Programming tumor evolution with selection gene drives to proactively combat drug resistance.

Most targeted anticancer therapies fail due to drug resistance evolution. Here we show that tumor evolution can be reproducibly redirected to engineer therapeutic opportunity, regardless of the exact ensemble of pre-existing genetic heterogeneity. We develop a selection gene drive system that is stably introduced into cancer cells and is composed of two genes, or switches, that couple an inducible fitness advantage with a shared fitness cost. Using stochastic models of evolutionary dynamics, we identify the design criteria for selection gene drives. We then build prototypes that harness the selective pressure of multiple approved tyrosine kinase inhibitors and employ therapeutic mechanisms as diverse as prodrug catalysis and immune activity induction. We show that selection gene drives can eradicate diverse forms of genetic resistance in vitro. Finally, we demonstrate that model-informed switch engagement effectively targets pre-existing resistance in mouse models of solid tumors. These results establish selection gene drives as a powerful framework for evolution-guided anticancer therapy.

High-Grade Glioma Recurrence Is Delayed in Hispanic Patients despite Severe Social Vulnerability: A Retrospective Cohort Study.

High-grade gliomas (HGGs; WHO grade III or IV) are the most common and lethal brain malignancy. Patients of Hispanic ethnicity are diagnosed with HGGs earlier than non-Hispanic patients, but they exhibit improved HGG survival following diagnosis. Either environmental or biological factors could explain this survival benefit. We aimed to determine if post-diagnosis advantages would still be present in Hispanic patients with high social vulnerability, an environmental condition predisposing patients to poor oncologic outcomes. HGG outcomes were retrospectively assessed in a cohort of 22 Hispanic patients and 33 non-Hispanic patients treated for HGGs from 2015 to 2020 at a single institution that serves a highly vulnerable region. Compared to non-Hispanic patients, Hispanic patients demonstrated higher social vulnerability index scores (96.8 + 0.7 vs. 76.3 + 4.6; *** p = 0.0002) and a 14-month longer interval between diagnosis and recurrence (19.7 + 5.9 (n = 13) vs. 5.5 + 0.6 months (n = 19); ** p = 0.001). In only those patients with more aggressive IDH-1 wildtype tumors (glioblastoma), Hispanic ethnicity still related to a longer time before recurrence (15.8 + 5.9 months (n = 9); 5.5 + 0.6 months (n = 18); * p = 0.034), and in a multivariate analysis, Hispanic ethnicity predicted time-to-recurrence (* p = 0.027) independent of patient age, functional status, MGMT gene methylation, or treatments received. Therefore, environmental factors, specifically social vulnerability, did not obscure the post-diagnosis benefits associated with Hispanic ethnicity. In future experiments, basic studies should be prioritized which investigate the cellular or genetic mechanisms underlying this ethnicity effect on HGG progression in the hopes of improving care for these devastating malignancies.

Novel biomarker discovery through comprehensive proteomic analysis of lupus mouse serum.

OBJECTIVES: The difficulty of monitoring organ-specific pathology in systemic lupus erythematosus (SLE) often complicates disease prognostication and treatment. Improved non-invasive biomarkers of active organ pathology, particularly lupus nephritis, would improve patient care. We sought to validate and apply a novel strategy to generate the first comprehensive serum proteome of a lupus mouse model and identify mechanism-linked lupus biomarker candidates for subsequent clinical investigation. METHODS: Serum levels of 1308 diverse proteins were measured in eight adult female MRL/lpr lupus mice and eight control MRL/mpj mice. ELISA validation confirmed fold increases. Protein enrichment analysis provided biological relevance to findings. Individual protein levels were correlated with measures of lymphoproliferative, humoral, and renal disease. RESULTS: Four hundred and six proteins were increased in MRL/lpr serum, including proteins increased in human SLE such as VCAM-1, L-selectin, TNFRI/II, TWEAK, CXCL13, MCP-1, IP-10, IL-10, and TARC. Newly validated proteins included IL-6, IL-17, and MDC. Results of pathway enrichment analysis, which revealed enhancement of cytokine signaling and immune cell migration, reinforced the similarity of the MRL/lpr disease to human pathology. Fifty-two proteins positively correlated with at least one measure of lupus-like disease. TECK, TSLP, PDGFR-alpha, and MDC were identified as novel candidate biomarkers of renal disease. CONCLUSIONS: We successfully validated a novel serum proteomic screening strategy in a spontaneous murine lupus model that highlighted potential new biomarkers. Importantly, we generated a comprehensive snapshot of the serum proteome which will enable identification of other candidates and serve as a reference for future mechanistic and therapeutic studies in lupus.

