Mitigating Organophosphate Nerve Agent, Soman (GD)-Induced Long-Term Neurotoxicity: Saracatinib, a Src Tyrosine Kinase Inhibitor, as a Potential Countermeasure

Background: Acute exposure to soman (GD), an organophosphate nerve agent (OPNA), irreversibly inhibits acetylcholinesterase (AChE), induces seizures, and could be fatal if not treated immediately. Existing medical countermeasures (MCMs- atropine, oximes, and benzodiazepines) mitigate the acute life-threatening cholinergic symptoms but have limited protection against long-term neurological damage in survivors. This indicates a need for an effective adjunct therapy to mitigate cognitive, behavioral, and brain pathology associated with OPNA exposure. Saracatinib (SAR), a selective Src tyrosine kinase inhibitor, has emerged as a potential candidate, given its protective properties in experimental models of excitotoxicity and neuroinflammation. Here, we evaluate the therapeutic efficacy of SAR in mitigating long-term neurological deficits triggered by acute exposure to soman in a rat model. Methods: Mixed-sex adult Sprague Dawley rats were exposed to soman (132 µg/kg, s.c.) and immediately treated with atropine (2 mg/kg, i.m.) and HI-6 (125 mg/kg, i.m.). Seizure severity was quantified for an hour before administering midazolam (3 mg/kg, i.m.). One-hour post-midazolam, SAR/vehicle was administered orally and daily for 18 weeks in the diet. After behavioral testing, brain MRI, and EEG acquisition, animals were perfused with 4% paraformaldehyde at 18 weeks post-soman. Serum and CSF were collected for nitro-oxidative markers and proinflammatory cytokines. Brains were processed for neuroinflammation and neurodegeneration markers. Results: SAR treatment attenuated the soman-induced anxiety/fear-like behavior and motor impairment and modulated the severity, frequency, and duration of seizures. Despite improved hippocampal functional connectivity (MRI), SAR did not reverse soman-induced learning and memory deficits at 5–7 weeks. However, 18 weeks of SAR treatment demonstrated anti-inflammatory and antioxidant properties, mitigated reactive gliosis and neurodegeneration, and partially protected somatostatin parvalbumin inhibitory neurons. The glial scars in the amygdala were reduced in SAR-treated animals compared to the vehicle-treated group. Conclusions: Long-term SAR treatment revealed disease-modifying effects by protecting the brain from soman-induced neuroinflammation and neurodegeneration, while also reducing seizure severity and modulating the frequency and duration of seizures. Furthermore, it provided partial protection against behavioral impairments and MRI deficits in the short term. These findings highlight the therapeutic potential of Src tyrosine kinase inhibition in addressing chronic neurotoxicity induced by soman.This is a preprint from Massey, Nyzil, Suraj S. Vasanthi, Claire Holtkamp, Christina Meyer, Nikhil S. Rao, Luis G. Gimenez-Lirola, Chong Wang et al. "Mitigating Organophosphate Nerve Agent, Soman (GD)-Induced Long-Term Neurotoxicity: Saracatinib, a Src Tyrosine Kinase Inhibitor, as a Potential Countermeasure." (2025). doi: https://doi.org/10.21203/rs.3.rs-6674766/v1

Enhanced mitochondrial fission inhibits triple-negative breast cancer cell migration through an ROS-dependent mechanism

Summary: Mitochondria produce reactive oxygen species (ROS), which function in signal transduction. Mitochondrial dynamics, encompassing morphological shifts between fission and fusion, can directly impact ROS levels in cancer cells. In this study, we identified an ROS-dependent mechanism for how enhanced mitochondrial fission inhibits triple negative breast cancer (TNBC) cell migration. We found that enforcing mitochondrial fission in TNBC resulted in an increase in intracellular ROS levels and reduced cell migration and the formation of actin-rich migratory structures. Consistent with mitochondrial fission, increasing ROS levels in cells inhibited cell migration. Conversely, reducing ROS levels with either a global or mitochondrially targeted scavenger overcame the inhibitory effects of mitochondrial fission. Mechanistically, we found that the ROS sensitive SHP-1/2 phosphatases partially regulate inhibitory effects of mitochondrial fission on TNBC migration. Overall, our work reveals the inhibitory effects of ROS in TNBC and supports mitochondrial dynamics as a potential therapeutic target for cancer

