tumour-opsonizing antibody and perhaps also via rigidity sensing. Cytoskeletal forces in phagocytosis and inhibitory 'self'-signalling are thus reviewed together with macrophage mechanosensing, which extends to regulating levels of SIRPα and the nuclear protein lamin A, which affects phenotype and cell trafficking. Considerations of such physical factors in cancer and the immune system can inform the design of new immunotherapies and help to refine existing therapies to improve safety and efficacy. This article is part of a discussion meeting issue 'Forces in cancer: interdisciplinary approaches in tumour mechanobiology'.

Multi-day rhythms modulate seizure risk in epilepsy.

Epilepsy is defined by the seemingly random occurrence of spontaneous seizures. The ability to anticipate seizures would enable preventative treatment strategies. A central but unresolved question concerns the relationship of seizure timing to fluctuating rates of interictal epileptiform discharges (here termed interictal epileptiform activity, IEA), a marker of brain irritability observed between seizures by electroencephalography (EEG). Here, in 37 subjects with an implanted brain stimulation device that detects IEA and seizures over years, we find that IEA oscillates with circadian and subject-specific multidien (multi-day) periods. Multidien periodicities, most commonly 20-30 days in duration, are robust and relatively stable for up to 10 years in men and women. We show that seizures occur preferentially during the rising phase of multidien IEA rhythms. Combining phase information from circadian and multidien IEA rhythms provides a novel biomarker for determining relative seizure risk with a large effect size in most subjects.

Anionic Polymer and Quantum Dot Excipients to Facilitate siRNA Release and Self-Reporting of Disassembly in Stimuli-Responsive Nanocarrier Formulations.

The incorporation of anionic excipients into polyplexes is a promising strategy for modulating siRNA binding versus release and integrating diagnostic capabilities; however, specific design criteria and structure-function relationships are needed to facilitate the development of nanocarrier-based theranostics. Herein, we incorporated poly(acrylic acid) (PAA) and quantum dot (QD) excipients into photolabile siRNA polyplexes to increase gene silencing efficiencies by up to 100% and enable self-reporting of nanocarrier disassembly. Our systematic approach identified the functional relationships between gene silencing and key parameters such as excipient loading fractions and molecular weights that facilitated the establishment of design rules for optimization of nanocarrier efficacy. For example, we found that PAA molecular weights ∼10-20× greater than that of the coencapsulated siRNA exhibited the most efficient release and silencing. Furthermore, siRNA release assays and RNAi modeling allowed us to generate a PAA "heat map" that predicted gene silencing a priori as a function of PAA molecular weight and loading fraction. QDs further promoted selective siRNA release and provided visual as well as Förster resonance energy transfer (FRET)-based monitoring of the dynamic changes in nanostructure in situ. Moreover, even with the addition of anionic components, our formulations exhibited substantially improved stability and shelf life relative to typical formulations, with complete stability after a week of storage and full activity in the presence of serum. Taken together, this study enabled synergistic improvements in siRNA release and diagnostic capabilities, along with the development of mechanistic insights that are critical for advancing the translation of nucleic acid theranostics into the clinic.