Progress and unmet needs in understanding fundamental mechanisms of autoimmunity.

The rising incidence of autoimmune diseases is straining the healthcare system's capacity to care for patients with autoimmunity. To further compound this growing crisis, this rise occurs at a time when virulent infectious diseases exacerbate pre-existing conditions. Despite some novel targeted therapies introduced over the preceding decades, current treatment strategies must often fall back on non-specific immunosuppression, inflicting its own toll on patient morbidity. To improve patient care, we must re-double our efforts to understand and target the fundamental mechanisms of autoimmune disease initiation and progression. Technologic innovations have recently accelerated our ability to discover key components of the processes leading to loss of tolerance and propagation of self-tissue damage in autoimmune conditions. The special issue "Cellular and Molecular Mechanisms of Autoimmunity" highlights many of these findings through primary research and review articles which detail advances in genetics, molecular processes, cellular functions, and host-pathogen interactions. Discussion of topics ranging from non-coding RNA and the complement cascade to T-cell aging and the microbiome uncovers exciting avenues for basic and clinical investigation. Importantly, the issue seeks to focus attention on both established and emerging mechanisms of autoimmunity to ultimately help improve the specificity, safety, and efficacy of treatments for this group of challenging immune disorders.

Noxious Stimulation Induces Acute Hemorrhage and Impairs Long-Term Recovery after Spinal Cord Injury (SCI) in Female Rats: Evidence Estrous Cycle May Have a Modulatory Effect.

Spinal cord injuries (SCIs) are often the result of traumatic accidents, which also produce multiple other injuries (polytrauma). Nociceptive input from associated injuries has been shown to significantly impair recovery post-SCI. Historically, work in our laboratory has focused exclusively on male animals; however, increasing incidence of SCI in females requires research to determine whether pain (nociceptive) input poses the same risk to their recovery. Some animal studies have shown that females demonstrate greater tissue preservation and better locomotor recovery post-SCI. Given this, we examined the effect of sex on SCI recovery in two pain models-intermittent electrical stimulation (shock) to the tail or capsaicin injection to the hindpaw. Female rats received a lower thoracic contusion injury and were exposed to noxious stimulation the next day. The acute effect of noxious input on cardiovascular function, locomotor performance, and hemorrhage were assessed. Treatment with capsaicin or noxious electrical stimulation disrupted locomotor performance, increased blood pressure, and disrupted stepping. Additional experiments examined the long-term consequences of noxious input, demonstrating that both noxious electrical stimulation and capsaicin impair long-term recovery in female rats. Interestingly, injury had a greater effect on behavioral performance when progesterone and estrogen were low (metestrus). Conversely, nociceptive input led to a greater disruption in locomotor performance and produced a greater rise in blood pressure in animals injured during estrus.

Hemorrhage and Locomotor Deficits Induced by Pain Input after Spinal Cord Injury Are Partially Mediated by Changes in Hemodynamics.

Nociceptive input diminishes recovery and increases lesion area after a spinal cord injury (SCI). Recent work has linked these effects to the expansion of hemorrhage at the site of injury. The current article examines whether these adverse effects are linked to a pain-induced rise in blood pressure (BP) and/or flow. Male rats with a low-thoracic SCI were treated with noxious input (electrical stimulation [shock] or capsaicin) soon after injury. Locomotor recovery and BP were assessed throughout. Tissues were collected 3 h, 24 h, or 21 days later. Both electrical stimulation and capsaicin undermined locomotor function and increased the area of hemorrhage. Changes in BP/flow varied depending on type of noxious input, with only shock producing changes in BP. Providing behavioral control over the termination of noxious stimulation attenuated the rise in BP and hemorrhage. Pretreatment with the α-1 adrenergic receptor inverse agonist, prazosin, reduced the stimulation-induced rise in BP and hemorrhage. Prazosin also attenuated the adverse effect that noxious stimulation has on long-term recovery. Administration of the adrenergic agonist, norepinephrine 1 day after injury induced an increase in BP and disrupted locomotor function, but had little effect on hemorrhage. Further, inducing a rise in BP/flow using norepinephrine undermined long-term recovery and increased tissue loss. Mediational analyses suggest that the pain-induced rise in blood flow may foster hemorrhage after SCI. Increased BP appears to act through an independent process to adversely affect locomotor performance, tissue sparing, and long-term recovery.

