Biology subjects

Roh-Johnson, M.

Publications and source records attributed to Roh-Johnson, M..

11 recordsLinked to original sources

bioRxiv2026

Mitochondrial transfer between breast cancer cells promotes ROS-dependent proliferation

Previously, we showed that macrophages transfer mitochondria to breast cancer cells, promoting proliferation in acceptor cancer cells. Transferred mitochondria were depolarized and accumulated reactive oxygen species (ROS), and the mitochondrial transfer-induced proliferation was dependent upon ROS signaling (Kidwell et al. 2023). Our unexpected findings supported a model in which transferred mitochondria act as a signal for proliferation in acceptor cancer cells rather than a direct source of increased bioenergetics. It remains unclear whether this unexpected signaling mechanism is unique to macrophages as the donor cell, or whether this mechanism applies to mitochondrial transfer between other cells within the tumor microenvironment. Here, we show that highly metastatic cancer cells transfer mitochondria to weakly metastatic cancer cells. These transferred mitochondria are depolarized, accumulate ROS, and promote ROS-dependent proliferation in acceptor cancer cells. Furthermore, we specifically attribute this proliferative phenotype to the transfer of mitochondria, as when we isolate mitochondria from highly metastatic cells and apply these purified mitochondria directly to weakly metastatic cells in culture, acceptor cancer cells that internalize the purified mitochondria exhibit increased proliferation in a ROS-dependent manner. These findings support mitochondrial transfer within the breast tumor microenvironment as a signaling axis for proliferation, regardless of donor cell identity.

cancer biology↗

bioRxiv2026

PD-L1 deletion or blockade regulate macrophage antigen presentation and checkpoint molecule surface levels

Macrophages in the tumor microenvironment are known to upregulate PD-L1 expression, thereby suppressing T cells through PD-1 ligation. However, the manner in which PD-L1 expression intrinsically impacts macrophages and their immunomodulatory phenotype is less clear. Clarifying this knowledge gap would yield insight into the mechanisms of immunosuppression within the tumor microenvironment. To characterize the macrophage intrinsic role of PD-L1, we used complementary genetic and pharmacological approaches by analyzing primary murine bone marrow-derived macrophages (BMDMs) with complete genetic PD-L1 deletion and wildtype BMDMs treated with anti-PD-L1 blocking antibodies. Macrophages were evaluated across naive, pro-inflammatory (M1), and tumor conditioned (TCM) polarization states in vitro. Unlike prior reports, neither genetic deletion nor antibody blockade dramatically altered the expression of macrophage polarization markers or in vitro phagocytic capacity. Both conditions consistently reduced surface levels of the M1-associated costimulatory molecule CD80, prompting further analysis of T cell interacting and antigen presenting proteins, in which we revealed disparate effects of genetic deletion and antibody blockade on the surface levels of MHCI, MHCII, PD-1, and PD-L2. These findings suggest that PD-L1 deletion and antibody-mediated blockade contribute to macrophage immune regulatory profiles in distinct manners. This difference supports a model in which PD-L1 functions in macrophages beyond its canonical role as a ligand for PD-1, influencing antigen presentation and checkpoint molecule levels and playing a broader role in immune regulation in the tumor microenvironment.

immunology↗

bioRxiv2026

NG2-targeting macrophages inhibit 3D invasion of patient-derived glioblastoma spheroids

