Dennis E. Discher

Abstract Extracellular matrix often accumulates in and around solid tumors, and such tumors also evolve diverse mutations that drive cancers, confound therapies, and modulate immune interactions. Across cancer types, we observe chromosome number changes associate with collagen-I levels, and our experiments show rare heritable chromosome losses are induced by a stiff 3D matrix around spheroids. Chromosome reporters (ChReporters) reveal losses in as few as ∼0.1% of cells, with a mechanism in spheroids based on distortion of mitotic spindles - which increases with knockdown of the candidate tumor suppressor myosin-II. Chromosomes mis-segregate into micronuclei that increase with matrix stiffness despite suppressed cell division. Drugs that increase micronuclei in 2D and that rely on an unperturbed spindle show no effect in 3D where the spindle is perturbed. Tumors in vivo that are surrounded by stiff collagen likewise show more but varied chromosome loss and slower growth than 2D cultures. High variance of ChReporter-negative colonies further illustrate increased heterogeneity with 3D matrix stiffness and heritable mutations per Luria-Delbruck theory. Physical learning models of evolving chromosome numbers in proliferating cells are developed and fit key statistical trends.Temperature is another physical stressor - as solid tumors tend to be warm - and we show it has similar outcomes as matrix physical properties. Heating is also now part of various therapies as are immune-engineering approaches. We take advantage of Macrophages that often pervade solid tumors where clusters of macrophages are sometimes seen and associate with longer survival of patients. However, clustering mechanisms, responses to stressor above, and impacts on key functions such as phagocytosis remain obscure. Under conditions that maximize cancer cell phagocytosis within cohesive tumors, we uncover pathways that favor dynamic clusters and find a colocalization of tumor-intrusive pseudopodia which we term “intrudopodia.” Cluster formation is favored by M1 macrophages after exposure to interferons and T cell-derived cytokines. M1 macrophages upregulate specific cell-cell adhesion receptors but suppress actomyosin contractility, with both pathways contributing to cluster formation and unleashing pseudopodia. Macrophage neighbors in tumor spheroids indeed coextend intrudopodia between cancer cell junctions—at least when phagocytosis conditions are maximized by checkpoint disruption and other strategies. Intrudopodia from neighbors help detach and individualize cancer cells for rapid engulfment. Cooperative phagocytosis thus overcomes solid tumor cohesion—and might explain why the macrophage clustering factor ITGAL associates with patient survival. Citation Format: Dennis Discher, Markus Sprenger, Joanna Georgiou, Tristan Marchena, Jude Khatib. Physically driven chromosome instabilities spur macrophages to attack cooperatively [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 766.

Heritable genetic changes continually arise in cancer, especially in solid tumors where cells are sometimes compressed. Rare heritable losses of chromosomes in live cells are quantified here with chromosome reporters (ChReporters), which reveal losses only after imposing a threshold level of confinement. Compression to ∼60% of interphase height ruptures few nuclei compared to deeper compression but perturbs mitotic spindles and prolongs pro/metaphase. Chromosome mis-segregation into micronuclei is discovered only after release from modest confinement, but arrest and death predominate. All such effects are phenocopied by nocodazole washout, which generates a "memory" of prolonged mitosis. The effects also differ from the rapid induction of micronuclei by a spindle-assembly checkpoint inhibitor and by a clinical CDK4/6 inhibitor of cell-cycle entry. Single-cell RNA sequencing confirms chromosome loss days after confinement and reveals dysregulation of chromosome-segregation pathways. Chromosome losses as mitotic memories of confinement ultimately address knowledge gaps in mechanobiology and cancer evolution.

