Biology subjects

Andrechak, J. C.

Publications and source records attributed to Andrechak, J. C..

5 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

Titrating CD47 by mismatch CRISPRi reveals incomplete repression can eliminate IgG-opsonized tumors but CD47 heterogeneity limits induction of anti-tumor IgG

Phagocytic elimination of solid tumors is an attractive mechanism for immunotherapy - particularly because of the possible induction of anti-cancer immunity. The phagocytic potential of macrophages is limited, however, by the CD47-SIRP checkpoint, and how much CD47 disruption is needed for efficacy remains unclear, even when tumors are opsonized by a pro-phagocytic antibody. Here, CRISPR-interference (CRISPRi) is applied with a large set of sgRNAs to produce a broad range of CD47 knockdowns in B16F10 melanoma, which is generally found to be resistant to the heavily studied PD-1 blockade. Guided by 3D immuno-tumoroid results, we identify a critical CD47 density below which macrophage-mediated phagocytosis dominates proliferation in the presence of an otherwise ineffective pro-phagocytic antibody (anti-Tyrp1). Growing tumors and immuno-tumoroids generally show selection for CD47-positive cells, but some mice reject tumors having >97% mean repression of CD47 or even having 80% repression - unless mixed with 50% repressed cells. Interestingly, long-term survivors have de novo pro-phagocytic IgG antibodies that increase in titer with depth of repression and also with early accumulation of tumor macrophages. Given well-known limitations of antibody permeation into solid tumors, our studies set a benchmark for anti-CD47 blockade and suggest deep disruption favors acquired immunity.

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↗

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↗