Biology subjects

Grimm, J.

Publications and source records attributed to Grimm, J..

16 recordsLinked to original sources

bioRxiv2026

Sustained Ion Imbalances Enhance Ferroptosis

Ferroptosis has attracted significant attention for its potential to treat drug resistant tumors, along with its role in neurodegeneration and ischemia. Several ferroptosis targets were identified in the past decade, yet it is also highly affected by cellular metabolism and epithelial or mesenchymal phenotypes which limits the utility of these targets across diverse cell and tissue types. To address this, we utilized chemo-resistant cancer cell lines with different metastatic potential and tissue origins and discovered a common role of cytoplasmic ion changes during ferroptosis. We observed significant changes in intracellular potassium, sodium and chloride levels upon induction of ferroptosis. Therefore, we screened several ion channel inhibitors and activators (i.e. modulators) and identified Kv7.2 and CFTR modulators influence ferroptosis execution despite the fact that these cell lines do not express these proteins. We demonstrated that clinically approved modulators of CFTR and Kv7.2 can inhibit ferroptosis which indicates that these inhibitors could be repurposed as treatments in ferroptosis-associated diseases such as neurodegeneration and kidney diseases independent of ion channel expression. These modulators also influenced potassium and chloride levels in different contexts indicating off-target effects may also lead to changes in ion levels and may serve as a ferroptosis target. Further, we demonstrated that sustained imbalance of sodium, potassium and chloride ions with ferroptosis inducer salinomycin and digoxin, a Na+/K+ ATPase inhibitor, enhanced ferroptosis. The combination of salinomycin and digoxin successfully enhanced ferroptotic cell death across both cell lines with different metabolic signatures in vitro, offering a novel ferroptosis target. This combination significantly reduced tumor growth in vivo compared with the single agent alone, offering a new therapeutic modality against drug resistant tumors.

cancer biology↗

bioRxiv2026

ENHANCING TUMOR PERFUSION AND NANOMEDICINE DELIVERY VIA ENDOGENOUS NITRIC OXIDE RELEASE BY METHYL PALMITATE NANOPARTICLES

Despite a few clinical successes, the efficacy of cancer nanomedicines remains limited by rapid clearance by the mononuclear phagocytic system and poor permeation across the abnormal tumor vasculature. We previously showed that methyl palmitate nanoparticles (MPN) can safely and reversibly inhibit the phagocytic activity of immune cells for several hours, thereby improving tumor accumulation and the efficacy of systemically administered nanomedicines. Here, we demonstrate that, on a shorter time scale, MPN can induce vasodilation, introducing an additional mechanism to enhance the accumulation of therapeutic agents within the malignant tissue. Upon internalization by macrophages and endothelial cells, MPN trigger the release of endogenous nitric oxide (NO), a key mediator of vasodilation, in a concentration-, and time-dependent manner. Following MPN administration, raster-scanning optoacoustic mesoscopy (RSOM) revealed vasodilation across multiple tissues, with the strongest effect observed in tumors. To assess enhanced tumor accumulation, we injected 70 kDa fluorescent dextran and demonstrated via histology a markedly increased fluorescence signal exclusively in MPN-treated tumors compared to controls 24 hours later. In addition, positron emission tomography (PET) imaging of 89Zr-labeled clinical iron oxide nanoparticles (Feraheme) showed significantly greater tumor accumulation after a 15-minute MPN pretreatment. Finally, general serum biochemistry panels and histological analyses of major organs in healthy mice revealed no toxicity following either single or repeated MPN dosing. Overall, this study demonstrates that MPN-induced vasodilation occurring within minutes enhances intra-tumoral deposition of macromolecules and small nanoparticles. Together with their longer-term effects on phagocytosis inhibition, these findings indicate that MPN can improve therapeutic delivery through complementary, time-dependent mechanisms that increase tumor perfusion and vascular permeability.

bioengineering↗

bioRxiv2024

In vivo multiplex imaging of dynamic neurochemical networks with designed far-red dopamine sensors