SUMMARY Heritable genetic changes continually arise in cancer, especially in solid tumors where cells sometimes appear compressed. Rare heritable losses of chromosomes in live cells are quantified here with chromosome reporters (ChReporters) that reveal similar levels of loss after imposing a threshold level of confinement. Compression to ∼60% of interphase height ruptures few nuclei compared to deeper compression but perturbs mitotic spindles and prolongs pro/metaphase. Chromosome mis-segregation into micronuclei is discovered only after release from modest confinement, but arrest and death predominate. All such effects are phenocopied by Nocodazole washout that generates a ‘memory’ of prolonged mitosis, and effects differ from the rapid induction of micronuclei by a spindle assembly checkpoint inhibitor and by a clinical CDK4/6-inhibitor of cell cycle entry. Single-cell-RNA-sequencing confirms chromosome loss days after confinement and reveals persistence of chromosome segregation pathways. Chromosome losses as mitotic memories of confinement ultimately address knowledge gaps in mechanobiology and cancer evolution.

As with many cell types, macrophages are sometimes filled with micron-sized lipid droplets (LD's), but effects on phagocytosis of other cells, particulates, and microbes remain unclear. Here, we show that LDs restructure the cytoskeleton but remain round, consistent with a high interfacial tension; functionally, LD's impair actomyosin-driven uptake, which proves independent of target size. Engulfment of targets starts at the apical surface, but LD's displace apical actomyosin to the basal cortex. Partial rescue occurs tissue-relevant compressive stresses which activate actomyosin. Macrophages that are densely filled with LD's or pre-engulfed rigid beads likewise activate actomyosin, which again rescues phagocytosis relative to sparsely loaded cells. As further evidence of LD rigidity, both LD's and rigid beads impede macrophage migration through small pores, and LD's pressed into a nucleus cause rapid focal rupture independent of actin. LD rigidity thus disrupts cytoskeleton organization and nucleus integrity, suppressing motility processes unless actomyosin is activated by cell compression or stretching.

Lipid nanoparticles (LNPs) have emerged as the dominant platform for RNA delivery, but they induce severe inflammation. Here we show that LNPs’ hallmark feature, endosomal escape, which is necessary for RNA expression, also triggers inflammation by causing endosomal membrane damage. Large, irreparable, endosomal holes are recognized by cytosolic proteins called galectins, which regulate downstream inflammation. We find that inhibition of galectins abrogates LNP-associated inflammation, both in vitro and in vivo. Moreover, we show that a unique class of ionizable lipids can create smaller endosomal holes, reparable by the endosomal sorting complex required for transport (ESCRT) pathway. Such lipids can produce high expression from cargo messenger RNA with minimal inflammation. Finally, we show that both galectin inhibition or ESCRT-recruiting ionizable lipids allow for treatment of highly inflammatory disease models by therapeutic mRNAs. These strategies should lead to safer non-inflammatory LNPs that can be generally used to treat inflammatory diseases. Preventing endosomal damage sensing or using lipids that create reparable endosomal holes reduces inflammation caused by RNA-lipid nanoparticles while enabling high RNA expression.

Macrophages often pervade solid tumors, and clusters of macrophages sometimes associate with longer survival of patients. However, clustering mechanisms and impacts on key functions such as phagocytosis remain obscure. Here, under conditions that maximize cancer cell phagocytosis within cohesive tumors, we uncover pathways that favor dynamic clusters and find a colocalization of tumor-intrusive pseudopodia which we term “intrudopodia.” Cluster formation over hours on low-adhesion substrates occurs after macrophage induction to a state colloquially referred to as M1 after exposure to interferons and T cell–derived cytokines. Clusters prove fluid on timescales of minutes and also sort from interleukin-4-treated, so-called M2 macrophages that tend to disperse. M1 macrophages upregulate specific cell–cell adhesion receptors but suppress actomyosin contractility, with both pathways contributing to cluster formation. Decreased cortical tension was not only reflected in a low level of nuclear lamin-A that downregulates cytoskeletal targets of serum response factor and tends to soften the nucleus but was also predicted to unleash pseudopodia. Macrophage neighbors in tumor spheroids indeed coextend intrudopodia between cancer cell junctions—at least when phagocytosis conditions are maximized. Intrudopodia from neighbors help detach and individualize cancer cells for rapid engulfment. Juxtaposition of a macrophage cluster with tumor cell nests defines a broad interface that minimizes cancer cell nearest neighbor interactions and maximizes coordination of macrophage intrudopodia. Cooperative phagocytosis thus overcomes solid tumor cohesion—and might explain why the macrophage clustering factor ITGAL associates with patient survival.