Contribution of Brain Processes to Tissue Loss After Spinal Cord Injury: Does a Pain-Induced Rise in Blood Pressure Fuel Hemorrhage?

Pain (nociceptive) input soon after spinal cord injury (SCI) expands the area of tissue loss (secondary injury) and impairs long-term recovery. Evidence suggests that nociceptive stimulation has this effect because it promotes acute hemorrhage. Disrupting communication with the brain blocks this effect. The current study examined whether rostral systems exacerbate tissue loss because pain input drives an increase in systolic blood pressure (BP) and flow that fuels blood infiltration. Rats received a moderate contusion injury to the lower thoracic (T12) spinal cord. Communication with rostral processes was disrupted by cutting the spinal cord 18 h later at T2. Noxious electrical stimulation (shock) applied to the tail (Experiment 1), or application of the irritant capsaicin to one hind paw (Experiment 2), increased hemorrhage at the site of injury. Shock, but not capsaicin, increased systolic BP and tail blood flow in sham-operated rats. Cutting communication with the brain blocked the shock-induced increase in systolic BP and tail blood flow. Experiment 3 examined the effect of artificially driving a rise in BP with norepinephrine (NE) in animals that received shock. Spinal transection attenuated hemorrhage in vehicle-treated rats. Treatment with NE drove a robust increase in BP and tail blood flow but did not increase the extent of hemorrhage. The results suggest pain input after SCI can engage rostral processes that fuel hemorrhage and drive sustained cardiovascular output. An increase in BP was not, however, necessary or sufficient to drive hemorrhage, implicating other brain-dependent processes.

High-affinity mutant Interleukin-13 targeted CAR T cells enhance delivery of clickable biodegradable fluorescent nanoparticles to glioblastoma.

Glioblastoma (GBM), the deadliest form of brain cancer, presents long-standing problems due to its localization. Chimeric antigen receptor (CAR) T cell immunotherapy has emerged as a powerful strategy to treat cancer. IL-13-receptor-α2 (IL13Rα2), present in over 75% of GBMs, has been recognized as an attractive candidate for anti-glioblastoma therapy. Here, we propose a novel multidisciplinary approach to target brain tumors using a combination of fluorescent, therapeutic nanoparticles and CAR T cells modified with a targeted-quadruple-mutant of IL13 (TQM-13) shown to have high binding affinity to IL13Rα2-expressing glioblastoma cells with low off-target toxicity. Azide-alkyne cycloaddition conjugation of nanoparticles to the surface of T cells allowed a facile, selective, and high-yielding clicking of the nanoparticles. Nanoparticles clicked onto T cells were retained for at least 8 days showing that the linkage is stable and promising a suitable time window for in vivo delivery. T cells clicked with doxorubicin-loaded nanoparticles showed a higher cytotoxic effect in vitro compared to bare T cells. In vitro and in vivo T cells expressing TQM-13 served as delivery shuttles for nanoparticles and significantly increased the number of nanoparticles reaching brain tumors compared to nanoparticles alone. This work represents a new platform to allow the delivery of therapeutic nanoparticles and T cells to solid tumors.

Brain-Dependent Processes Fuel Pain-Induced Hemorrhage After Spinal Cord Injury.

Pain (nociceptive) input caudal to a spinal contusion injury can undermine long-term recovery and increase tissue loss (secondary injury). Prior work suggests that nociceptive stimulation has this effect because it fosters the breakdown of the blood-spinal cord barrier (BSCB) at the site of injury, allowing blood to infiltrate the tissue. The present study examined whether these effects impact tissue rostral and caudal to the site of injury. In addition, the study evaluated whether cutting communication with the brain, by means of a rostral transection, affects the development of hemorrhage. Eighteen hours after rats received a lower thoracic (T11-12) contusion injury, half underwent a spinal transection at T2. Noxious electrical stimulation (shock) was applied 6 h later. Cellular assays showed that, in non-transected rats, nociceptive stimulation increased hemoglobin content, activated pro-inflammatory cytokines and engaged signals related to cell death at the site of injury. These effects were not observed in transected animals. In the next experiment, the spinal transection was performed at the time of contusion injury. Nociceptive stimulation was applied 24 h later and tissue was sectioned for microscopy. In non-transected rats, nociceptive stimulation increased the area of hemorrhage and this effect was blocked by spinal transection. These findings imply that the adverse effect of noxious stimulation depends upon spared ascending fibers and the activation of rostral (brain) systems. If true, stimulation should induce less hemorrhage after a severe contusion injury that blocks transmission to the brain. To test this, rats were given a mild, moderate, or severe, injury and electrical stimulation was applied 24 h later. Histological analyses of longitudinal sections showed that nociceptive stimulation triggered less hemorrhage after a severe contusion injury. The results suggest that brain-dependent processes drive pain-induced hemorrhage after spinal cord injury (SCI).