Neurochemical signals like dopamine (DA) play a crucial role in a variety of brain functions through intricate interactions with other neuromodulators and intracellular signaling pathways. However, studying these complex networks has been hindered by the challenge of detecting multiple neurochemicals in vivo simultaneously. To overcome this limitation, we developed a single-protein chemigenetic DA sensor, HaloDA1.0, which combines a cpHaloTag-chemical dye approach with the G protein-coupled receptor activation-based (GRAB) strategy, providing high sensitivity for DA, sub-second response kinetics, and an extensive spectral range from far-red to near-infrared. When used together with existing green and red fluorescent neuromodulator sensors, Ca2+ indicators, cAMP sensors, and optogenetic tools, HaloDA1.0 provides high versatility for multiplex imaging in cultured neurons, brain slices, and behaving animals, facilitating in-depth studies of dynamic neurochemical networks.

neuroscience↗

bioRxiv2024

In vivo imaging of ferroptosis through nanodynamic changes in lipid membranes

Ferroptosis emerged as a cell death modality for drug resistant cancer cells, but there are currently no available biomarkers for imaging ferroptosis based therapies. To address this gab, we evaluated the nanodynamic changes in lipid membranes occurring during cell death to explore potential targeting opportunities to image cell death. We nano-sized gaps at late stages of ferroptosis can serve as entry points for dyes that can bind to cellular structures. These changes were accompanied with cellular signaling components similar to platelet activation, with phosphatidyl serine emerging on the surface of the cells and therefore as a potential target for imaging of programed cell death, including ferroptosis. Taking advantage of these changes in cell membrane dynamics, we employed a novel tumor-seeking dye CJ215 that can label apoptotic cells as recently described by us. We show that CJ215 accumulates in ferroptotic cells both in vitro and in vivo by binding to phosphatidyl serine, a process that is prevented by inhibition of ferroptosis. Since phosphatidyl serine exposure also occurs during apoptosis, CJ215 can serve to image both apoptosis and ferroptosis based therapy.

cancer biology↗

bioRxiv2023

Nanoscale imaging reveals the mechanisms of ER-to-Golgi transport via a dynamic tubular-vesicular network

The endoplasmic reticulum (ER) and the Golgi apparatus are the first sorting stations along the secretory pathway of mammalian cells and have a crucial role in protein quality control and cellular homeostasis. While machinery components mediating ER-to-Golgi transport have been mapped, it is unclear how exchange between the two closely juxtaposed organelles is coordinated in living cells. Here, using gene editing to tag machinery components, live-cell confocal and stimulated emission depletion (STED) super-resolution microscopy, we show that ER-to-Golgi transport occurs via a dynamic network of tubules positive for the small GTPase ARF4. swCOPI machinery is tightly associated to this network and moves with tubular-vesicular structures. Strikingly, the ARF4 network appears to be continuous with the ER and ARF4 tubules remodel around static ER exit sites (ERES) defined by COPII machinery. We were further able to dissect the steps of ER-to-Golgi transport with functional trafficking assays. A wave of cargo released from the ER percolates through peripheral and Golgi-tethered ARF4 structures before filling the cis-Golgi. Perturbation via acute degradation of ARF4 shows an active regulatory role for the GTPase and COPI in anterograde transport. Our data supports a model in which anterograde ER-to-Golgi transport occurs via an ARF4 tubular-vesicular network directly connecting the ER and Golgi-associated pre-cisternae.

cell biology↗

bioRxiv2023

Vascularized tumor on a microfluidic chip to study mechanisms promoting tumor neovascularization and vascular targeted therapies

The cascade of events leading to tumor formation includes induction of a tumor supporting neovasculature as a primary hallmark of cancer. Developing vasculature is difficult to evaluate in vivo but can be captured using microfluidic chip technology and patient derived cells. Herein, we established an on chip approach to investigate the mechanisms promoting tumor vascularization and vascular targeted therapies via co-culture of metastatic renal cell carcinoma spheroids and endothelial cells in a 3D environment. Our model permitted real-time, high-resolution observation and assessment of tumor-induced angiogenesis, where endothelial cells sprout towards the tumor and mimic a vascular network. Bevacizumab, an angiogenic inhibitor, disrupted interactions between vessels and tumors, destroying the vascular network. The on chip approach enabled assessment of endothelial cell biology, vessels functionality, drug delivery, and molecular expression of PSMA. Finally, observations in the vascularized tumor on chip permitted direct and conclusive quantification of this therapy in weeks as opposed to months in a comparable animal model. TeaserVascularized tumor on microfluidic chip provides opportunity to study targeted therapies and improves preclinical drug discovery.