Abstract Sequencing of many types of tumors shows that solid tumors are far more mutated than liquid tumors and soft tumors such as brain tumors, but it is unclear whether the differences are caused by induction of more mutations or subsequent selective pressures. Our pan-cancer analyses of clinical data shows increased chromosomal changes associate with collagen-I and further shows that genetic variance between patients also increases with the mean for a given tumor type. Using a 3D hydrogel-based model with tunable stiffness and of cancer spheroids, we discover that matrix stiffness increases mitotic aberrations including micronuclei and also increases chromosome loss as detected using live cell chromosome reporters (ChReporters). However, stiffness suppresses growth and size variation, which indicates more mistakes per division. Our approach is >10-fold more sensitive than sequencing approaches, and we quantify colonies of ChReporter-negative cells within cancer spheroids. Our results are consistent with Luria-Delbruck’s theory of heritable genetic changes which predicts inter-spheroid variance greatly exceeds Poisson statistics. Knockdown of myosin-II, a well-known mechanosensor, increases chromosomal loss and variance without affecting spheroid growth, consistent with a tumor suppressor role. Various drug strategies provide deeper insight into mechanisms, and use of clinically deployed CDK4/6-inhibitor reveals this drug on its own drives micronuclei formation and genetic variation. Citation Format: Dennis E. Discher, Markus Sprenger. Solid tumor microenvironments cause more mutations than liquid or soft tumor microenvironments -- even before selection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2669.

Abstract Macrophages often pervade solid tumors, but observations that macrophage clusters might associate with patient survival have remained largely unexplored. We observe dynamic macrophage clusters in tumors under conditions that maximize cancer cell phagocytosis, and our reductionist approaches to cluster formation reveal pathways and roles for tumor-intrusive pseudopodia. Aggregates form over hours on low-adhesion substrates after ‘M1’ polarization of macrophages with interferons, including Tcell-derived cytokines, and yet clusters prove fluid on timescales of minutes. Clusters also sort from M2 macrophages which are induced by an interleukin and that disperse on the same substrates. M1’s upregulate 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 intrusive pseudopodia or ‘intrudopodia’ in 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. Citation Format: Dennis E. Discher, Lawrence Dooling. Clustered macrophages cooperate to eliminate tumors via coordinated intrudopodia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7465.

Abstract Solid tumors often have an abundance of collagen-I that stiffens the tissue, and they are invariably driven by mutations that include chromosome losses and gains. These observations are linked here by showing that 3D matrix stiffness induces heritable changes to a cell ’ s DNA. We use live-cell chromosome reporters (ChReporters) and hydrogels of tunable stiffness to show mitotic compression, micronuclei counts, ChReporter losses and heterogeneity all increase as functions of stiffness. Increased mistakes occur despite suppressed cell division in stiff matrix and minimal size variation between spheroids. Colonies of ChReporter-negative cells within cancer spheroids align with Luria-Delbruck ’ s seminal theory for heritable mutations, which predicts inter-spheroid variances that exceed Poisson statistics. Suppression of the contractility motor Myosin-II also increases chromosome loss in 3D but not 2D and does not affect spheroid growth – thus clarifying Myosin-II ’ s putative role as a tumor suppressor. Consistent with experiments, pan-cancer analyses of clinical data associates chromosome losses and gains with collagen-I levels and genetic variation. Stiff extracellular matrix thus drives mechano-evolution of solid tumors as a Darwin-Lamarck process with heterogeneity that complicates therapy.

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.

Chromosomal instability induced in solid tumors can combine with macrophages that are made highly phagocytic to thereby initiate elimination of and immunity against poorly immunogenic tumors in immunocompetent mice.

