Biology subjects

Dooling, L. J.

Publications and source records attributed to Dooling, L. J..

7 recordsLinked to original sources

bioRxiv2024

Clustered macrophages cooperate to eliminate tumors via coordinated intrudopodia

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.

bioengineering↗

bioRxiv2023

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-SIRP 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-SIRP 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.

cancer biology↗

bioRxiv2022

Differential roles of extracellular matrix proteins on primary and metastatic pancreatic cancer cells

Pancreatic cancer 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 non-malignant 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.

cancer biology↗

bioRxiv2022

Cooperative phagocytosis underlies macrophage immunotherapy of solid tumours and initiates a broad anti-tumour IgG response

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 tumours 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.

bioengineering↗

bioRxiv2021

Gaussian curvature dilutes the nuclear lamina, favoring nuclear rupture, especially at high strain rate

Nuclear rupture has long been associated with deficits or defects in lamins, with recent results also indicating a role for actomyosin stress, but key physical determinants of rupture remain unclear. Here, lamin-B stably interacts with the nuclear membrane at sites of low Gaussian curvature yet dilutes at high-curvature to favor rupture, whereas lamin-A depletes similarly but only at high strain-rates. Live cell imaging of lamin-B1 gene-edited cancer cells is complemented by fixed-cell imaging of ruptured nuclei in: iPS-derived cells from progeria patients, cells within beating chick embryo hearts, and cancer cells that develop multiple ruptures in migrating through small pores. Dilution and curvature-dependent rupture fit a parsimonious model of a stiff filament that detaches from a curved surface, suggesting an elastic-type response of lamin-B, but rupture is also modestly suppressed by inhibiting myosin-II and by hypotonic stress, which slow the strain rates. Lamin-A dilution and nuclear rupture likelihood indeed increase above a threshold rate of pulling into small pipettes, suggesting a viscoplastic coupling to the envelope for protection against nuclear rupture. Summary statementHigh nuclear curvature drives lamina dilution and nuclear envelope rupture even when myosin stress is inhibited. Stiff filaments generally dilute from sites of high Gaussian curvature, providing mathematical fits of experiments.

biophysics↗

bioRxiv2018

Live cell monitoring for factors affecting genome variation

Cancer cells and pluripotent stem cells frequently exhibit gains or losses of entire chromosomes and chromosome segments, and the typical terminal analyses of genomes suggest this aneuploidy is ongoing and particularly variable in solid tumors. Here, we quantify aneuploidy-inducing perturbations by live cell fluorescence monitoring for changes in chromosome-5 in a lung cancer line and in normal diploid iPS cells. Inhibition of the spindle assembly checkpoint (SAC) and knockdown of DNA repair factors cause chromosome mis-segregation to increase several-fold above a low baseline level, and both perturbations also generate several-fold more rare fluorescent-null cells. Loss of chromosome-5 is confirmed by single cell karyotyping, SNP arrays on stable isolated clones, and downregulated expression of genes on chromosome-5 in single cell transcriptomics. The iPS cells also show loss of fluorescence in infrequent cells after SAC inhibition and upon growth as teratomas in mice. Viability, selection, and altered expression can thus be tracked to reveal molecular mechanisms in aneuploidy.

genomics↗

bioRxiv2018

Rescue of DNA damage in cells after constricted migration reveals bimodal mechano-regulation of cell cycle

Migration through constrictions can clearly rupture nuclei and mis-localize nuclear proteins but damage to DNA remains uncertain as does any effect on cell cycle. Here, myosin-II inhibition rescues rupture and partially rescues the DNA damage marker {gamma}H2AX, but an apparent delay in cell cycle is unaffected. Co-overexpression of multiple DNA repair factors and antioxidant inhibition of break formation also have partial effects, independent of rupture. However, there seems to be a bimodal dependence of cell cycle on DNA damage. Migration through custom-etched pores yields the same bimodal, with ~4-m pores causing intermediate levels of damage and cell cycle delay. Micronuclei (generated in faulty division) of the smallest diameter appear similar to ruptured nuclei, with high DNA damage and entry of chromatin-binding cGAS (cyclic-GMP-AMP-synthase) from cytoplasm but low repair factor levels. Increased genomic variation after constricted migration is quantified in expanding clones and is consistent with (mis)repair of excess DNA damage and subsequent proliferation.

cell biology↗