cancer biology↗

bioRxiv2023

Radiances of Cerenkov-Emitting Isotopes on the IVIS

Cerenkov (or Cherenkov) luminescence occurs when charged particles exceed the phase velocity of a given medium. Cerenkov has gained interest in preclinical space as well as in clinical trials for optical visualization of numerous radionuclides. However, Cerenkov intensity has to be inferred from alternative databases with energy emission spectra, or theoretical fluence estimates. Here we present the largest experimental dataset of Cerenkov emitting isotopes recorded using the IVIS optical imaging system. We report Cerenkov measurements spanning orders of magnitude normalized to the activity concentration for 21 Cerenkov emitting isotopes, covering electron, alpha, beta minus, and positron emissions. Isotopes measured include Carbon-11, Fluorine-18, Phosphorous-32, Scandium-47, Copper-64, Copper-67, Gallium-68, Arsenic-72, Bromine-76, Yttrium-86, Zirconium-89, Yttrium-90, Iodine-124, Iodine-131, Cerium-134, Lutetium-177, Lead-203, Lead-212, Radium-223, Actinium-225, and Thorium-227. We hope this updating resource will serve as a rank ordering for comparing isotopes for Cerenkov luminescence in the visible window and serve as a rule of thumb for comparing Cerenkov intensities in vitro and in vivo. MethodsAll Cerenkov emitting radionuclides were either produced at Memorial Sloan Kettering Cancer Center (Carbon-11, 11C; Fluorine-18, 18F; Iodine-124, 124I), from commercial sources such as Perkin Elmer (Phosphorous-32, 32P; Yttrium-90, 90Y), Bayer (Radium-223, 223Ra, Xofigo), 3D-Imaging (Zirconium-89, 89Zr), Nuclear Diagnostic Products (Iodine-131, 131I), or from academic collaborators at Washington University at St. Louis (Copper-64, 64Cu), University of Wisconsin (Bromine-76, 76Br), MD Anderson Cancer Center (Yttrium-86, 86Y), Brookhaven National Laboratory (Arsenic-72, 72As; Thorium-227, 227Th), or Oak Ridge National Laboratory (Cerium-134, 134Ce, Actinium-225, 225Ac), and Viewpoint Molecular Targeting (Lead-203, 203Pb; Lead 212, 212Pb). All isotopes were diluted in triplicate on a black bottomed corning 96 well plate to several activity concentrations ranging from 0.1-250 Ci in 100-200 L of Phosphate Buffered Saline. Cerenkov imaging was acquired on a single Perkin-Elmer Spectrum In-Vivo Imaging System (IVIS) at field of view c with exposures ranging up to 15 minutes or lower provided no part of the image intensity was saturated, or that the activity significantly changed during the exposure. Experimental radiances on the IVIS were calculated from regions of interest drown over each 96 well, and then normalized for the activity present in the well, and the volume the isotope was diluted into.

bioengineering↗

bioRxiv2022

Preclinical shortwave infrared tumor screening and resection via pHLIP ICG under ambient lighting conditions

There is a critical need to improve optical imaging that will lead to its widespread acceptance for routine clinical procedures. Shortwave infrared (SWIR, 900-1700nm) imaging has demonstrated clear advantages over visible and near-infrared imaging (reduced autofluorescence with improved contrast, resolution, and sensitivity at tissue depth). Here we show that the previously reported compound, pH low insertion peptide (pHLIP) conjugated to indocyanine green (ICG, pHLIP ICG) currently in clinical trials, serves as an excellent candidate for SWIR imaging protocols. SWIRs increased sensitivity enabled preclinical tumor screening and resection at exposure times as low as 0.1 ms with acceptable signal-to-noise and contrast-to-noise ratios. Imaging was performed under ambient lighting conditions, and SWIRs sensitivity enabled an extended surgical resection window up to 96 hrs post injection in an orthotopic breast cancer mouse model. This work provides a direct precedent for the clinical translation of SWIR pHLIP ICG imaging for cancer resection. One Sentence SummarySWIR imaging under ambient lighting is highly sensitive to pHLIP ICG, a cancer targeting fluorescent agent currently under clinical investigation.