Inhibiting CXCR4 reduces immunosuppressive effects of myeloid cells in breast cancer immunotherapy

Abstract Patients with triple negative breast cancer (TNBC) show only modest response rates to immune checkpoint inhibitor immunotherapy, motivating ongoing efforts to identify approaches to boost efficacy. Using an immunocompetent mouse model of TNBC, we investigated combination therapy with an anti-PD-1 immunotherapy antibody plus balixafortide, a cyclic peptide inhibitor of CXCR4. Cell-based assays demonstrated that balixafortide functions as an inverse agonist, establishing a mode of action distinct from most compounds targeting CXCR4. Combination anti-PD-1 plus balixafortide significantly reduced growth of orthotopic tumors and extended overall survival relative to single agent therapy or vehicle. Adding balixafortide to anti-PD-1 increased numbers of tertiary lymphoid structures, a marker of local tumor immune responses associated with favorable response to immunotherapy in TNBC. Single cell RNA sequencing revealed that combination anti-PD-1 plus balixafortide reduced T cell exhaustion and increased markers of effector T cell activity. Combination therapy also reduced signatures of immunosuppressive myeloid derived suppressor cells (MDSCs) in tumors. MDSCs isolated from mice treated with anti-PD-1 plus balixafortide showed reduced inhibition of T cell proliferation following ex vivo stimulation. These studies demonstrate that combining inhibition of CXCR4 with anti-PD-1 to enhances responses to checkpoint inhibitor immunotherapy in TNBC, supporting future clinical trials

Ultrasound-Induced Mechanical Compaction in Acoustically Responsive Scaffolds Promotes Spatiotemporally Modulated Signaling in Triple Negative Breast Cancer

Cancer cells continually sense and respond to mechanical cues from the extracellular matrix (ECM). Interaction with the ECM can alter intracellular signaling cascades, leading to changes in processes that promote cancer cell growth, migration, and survival. The present study used a recently developed composite hydrogel composed of a fibrin matrix and phase-shift emulsion, termed an acoustically responsive scaffold (ARS), to investigate effects of local mechanical properties on breast cancer cell signaling. Treatment of ARSs with focused ultrasound drives acoustic droplet vaporization (ADV) in a spatiotemporally controlled manner, inducing local compaction and stiffening of the fibrin matrix adjacent to the matrix–bubble interface. Combining ARSs and live single cell imaging of triple-negative breast cancer cells, it is discovered that both basal and growth-factor stimulated activities of protein kinase B (also known as Akt) and extracellular signal-regulated kinase (ERK), two major kinases driving cancer progression, negatively correlate with increasing distance from the ADV-induced bubble both in vitro and in a mouse model. Together, these data demonstrate that local changes in ECM compaction regulate Akt and ERK signaling in breast cancer and support further applications of the novel ARS technology to analyze spatial and temporal effects of ECM mechanics on cell signaling and cancer biology.The study uses a smart hydrogel system with focused ultrasound for precise temporal and spatial control of tissue compaction. Incorporating breast cancer cells into this hydrogel system reveals that ultrasound-triggered increases in compaction of extracellular matrix promotes signaling through pathways known to drive proliferation and aggressive features in breast cancer and other malignancies.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/172808/1/adhm202101672_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/172808/2/adhm202101672.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/172808/3/adhm202101672-sup-0001-SuppMat.pd

Figure S6 from Bone Marrow Mesenchymal Stem Cells Induce Metabolic Plasticity in Estrogen Receptor–Positive Breast Cancer