Engineering macrophages with chimeric antigen receptors is emerging as a promising cancer therapeutic. Chimeric antigen receptor-expressing macrophages (CAR-Ms) engineered to recognize tumor-specific antigens have been shown to inhibit tumor growth and activate adaptive immune responses, leading to robust tumor control in animal studies. Based on this work, clinical trials have been initiated. While the trials have shown promise, challenges remain. The dynamic interactions between CAR-Ms and cancer cells and the exact mechanisms driving anti-tumor effects remain poorly defined. Defining the dynamic interactions between CAR-Ms and cancer cells will provide critical insights for optimizing future CAR-M design and improving therapeutic efficacy. We sought to directly visualize CAR-M interactions with glioblastoma cells at high-resolution and in real-time using CAR-Ms engineered to recognize Neural-Glial Antigen 2 (NG2), an antigen expressed on glioblastoma cells. Using patient-derived glioblastoma cells, we formed glioblastoma spheroids and embedded them in a 3D matrix together with CAR-Ms. Using time-lapse microscopy, as expected, we found that NG2-targeting CAR-Ms engulfed glioblastoma cells. However, excitingly, we found that NG2-targeting CAR-Ms blocked >85% of glioblastoma cell invasion in 3D. This inhibition of glioblastoma invasion was not due to a significant change in CAR-M polarization states. Together, these data suggest that NG2-targeting CAR-Ms both engulf glioblastoma cells and block glioblastoma invasive behavior.

cancer biology↗

bioRxiv2025

Differential PaxillinB dynamics at Dictyostelium cell-substrate adhesions

Adhesion-based migration is regulated by focal adhesions, multi-protein nanostructures linking the intracellular cytoskeleton to the extracellular substrate. Efficient adhesion-based migration has been shown to be regulated by focal adhesion dynamics such as lifetime, size and turnover, which in turn are influenced by the molecular composition of focal adhesions. We recently identified the formation of cell-substrate adhesion populations in Dictyostelium discoideum with differing molecular compositions, but it is unclear how these distinct compositions influence Dictyostelium adhesion dynamics and cell migration. Here, we further investigate the role of molecular composition on Dictyostelium adhesion lifetime and protein turnover during cell migration. We show that co-localization of VinculinB to PaxillinB-positive cell-substrate adhesions increases adhesion lifetime without changing PaxillinB turnover. We further show that perturbing cell-substrate adhesion composition with a PaxillinB N-terminal truncation increases adhesion lifetime and decreases PaxillinB turnover at adhesions. These findings suggest that similar to mammalian focal adhesions, molecular composition of Dictyostelium cell-substrate adhesion regulates their adhesion lifetimes and protein turnover, providing insight into how cell-substate adhesions function during Dictyostelium cell migration. SUMMARY STATEMENTFierro Morales and Roh-Johnson demonstrate that Dictyostelium PaxillinB, a homolog of the core focal adhesion protein Paxillin, exhibits differential dynamics in distinct populations of cell-substrate adhesions.

evolutionary biology↗

bioRxiv2024

CSPG4-targeting CAR-macrophages inhibit melanoma growth

Chimeric antigen receptor (CAR) T-cell therapy has revolutionized the treatment of hematological malignancies but has been clinically less effective in solid tumors. Engineering macrophages with CARs has emerged as a promising approach to overcome some of the challenges faced by CAR-T cells due to the macrophages ability to easily infiltrate tumors, phagocytose their targets, and reprogram the immune response. We engineered CAR-macrophages (CAR-Ms) to target chondroitin sulfate proteoglycan 4 (CSPG4), an antigen expressed in melanoma, and several other solid tumors. CSPG4-targeting CAR-Ms exhibited specific phagocytosis of CSPG4-expressing melanoma cells. Combining CSPG4-targeting CAR-Ms with CD47 blocking antibodies synergistically enhanced CAR-M-mediated phagocytosis and effectively inhibited melanoma spheroid growth in 3D. Furthermore, CSPG4-targeting CAR-Ms inhibited melanoma tumor growth in mouse models. These results suggest that CSPG4-targeting CAR-M immunotherapy is a promising solid tumor immunotherapy approach for treating melanoma. STATEMENT OF SIGNIFICANCEWe engineered macrophages with CARs as an alternative approach for solid tumor treatment. CAR-macrophages (CAR-Ms) targeting CSPG4, an antigen expressed in melanoma and other solid tumors, phagocytosed melanoma cells and inhibited melanoma growth in vivo. Thus, CSPG4-targeting CAR-Ms may be a promising strategy to treat patients with CSPG4-expressing tumors.