Abstract Lipid nanoparticles (LNPs) have emerged as the dominant platform for RNA delivery, based on their success in the COVID-19 vaccines and late-stage clinical studies in other indications. However, we and others have shown that LNPs induce severe inflammation, and massively aggravate pre-existing inflammation. Here, using structure-function screening of lipids and analyses of signaling pathways, we elucidate the mechanisms of LNP-associated inflammation and demonstrate solutions. We show that LNPs’ hallmark feature, endosomal escape, which is necessary for RNA expression, also directly triggers inflammation by causing endosomal membrane damage. Large, irreparable, endosomal holes are recognized by cytosolic proteins called galectins, which bind to sugars on the inner endosomal membrane and then regulate downstream inflammation. We find that inhibition of galectins abrogates LNP-associated inflammation, both in vitro and in vivo . We show that rapidly biodegradable ionizable lipids can preferentially create endosomal holes that are smaller in size and reparable by the endosomal sorting complex required for transport (ESCRT) pathway. Ionizable lipids producing such ESCRT-recruiting endosomal holes can produce high expression from cargo mRNA with minimal inflammation. Finally, we show that both routes to non-inflammatory LNPs, either galectin inhibition or ESCRT-recruiting ionizable lipids, are compatible with therapeutic mRNAs that ameliorate inflammation in disease models. LNPs without galectin inhibition or biodegradable ionizable lipids lead to severe exacerbation of inflammation in these models. In summary, endosomal escape induces endosomal membrane damage that can lead to inflammation. However, the inflammation can be controlled by inhibiting galectins (large hole detectors) or by using biodegradable lipids, which create smaller holes that are reparable by the ESCRT pathway. These strategies should lead to generally safer LNPs that can be used to treat inflammatory diseases.

Abstract Solid tumors generally exhibit chromosome copy number variation caused by chromosomal instability (CIN) in mitosis, and the resulting aneuploidy associates with poor prognosis in various cancer types and poor Tcell checkpoint blockade response in melanoma. In such contexts, however, macrophages and the SIRPa-CD47 checkpoint are understudied. Here, CIN is induced pharmacologically in poorly immunogenic B16F10 mouse melanoma cells, generating micronuclei plus diverse aneuploidy and skewing macrophages towards an anti-cancer phenotype based on markers and short-term tumor studies. Mice bearing CIN-afflicted tumors with wild-type CD47 levels survive only slightly longer compared to chromosomally stable controls, but long-term survival can be maximized when anti-tumor IgG opsonization is combined with adoptive transfer of macrophages with SIRPa blockade or with CD47 knockout of the B16F10. Multi-epitope, de novo anti-cancer IgG in survivors promote phagocytosis of CD47 knockout B16F10 cells by macrophages and suppress tumoroids in vitro and growth of tumors in vivo. An unexpected benefit of pairing CIN with maximal macrophage anti-cancer activity is thus an anti-cancer vaccination-like response that can lead to durable cures and potentiate cell-mediated acquired immunity. Citation Format: Dennis E. Discher. Chromosomal instability leads to durable tumor suppression upon macrophage-checkpoint disruption, with induction of anti-tumor IgG [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 417.

Abstract 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.

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.

Chromosome numbers often change dynamically in tumors and cultured cells, which complicates therapy as well as understanding genotype-mechanotype relationships. Here we use a live-cell “ChReporter” method to identify cells with a single chromosomal loss in efforts to better understand differences in cell shape, motility, and growth. We focus on a standard cancer line and first show clonal populations that retain the ChReporter exhibit large differences in cell and nuclear morphology as well as motility. Phenotype metrics follow simple rules, including migratory persistence scaling with speed, and cytoskeletal differences are evident from drug responses, imaging, and single-cell RNA sequencing. However, mechanotype–genotype relationships between fluorescent ChReporter-positive clones proved complex and motivated comparisons of clones that differ only in loss or retention of a Chromosome-5 ChReporter. When lost, fluorescence-null cells show low expression of Chromosome-5 genes, including a key tumor suppressor APC that regulates microtubules and proliferation. Colonies are compact, nuclei are rounded, and cells proliferate more, with drug results implicating APC, and patient survival data indicating an association in multiple tumor-types. Visual identification of genotype with ChReporters can thus help clarify mechanotype and mechano-evolution.