Figure S6. Flow cytometry data for CSC stains. Cell type of interest is bolded in co-culture comparisons. A) Representative gating scheme for ALDH activity, CD24, and CD44 analyses. B-D) Flow cytometry data for MCF7 (B), T47D (C), and HCC1428 (HCC, D) cells in monoculture versus co-culture with MSCs stained for ALDH activity, CD24, and CD44. N > 10,000 cells.</p

Figure S7 from Bone Marrow Mesenchymal Stem Cells Induce Metabolic Plasticity in Estrogen Receptor–Positive Breast Cancer

Figure S7. Inhibition of monocarboxylate transporters regulates intracellular and extracellular lactate. Western blot shows expression of MCT4 in monocultures of MCF7, T47D, HS5, and HS27a cells. Images are from adjacent lanes on one gel and non-adjacent lanes from a second gel. We show β-actin as a loading control. B,C) Graphs show mean values ± standard deviation for relative concentrations of intracellular (B) and extracellular (C) lactate in monocultures of MCF7 cells or co-cultures of MCF7 and HS5 cells treated with 10 µM syrosingopine, an inhibitor of MCT1/4, or vehicle for 3 days. *** p<0.0001 using paired t tests for each comparison.</p

Figure S7 from Bone Marrow Mesenchymal Stem Cells Induce Metabolic Plasticity in Estrogen Receptor–Positive Breast Cancer

Figure S7. Inhibition of monocarboxylate transporters regulates intracellular and extracellular lactate. Western blot shows expression of MCT4 in monocultures of MCF7, T47D, HS5, and HS27a cells. Images are from adjacent lanes on one gel and non-adjacent lanes from a second gel. We show β-actin as a loading control. B,C) Graphs show mean values ± standard deviation for relative concentrations of intracellular (B) and extracellular (C) lactate in monocultures of MCF7 cells or co-cultures of MCF7 and HS5 cells treated with 10 µM syrosingopine, an inhibitor of MCT1/4, or vehicle for 3 days. *** p<0.0001 using paired t tests for each comparison.</p

Figure S6 from Bone Marrow Mesenchymal Stem Cells Induce Metabolic Plasticity in Estrogen Receptor–Positive Breast Cancer

Figure S6. Flow cytometry data for CSC stains. Cell type of interest is bolded in co-culture comparisons. A) Representative gating scheme for ALDH activity, CD24, and CD44 analyses. B-D) Flow cytometry data for MCF7 (B), T47D (C), and HCC1428 (HCC, D) cells in monoculture versus co-culture with MSCs stained for ALDH activity, CD24, and CD44. N > 10,000 cells.</p

Figure S2 from Bone Marrow Mesenchymal Stem Cells Induce Metabolic Plasticity in Estrogen Receptor–Positive Breast Cancer

Figure S2. NADH lifetime and ROS quantification for ER+ breast cancer cells with CM and MSCs in monoculture and co-culture. Cell type of interest is bolded in co-culture comparisons. A-B) Histograms of NADH lifetime for HS5 (A) and HS27a (B) in monoculture or co-culture with MCF7, T47D, and HCC1428 (HCC) ER+ breast cancer cells. C-E) Histograms of NADH lifetime for MCF7 (C), T47D (D), and HCC1428 (HCC, E) cells in co-culture and monoculture with control or CM from HS5 or HS27a MSCs. Dashed lines represent cancer cells in CM and solid lines represent cancer cells in monoculture or co-culture with MSCs. N>100 cells.</p

Figure S3 from Bone Marrow Mesenchymal Stem Cells Induce Metabolic Plasticity in Estrogen Receptor–Positive Breast Cancer

Figure S3. ROS quantification for MSCs in monoculture and co-culture. Flow cytometry data for ROS detected by CellROX Green in HS5 and HS27a MSCs in monoculture or co-culture with MCF7 (A), T47D (B), or HCC1428 (C) ER+ breast cancer cells. Solid and dashed lines depict MSCs in monoculture co-culture, respectively. Cell type of interest is bolded in co-culture comparisons. N>10,000 cells.</p