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.

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. 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.

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.

Macrophages are potential effector cells in immunotherapy against solid tumors, but any phagocytosis requires macrophage-cancer cell interactions to out-compete cohesive interactions between tumor cells (e.g., cadherins). It also requires overcoming a "don't eat me" signal from CD47. We engineered conditionally immortalized macrophages (CIMs) with deletion of the CD47-binding inhibitory receptor SIRPα, and we first show such SIRPαKO-CIMs readily engulf added suspensions of IgG-opsonized melanoma cells in a standard 2D assay.

Cancer progression is highly associated with chromosome instability arising from mitotic errors, but the underlying mechanisms are difficult to explain simply by mutations in oncogenes or tumor suppressor genes. Physical constraints, such as extracellular constraints by a stiff tumor microenvironment, can also cause mitotic errors independent of genetic defects. Hepatic cells provide a natural system to study the impacts of physical constraints on mitosis because they deposit intracellular lipid droplets and extracellular matrices during cancer progression.

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.

Differential adhesion and contractility drive cell sorting in diverse systems such as embryonic germ layer cells and cancer cell mixtures, but similar behavior among immune cells is underexplored despite major alterations in adhesive protein repertoires and cytoskeleton when activated. We observed a strong tendency of macrophages to aggregate and segregate from melanoma cells in cohesive tumoroids, but only under pro-phagocytic conditions in which macrophage Fc receptors were activated by IgG opsonization of the melanoma cells and the "don't eat me" CD47-SIPRα checkpoint was inhibited.

Pancreatic ductal adenocarcinoma (PDAC) is reported to be the third highest cause of cancer-related deaths in the United States. PDAC is known for its high proportion of stroma, which accounts for 90% of the tumor mass. The stroma is made up of extracellular matrix (ECM) and nonmalignant cells such as inflammatory cells, cancer-associated fibroblasts, and lymphatic and blood vessels. Here, we decoupled the effects of the ECM on PDAC cell lines by culturing cells on surfaces coated with different ECM proteins. Our data show that the primary tumor-derived cell lines have different morphology depending on the ECM proteins on which they are cultured, while metastatic lesion-derived PDAC lines’ morphology does not change with respect to the different ECM proteins. Similarly, ECM proteins modulate the proliferation rate and the gemcitabine sensitivity of the primary tumor PDAC cell lines, but not the metastatic PDAC lines. Lastly, transcriptomics analysis of the primary tumor PDAC cells cultured on different ECM proteins reveals the regulation of various pathways, such as cell cycle, cell-adhesion molecules, and focal adhesion, including the regulation of several integrin genes that are essential for ECM recognition.

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 nucleus in many cell types is a stiff organelle, and yet fat-filled lipid droplets (FD's) in the cytoplasm can indent and displace the nucleus. FD's are phase-separated liquids with a poorly understood interfacial tension that determines how FD's interact with other organelles. Here, micron-sized FD's remain spherical while deforming both the nuclear lamina and peri-nuclear actomyosin. Nuclear lamins are intermediate filaments with a persistence length in the range of ∼0.2-1.5 μm with Lamin-B1 directly attached to the inner nuclear membrane via farnesylation groups that are lacking in mature lamin-A,C. Such rigidity can compromise the attachment of Lamin-B1 to the membrane in cases of high Gaussian curvature as seen through local dilution of Lamin-B1 at sites of indentation which is independent of Lamin-A,C. A single filament model of curvature-driven membrane detachment fits to Lamin-B1's relative density with the association energy being the key parameter to fit the experimental data. Lamin-B1 depletion triggers rupture as indicated by persistent, local accumulation of cytosolic DNA sensor cGAS at the nuclear boundary. FD-nucleus interactions also initiate rapid mis-localization of the DNA repair factor KU80, confirming nuclear rupture, and associate with heightened DNA damage and perturbed cell cycle. Similar results are evident in FD-laden cells after constricted 3D-migration, which is impeded by FD's. Spherical shapes of small FD's are consistent with a high interfacial tension that we measure for FD's mechanically isolated from fresh adipose tissue as ∼40 mN/m - which is far higher than other liquid condensates, but typical of oils in water and sufficiently rigid to disrupt cell structures.

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.

ABSTRACTMatrix 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 seenin vivoand killsin 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.

A cell's mechanical environment can have many effects, but whether it impacts a cell's DNA sequence has remained unclear. To investigate this, we developed a live-cell method to measure changes in chromosome numbers. We edited constitutive genes with GFP/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 in 3D-compression and 2D-confinement, but not in standard 2D-culture. ChReporter loss 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, as expected, but not in 3D-compression, suggesting a SAC perturbation. Thus, confinement and myosin-II affect DNA sequence and mechano-evolution, and ChReporters enable diverse studies of viable genetic changes.

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.

Macrophages are abundant in solid tumours and typically associate with poor prognosis, but macrophage clusters in tumour nests have also been reported as beneficial even though dispersed macrophages would have more contacts with cancer cells. Here, by maximizing both phagocytic activity and macrophage numbers, we discover cooperative phagocytosis by low entropy clusters in rapidly growing engineered immuno-tumouroids. The results fit the calculus of proliferation-versus-engulfment, and rheological measurements and molecular perturbations provide a basis for understanding phagocytic disruption of a tumour’s cohesive forces in soft cellular phases. The perturbations underscore the utility of suppressing a macrophage checkpoint in combination with an otherwise ineffective tumour-opsonizing monoclonal antibody, and the approach translates in vivo to tumour elimination that durably protects mice from re-challenge and metastasis. Adoptive transfer of engineered macrophages increases the fraction of mice that eliminate tumours and potentially overcomes checkpoint blockade challenges in solid tumours like insufficient permeation of blocking antibodies and on-target, off-tumour binding. Finally, anti-cancer IgG induced in vivo are tumour-specific but multi-epitope and contribute to a phagocytic feedback that drives macrophage clustering in vitro . Given that solid tumours remain challenging for immunotherapies, durable anti-tumour responses here illustrate unexpected advantages in maximizing net phagocytic activity.

Data for the Cancers (2022) publication: CD47-SIRPα checkpoint disruption in metastases requires tumor-targeting antibody for molecular and engineered macrophage therapies

Fibrillar proteins are principal components of extracellular matrix (ECM) that confer mechanical properties to tissues. Fibrosis can result from wound repair in nearly every tissue in adults, and it associates with increased ECM density and crosslinking as well as increased tissue stiffness. Such fibrotic tissues are a major biomedical challenge, and an emerging view posits that the altered mechanical environment supports both synthetic and contractile myofibroblasts in a state of persistent activation. Here, we review the matrisome in several fibrotic diseases, as well as normal tissues, with a focus on physicochemical properties. Stiffness generally increases with the abundance of fibrillar collagens, the major constituent of ECM, with similar mathematical trends for fibrosis as well as adult tissues from soft brain to stiff bone and heart development. Changes in expression of other core matrisome and matrisome-associated proteins or proteoglycans contribute to tissue stiffening in fibrosis by organizing collagen, crosslinking ECM, and facilitating adhesion of myofibroblasts. Understanding how ECM composition and mechanics coevolve during fibrosis can lead to better models and help with antifibrotic therapies.