Abstract Polymer network properties such as stiffness often exhibit characteristic power laws in polymer density and other parameters. However, it remains unclear whether diverse animal tissues, composed of many distinct polymers, exhibit such scaling. Here, we examined many diverse tissues from adult mouse and embryonic chick to determine if stiffness ( E tissue ) follows a power law in relation to the most abundant animal protein, Collagen-I, even with molecular perturbations. We quantified fibrillar collagen in intact tissue by second harmonic generation (SHG) imaging and from tissue extracts by mass spectrometry (MS), and collagenase-mediated decreases were also tracked. Pan-tissue power laws for tissue stiffness versus Collagen-I levels measured by SHG or MS exhibit sub-linear scaling that aligns with results from cellularized gels of Collagen-I but not acellular gels. Inhibition of cellular myosin-II based contraction fits the scaling, and combination with inhibitors of matrix metalloproteinases (MMPs) show collagenase activity is strain - not stress- suppressed in tissues, consistent with past studies of gels and fibrils. Beating embryonic hearts and tendons, which differ in both collagen levels and stiffness by >1000-fold, similarly suppressed collagenases at physiological strains of ∼5%, with fiber-orientation regulating degradation. Scaling of E tissue based on ‘use-it-or-lose-it’ kinetics provides insight into scaling of organ size, microgravity effects, and regeneration processes while suggesting contractility-driven therapeutics.

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.

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.

Abstract 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. Macrophages, particularly, have been understudied in the CIN context. Here, through MPS1 inhibition-induced CIN in poorly immunogenic B16F10 mouse melanoma, we find that CIN- afflicted cancer cells skew macrophages towards an anti-cancer phenotype while also pushing them away from a pro-cancer one. We confirm these findings via RNA-sequencing, protein expression, and short-term tumor studies. These results further translate to in vivo efficacy in suppressing tumor growth: Mice can survive challenges of CIN-afflicted tumors. Long-term survival, however, is dependent on CD47 expression and IgG opsonization. Mice bearing CIN- afflicted tumors with wild-type CD47 levels see prolonged survival compared to their chromosomally stable counterparts, but all succumb. Mice bearing CIN-afflicted CD47 knockout tumors, however, show 28% long-term survival. When CD47 knockout was further paired with IgG opsonization, survival rate increased to 97%. Successful rejection and clearance of CIN- afflicted tumors induced de novo anti-cancer IgG antibodies that were multi-epitope and functionally promoted macrophage-mediated phagocytosis. These de novo IgG antibodies could also suppress in vitro tumoroid and in vivo tumor growth in a CD47 knockout context. 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 durable cures and further potentiate cell-mediated acquired immunity.

Chromosome gains or losses often lead to copy number variations (CNV) and loss of heterozygosity (LOH). Both quantities are low in hematologic “liquid” cancers versus solid tumors in data of The Cancer Genome Atlas (TCGA) that also shows the fraction of a genome affected by LOH is ~ one-half of that with CNV. Suspension cultures of p53-null THP-1 leukemia-derived cells conform to these trends, despite novel evidence here of genetic heterogeneity and transiently elevated CNV after perturbation. Single-cell DNAseq indeed reveals at least 8 distinct THP-1 aneuploid clones with further intra-clonal variation, suggesting ongoing genetic evolution. Importantly, acute inhibition of the mitotic spindle assembly checkpoint (SAC) produces CNV levels that are typical of high-CNV solid tumors, with subsequent cell death and down-selection to novel CNV. Pan-cancer analyses show p53 inactivation associates with aneuploidy, but leukemias exhibit a weaker trend even though p53 inactivation correlates with poor survival. Overexpression of p53 in THP-1 does not rescue established aneuploidy or LOH but slightly increases cell death under oxidative or confinement stress, and triggers p21, a key p53 target, but without affecting net growth. Our results suggest that factors other than p53 exert stronger pressures against aneuploidy in liquid cancers, and identifying such CNV suppressors could be useful across liquid and solid tumor types.