cancer biology↗

bioRxiv2022

PSMA-bearing extracellular vesicles secreted from prostate cancer convert the microenvironment to a tumor-supporting, pro-angiogenic state.

Extracellular vesicles (EV) are comprised of vesicles budding from cell membranes and smaller intracellular vesicles shed by cells. EV play a role in remodeling the tumor microenvironment (TME) and support tumor progression. Prostate-specific membrane antigen (PSMA) is a transmembrane glycoprotein with a carboxypeptidase function, frequently associated with poor clinical prognosis in prostate cancer (PCa). We previously identified an oncogenic PSMA signaling function in prostate cancer. Others demonstrated that EV isolated from the plasma of patients with high-grade PCa carry PSMA, but so far no pathophysiological effect has been associated with PSMA-bearing EV. Here we demonstrate that EV from PCa cells are able to transfer PSMA and its functionality to cells in the TME. The consequence of that EV-mediated PSMA transfer is an acute to long-term increased secretion of vascular endothelial growth factor-A (VEGF-A), angiogenin, pro-angiogenic and pro-lymphangiogenic mediators and increased 4E binding protein 1 (4EBP-1) phosphorylation in tumors. We compare EV from PCa cells with or without PSMA expression to address the role of PSMA-bearing EV in promoting pro-tumoral changes in the TME using classical molecular biology and novel molecular imaging approaches.

cancer biology↗

bioRxiv2022

Combining LSD1 and JAK-STAT inhibition targets Down syndrome-associated myeloid leukemia at its core

Children with Down syndrome (DS) are predisposed to developing megakaryoblastic leukemia (ML-DS) and often experience severe toxicities from chemotherapy, highlighting the need for targeted therapies with beneficial risk profiles. The genomic landscape of ML-DS is characterized by a combination of mutations in signaling pathway genes and epigenetic modifiers, while aberrant lysine specific demethylase 1 (LSD1) and JAK-STAT activation have both been implicated in leukemogenesis. Here, we demonstrate that combined LSD1 and JAK1/2 inhibition exerts synergistic anti-leukemic effects specifically in ML-DS, both in vitro and in patient derived xenografts in vivo. The JAK1/2 inhibitor ruxolitinib enhanced the LSD1 inhibitor-induced differentiation, proliferation arrest and apoptosis in patient-derived leukemic blasts. At the transcriptional level, the combination synergistically repressed gene expression signatures essential for cell division. We further observed an immunogenic gene expression pattern in the form of increased cytokine signaling, which - by sensitizing ML-DS blasts to the JAK-STAT signaling blockade induced by ruxolitinib - could explain the increased susceptibility of ML-DS blasts to combination therapy. Taken together, we establish combined LSD1 and JAK-STAT inhibition as an efficacious therapeutic regimen specifically designed to target important steps in ML-DS leukemogenesis, paving the way for targeted therapies in this entity.

cancer biology↗

bioRxiv2022

Comprehensive preclinical evaluation of human-derived anti-poly-GA antibodies in cellular and animal models of C9ORF72 disease