cancer biology↗

bioRxiv2024

Macrophage subtypes inhibit breast cancer proliferation in culture

Macrophages are a highly plastic cell type that adopt distinct subtypes and functional states depending on environmental cues. These functional states can vary wildly, with distinct macrophages capable of displaying opposing functions. We sought to understand how macrophage subtypes that exist on two ends of a spectrum influence the function of other cells. We used a co-culture system with primary human macrophages to probe the effects of macrophage subtypes on breast cancer cell proliferation. Our studies revealed a surprising phenotype in which both macrophage subtypes inhibited cancer cell proliferation compared to cancer cells alone. Of particular interest, using two different proliferation assays with two different breast cancer cell lines, we showed that differentiating macrophages into a "pro-tumor" subtype inhibited breast cancer cell proliferation. These findings are inconsistent with the prevailing interpretation that "pro-tumor" macrophages promote cancer cell proliferation and suggest a re-evaluation of how these interpretations are made.

cell biology↗

bioRxiv2024

Reduced PaxillinB localization to cell-substrate adhesions promotes cell migration in Dictyostelium

Many cells adhere to the extracellular matrix for efficient cell migration. This adhesion is mediated by focal adhesions, a protein complex linking the extracellular matrix to the intracellular cytoskeleton. Focal adhesions have been studied extensively in Metazoan mesenchymal cells, but recent research in physiological contexts and amoeboid cells suggest that focal adhesion regulation differs from the mesenchymal focal adhesion paradigm. While focal adhesion machinery predates the origin of Metazoans, focal adhesion formation and regulation during non-Metazoan cell migration is largely unexplored. We used Dictyostelium discoideum to investigate novel mechanisms and the evolution of focal adhesion regulation, as Dictyostelium are non-Metazoans that form cell-substrate adhesion structures for migration. We show that PaxillinB, the Dictyostelium homologue of Paxillin, localizes to dynamic cell-substrate adhesions. As expected, PaxillinB mutations decreased the number of cell-substrate adhesions. Unexpectedly, however, decreased cell-substrate adhesion number led to an increase in cell migration speed. These findings are in direct contrast to Paxillin function at focal adhesions and regulation of cell migration in mammalian cells, challenging the established focal adhesion model and providing insight into the evolution of cell-substrate adhesions and Paxillin function during cell migration. Significance StatementO_LIFocal adhesions are understudied in non-mesenchymal, non-Metazoan systems. C_LIO_LIThe authors characterize PaxillinB, the Dictyostelium homologue of Paxillin, during Dictyostelium cell migration. Reducing cell-substrate adhesion number via PaxillinB mutations lead to decreased cell adhesion and, surprisingly, increased cell migration speeds. C_LIO_LIThese findings suggest Dictyostelium cells form cell-substrate adhesions that act as molecular "brakes" during cell migration and provides unique insight into the evolution of Paxillin as a regulator of cell-substrate adhesion function and formation during cell migration. C_LI

cell biology↗

bioRxiv2022

Focal adhesion-based cell migration is differentially regulated in vivo versus in vitro by Paxillin phosphorylation