The nucleus in many cell types is a stiff organelle, but fat-filled lipid droplets (FDs) in cytoplasm are seen to indent and displace the nucleus. FDs are phase-separated liquids with a poorly understood interfacial tension γ that determines how FDs interact with other organelles. Here, micron-sized FDs remain spherical as they indent peri-nuclear actomyosin and the nucleus, while causing local dilution of Lamin-B1 independent of Lamin-A,C and sometimes triggering nuclear rupture. Focal accumulation of the cytosolic DNA sensor cGAS at the rupture site is accompanied by sustained mislocalization of DNA repair factors to cytoplasm, increased DNA damage, and delayed cell cycle. Macrophages show FDs and engulfed rigid beads cause similar indentation dilution. Spherical shapes of small FDs indicate a high γ, which we measure for FDs mechanically isolated from fresh adipose tissue as ∼40 mN/m. This value is far higher than that of protein condensates, but typical of oils in water and sufficiently rigid to perturb cell structures including nuclei.

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.

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. Durable anti-tumour responses can be triggered by maximizing the cooperative phagocytic potency of macrophages through the disruption of the CD47–SIRPα macrophage checkpoint and by delivering a tumour-opsonizing monoclonal antibody.

Abstract 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.

Abstract Macrophages are abundant in many solid tumors and are potential effector cells for monoclonal antibody-based immunotherapies because they can in principle phagocytose IgG-opsonized cancer cells. However, it remains unknown whether phagocytic macrophages can overcome the cohesive forces between cancer cells in solid tumors. We show that maximum phagocytosis of B16 melanoma tumors and ‘tumoroids’ requires IgG-opsonization and very deep suppression of the macrophage checkpoint ligand CD47. Under these conditions, phagocytic macrophages cluster or ‘phase separate’, and the elimination exhibits clear cooperativity with respect to macrophage number and phagocytic activity. In vitro disruption of phagocytic clusters specifically implicates Mg2+ and macrophage integrins. Cytokine treatments and other inhibitors further implicate roles for integrins and phagocytic surface receptors. Although tumor-associated macrophages correlate with negative prognoses in many cancers, our findings are among the first to reveal advantages of high macrophage numbers, macrophage clusters, and resulting cooperative effects when combined with opsonizing antibodies and CD47 disruption. Lastly, the challenging B16 tumors are durably eliminated by the approach and induce de novo IgG's that also drive anti-cancer phagocytosis. Citation Format: Dennis E. Discher. Phagocytosis of solid tumors favors macrophage clusters and cooperativity in tumor elimination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1346.

ABSTRACT Matrix around cells exerts many effects, some of which depend on the putative tumor suppressor Myosin-II, but whether such factors affect DNA sequences in a cell remains unclear. Here, live-cell monitoring of changes to chromosome copy numbers is developed and studied under diverse perturbations, including Myosin-II inhibition in confined mitosis. Squeezing of mitotic cells is seen in vivo and kills in vitro , but stem cells and cancer cells that survive show heritable loss of mono-allelic GFP/RFP-tagged constitutive genes that function as novel Chromosome-reporters (ChReporters). Myosin-II suppression increases such loss in 3D & 2D confinement but not in standard 2D, with “lethal” multipolar divisions proving myosin-dependent. Viable chromosome loss after confined mitosis associates more with mis-segregation than with multipolars or division number. Solid human tumors and teratomas in mice also show ChReporter loss and a confinement-signature of Myosin-II suppression, although losses are selected against in 2D culture. Heritable loss in rigid-confinement also appears independent of a spindle assembly checkpoint that functions in 2D. Confinement and myosin-II thus regulate pathways of heritable mechanogenetic change.