Hexanucleotide G4C2 repeat expansions in the C9ORF72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Dipeptide repeat proteins (DPRs) generated by translation of repeat-containing RNAs show toxic effects in vivo as well as in vitro and are key targets for therapeutic intervention. We generated human antibodies that bind DPRs with high affinity and specificity. Anti-GA antibodies engaged extra- and intracellular poly-GA and reduced aggregate formation in a poly-GA over-expressing human cell line. However, antibody treatment in human neuronal cultures synthesizing exogenous poly-GA resulted in the formation of large extracellular immune complexes and did not affect accumulation of intracellular poly-GA aggregates. Treatment with antibodies was also shown to directly alter the morphological and biochemical properties of poly-GA and to shift poly-GA/antibody complexes to more rapidly sedimenting ones. These alterations were not observed with poly-GP and have important implications for accurate measurement of poly-GA levels including the need to evaluate all centrifugation fractions and disrupt the interaction between treatment antibodies and poly-GA by denaturation. Targeting poly-GA and poly-GP in two mouse models expressing G4C2 repeats by systemic antibody delivery for up to 16 months was well-tolerated and led to measurable brain penetration of antibodies. Long term treatment with anti-GA antibodies produced improvement in an open field movement test in aged C9ORF72450 mice. However, chronic administration of anti-GA antibodies in AAV-(G4C2)149 mice was associated with increased levels of poly-GA detected by immunoassay and did not significantly reduce poly-GA aggregates or alleviate disease progression in this model. SignificanceImmunotherapy has been proposed for neurodegenerative disorders including Alzheimers or Parkinsons diseases. Recent reports using antibodies against poly-GA or active immunization suggested similar immunotherapy in ALS/FTD caused by repeat expansion in the C9ORF72 gene (1, 2). Here, we systematically characterized human antibodies against multiple DPR species and tested the biological effects of antibodies targeting poly-GA in different cellular and mouse models. Target engagement was shown in three independent cellular models. Anti-GA antibodies reduced the number of intracellular poly-GA aggregates in human T98G cells but not in cultured human neurons. Whereas chronic anti-GA treatment in BAC C9ORF72450 mice did not impact poly-GA levels and modestly improved one behavioral phenotype, poly-GA levels detected by immunoassays were increased and disease progression was unaltered in AAV-(G4C2)149 mice.

neuroscience↗

bioRxiv2020

The inner membrane protein YhdP modulates the rate of anterograde phospholipid flow in Escherichia coli

The outer membrane (OM) of Gram-negative bacteria is a selective permeability barrier that allows uptake of nutrients while simultaneously protecting the cell from harmful compounds. The basic pathways and molecular machinery responsible for transporting lipopolysaccharides (LPS), lipoproteins, and {beta}-barrel proteins to the OM have been identified, but very little is known about phospholipid (PL) transport. To identify genes capable of affecting PL transport, we screened for genetic interactions with mlaA*, a mutant in which anterograde PL transport causes the inner membrane (IM) to shrink and eventually rupture; characterization of mlaA*-mediated lysis suggested that PL transport can occur via a high-flux, diffusive flow mechanism. We found that YhdP, an IM protein involved in maintaining the OM permeability barrier, modulates the rate of PL transport during mlaA*-mediated lysis. Deletion of yhdP from mlaA* reduced the rate of IM transport to the OM by 50%, slowing shrinkage of the IM and delaying lysis. As a result, the weakened OM of {Delta}ydhP cells was further compromised and ruptured before the IM during mlaA*-mediated death. These findings demonstrate the existence of a high-flux, diffusive pathway for PL flow in Escherichia coli that is modulated by YhdP. Significance StatementThe outer membrane (OM) of Gram-negative bacteria serves as a barrier that protects cells from harmful chemical compounds, including many antibiotics. Understanding how bacteria build this barrier is an important step in engineering strategies to circumvent it. A long-standing mystery in the field is how phospholipids (PLs) are transported from the inner membrane (IM) to the OM. We previously discovered that a mutation in the gene mlaA causes rapid flow of PLs to the OM, eventually resulting in IM rupture. Here, we found that deletion of the gene yhdP delayed cell death in the mlaA mutant by slowing flow of PLs to the OM. These findings reveal a high-flux, diffusive pathway for PL transport in Gram-negative bacteria modulated by YhdP.