Focal adhesions are important subcellular structures that physically link the cell to the extracellular matrix (ECM), thus facilitating efficient cell migration. Although in vitro cell culture studies have provided a wealth of information regarding focal adhesion biology, it is critical to understand how focal adhesions are dynamically regulated in their native environment. We developed a zebrafish transplantation system in which we could efficiently visualize focal adhesion structures during single cell migration in vivo with high-resolution live cell imaging. By comparing focal adhesions between this in vivo system and the traditional in vitro cell culture model, we show differential regulation of a core focal adhesion protein, Paxillin. We find that a key site of phosphoregulation on Paxillin, tyrosine 118 (Y118), exhibits reduced phosphorylation in migrating cells in vivo in both zebrafish and mouse melanoma models, contrary to the pivotal role for this phosphorylation event in cell culture studies. Furthermore, direct modulation of this residue by site directed mutagenesis leads to opposite cell migration phenotypes in vivo versus in vitro in both migrating cancer cells and macrophages. Unexpectedly, expression of a non-phosphorylatable version of Y118-Paxillin promotes cell migration in vivo, despite inhibiting cell migration in the in vitro cell culture conditions. To further understand the mechanism of this regulation, we find that the upstream kinase, focal adhesion kinase (FAK), is downregulated in cells in vivo, and that cells expressing non-phosphorylatable Y118-Paxillin exhibit increased interactions between Paxillin and CRKII, an adaptor protein known to promote cell migration signaling. Collectively, our findings provide significant new insight into how focal adhesions are regulated in cells migrating in their native environment.

cell biology↗

bioRxiv2021

Laterally transferred macrophage mitochondria acts as a signaling source promoting cancer cell proliferation

Lateral transfer of mitochondria occurs in many physiological and pathological conditions. Given that mitochondria provide essential energy for cellular activities, mitochondrial transfer is currently thought to promote the rescue of damaged cells. We report that mitochondrial transfer occurs between macrophages and breast cancer cells, leading to increased cancer cell proliferation. Unexpectedly, transferred macrophage mitochondria are dysfunctional, lacking mitochondrial membrane potential. Rather than performing essential mitochondrial activities, transferred mitochondria accumulate reactive oxygen species which activates ERK signaling, indicating that transferred mitochondria act as a signaling source that promotes cancer cell proliferation. We also demonstrate that pro-tumorigenic M2-like macrophages exhibit increased mitochondrial transfer to cancer cells. Collectively, our findings reveal how mitochondrial transfer is regulated and leads to sustained functional changes in recipient cells. One-Sentence SummaryLateral transfer of macrophage mitochondria acts as a ROS signaling source, regulating cancer cell proliferation through ERK signaling.

cancer biology↗

bioRxiv2020

Worrying drives cell migration in mechanically unrestrained environments

Migratory cells often encounter crowded microenvironments through which they must find or make a path. Amoeboid cells are thought to find a path by deforming their bodies to squeeze through tight spaces. Yet many amoeboid cells seem to maintain a near spherical morphology as they move. To examine this unexplored mechanism of migration, we visualized amoeboid melanoma cells in dense environments and found that they carve a path via bleb-driven mechanical degradation of extracellular matrix components without proteolytic degradation. Interactions between adhesions and collagen at the cell front induce a signaling cascade that promotes bleb enlargement via branched actin polymerization. Large blebs abrade collagen, creating feedback between extracellular matrix structure, cell morphology and polarization that enables both path generation and persistent movement.

cell biology↗

bioRxiv2020

Msp1/ATAD1 restores mitochondrial function in Zellweger Spectrum Disease

Peroxisomal Biogenesis Disorders (PBDs) are a class of inherited metabolic disorders with profound neurological and other phenotypes. The most severe PBDs are caused by mutations in peroxin genes, which result in nonfunctional peroxisomes typically through impaired protein import. In order to better understand the molecular causes of Zellweger Spectrum Disease (ZSD) -the most severe PBDs -, we investigated the fate of peroxisomal mRNAs and proteins in ZSD model systems. We found that loss of peroxisomal import has no effect on peroxin mRNA expression or translational efficiency. Instead, peroxin proteins--still produced at high levels-- aberrantly accumulate on the mitochondrial membrane, impairing respiration and ATP generation. Finally, we rescued mitochondrial function in fibroblasts derived from human patients with ZSD by overexpressing ATAD1, an AAA-ATPase that functions in mitochondrial quality control. These findings might provide a new focus of PBD therapies in supporting quality control pathways that protect mitochondrial function.

cell biology↗