molecular biology↗

bioRxiv2020

Optoacoustic imaging of GLP-1 Receptor with a near-infrared exendin-4 analog

Limitations in current imaging tools have long challenged the imaging of small pancreatic islets in animal models. Here, we report the first development and in vivo validation testing of a broad spectrum and high absorbance near infrared optoacoustic contrast agent, E4x12-Cy7. Our near infrared tracer (E4x12-Cy7) is based on the amino acid sequence of exendin-4 and targets the glucagon-like peptide-1 receptor (GLP-1R). Cell assays confirmed that E4x12-Cy7 has a high binding affinity (IC50 = 4.6 {+/-} 0.8 nM). Using the multi-spectral optoacoustic tomography (MSOT), we imaged E4x12-Cy7 and optoacoustically visualized {beta}-cell insulinoma xenografts in vivo for the first time. In the future, similar optoacoustic tracers that are specific for {beta}-cells and combines optoacoustic and fluorescence imaging modalities could prove to be important tools for monitoring the pancreas for the progression of diabetes.

bioengineering↗

bioRxiv2018

Bioorthogonal labeling with tetrazine-dyes forsuper-resolution microscopy

Genetic code expansion (GCE) technology allows the specific incorporation of functionalized noncanonical amino acids (ncAAs) into proteins. Here, we investigated the Diels-Alder reaction between trans-cyclooct-2-ene (TCO)-modified ncAAs, and 22 known and novel 1,2,4,5-tetrazine-dye conjugates spanning the entire visible wavelength range. A hallmark of this reaction is its fluorogenicity - the tetrazine moiety can elicit substantial quenching of the dye. We discovered that photoinduced electron transfer (PET) from the excited dye to tetrazine as the main quenching mechanism in red-absorbing oxazine and rhodamine derivatives. Upon reaction with dienophiles quenching interactions are reduced resulting in a considerable increase in fluorescence intensity. Efficient and specific labeling of all tetrazine-dyes investigated permits super-resolution microscopy with high signal-to-noise ratio even at the single-molecule level. The different cell permeability of tetrazine-dyes can be used advantageously for specific intra- and extracellular labeling of proteins and highly sensitive fluorescence imaging experiments in fixed and living cells.

biochemistry↗

bioRxiv2017

Cohesin Can Remain Associated With Chromosomes During DNA Replication

To ensure disjunction to opposite poles during anaphase, sister chromatids must be held together following DNA replication. This is mediated by cohesin, which is thought to entrap sister DNAs inside a tripartite ring composed of its Smc and kleisin (Scc1) subunits. How such structures are created during S phase is poorly understood, in particular whether they are derived from complexes that had entrapped DNAs prior to replication. To address this, we used selective photobleaching to determine whether cohesin associated with chromatin in G1 persists in situ after replication. We used unlabelled HaloTag ligand following fluorescent labelling to block newly synthesised Halo-tagged Scc1 protein from incorporating fluorescent dye (pulse-chase or pcFRAP). In cells whose cohesin turnover is inactivated by deletion of WAPL, Scc1 remains associated with chromatin throughout S phase. These findings suggest that cohesion might be generated by cohesin that is already bound to unreplicated DNA.

cell biology↗

bioRxiv2017

Stochastic Protein Labeling Enables Long-term Single Molecule Observation In Vivo

Our ability to unambiguously image and track individual molecules in live cells is limited by packing of multiple copies of labeled molecules within the resolution limit. Here we devise a universal genetic strategy to precisely control copy number of fluorescently labeled molecules in a cell. This system has a dynamic titration range of >10,000 fold, enabling sparse labeling of proteins expressed at different abundance levels. Combined with photostable labels, this system extends the duration of automated single-molecule tracking by 2 orders of magnitude. We demonstrate long-term imaging of synaptic vesicle dynamics in cultured neurons as well as in intact zebrafish. We found axon initial segment utilizes a waterfall mechanism gating synaptic vesicle transport polarity by promoting anterograde transport processivity. Long-time observation also reveals that transcription factor hops between clustered binding sites in spatially-restricted sub-nuclear regions, suggesting that topological structures in the nucleus shape local gene activities by a sequestering mechanism. This strategy thus greatly expands the spatiotemporal length scales of live-cell single-molecule measurements, enabling new experiments to quantitatively understand complex control of molecular dynamics in vivo.

biophysics↗