NF-kB signaling in myeloid cells mediates the pathogenesis of immune-mediated nephritis.

Immune-mediated glomerulonephritis is a serious end organ pathology that commonly affects patients with systemic lupus erythematosus (SLE). A classic murine model used to study lupus nephritis (LN) is nephrotoxic serum nephritis (NTN), in which mice are passively transferred nephrotoxic antibodies. We have previously shown that macrophages are important in the pathogenesis of LN. To further investigate the mechanism by which macrophages contribute to the pathogenic process, and to determine if this contribution is mediated by NF-κB signaling, we created B6 mice which had RelA knocked out in myeloid cells, thus inhibiting classical NF-κB signaling in this cell lineage. We induced NTN in this strain to assess the importance of macrophage derived NF-κB signaling in contributing to disease progression. Myeloid cell RelA knock out (KO) mice injected with nephrotoxic serum had significantly attenuated proteinuria, lower BUN levels, and improved renal histopathology compared to control injected wildtype B6 mice (WT). Inhibiting myeloid NF-κB signaling also decreased inflammatory modulators within the kidneys. We found significant decreases of IL-1a, IFNg, and IL-6 in kidneys from KO mice, but higher IL-10 expression. Flow cytometry revealed decreased numbers of kidney infiltrating classically activated macrophages in KO mice as well. Our results indicate that macrophage NF-κB signaling is instrumental in the contribution of this cell type to the pathogenesis of NTN. While approaches which decrease macrophage numbers can be effective in immune mediated nephritis, more targeted treatments directed at modulating macrophage signaling and/or function could be beneficial, at least in the early stages of disease.

Pain Input After Spinal Cord Injury (SCI) Undermines Long-Term Recovery and Engages Signal Pathways That Promote Cell Death.

Pain (nociceptive) input caudal to a spinal contusion injury increases tissue loss and impairs long-term recovery. It was hypothesized that noxious stimulation has this effect because it engages unmyelinated pain (C) fibers that produce a state of over-excitation in central pathways. The present article explored this issue by assessing the effect of capsaicin, which activates C-fibers that express the transient receptor potential vanilloid receptor-1 (TRPV1). Rats received a lower thoracic (T11) contusion injury and capsaicin was applied to one hind paw the next day. For comparison, other animals received noxious electrical stimulation at an intensity that engages C fibers. Both forms of stimulation elicited similar levels of c-fos mRNA expression, a cellular marker of nociceptive activation, and impaired long-term behavioral recovery. Cellular assays were then performed to compare the acute effect of shock and capsaicin treatment. Both forms of noxious stimulation increased expression of tumor necrosis factor (TNF) and caspase-3, which promotes apoptotic cell death. Shock, but not capsaicin, enhanced expression of signals related to pyroptotic cell death [caspase-1, inteleukin-1 beta (IL-1ß)]. Pyroptosis has been linked to the activation of the P2X7 receptor and the outward flow of adenosine triphosphate (ATP) through the pannexin-1 channel. Blocking the P2X7 receptor with Brilliant Blue G (BBG) reduced the expression of signals related to pyroptotic cell death in contused rats that had received shock. Blocking the pannexin-1 channel with probenecid paradoxically had the opposite effect. BBG enhanced long-term recovery and lowered reactivity to mechanical stimulation applied to the girdle region (an index of chronic pain), but did not block the adverse effect of nociceptive stimulation. The results suggest that C-fiber input after injury impairs long-term recovery and that this effect may arise because it induces apoptotic cell death.

Pain Input Impairs Recovery after Spinal Cord Injury: Treatment with Lidocaine.

More than 90% of spinal cord injuries are caused by traumatic accidents and are often associated with other tissue damage (polytrauma) that can provide a source of continued pain input during recovery. In a clinically relevant spinal cord contusion injury model, prior work has shown that noxious stimulation at an intensity that engages pain (C) fibers soon after injury augments secondary injury and impairs functional recovery. Noxious input increases the expression of pro-inflammatory cytokines (interleukin 1ß and 18), cellular signals associated with cell death (caspase 3 and 8), and physiological signs of hemorrhage. Here, it is shown that reducing neural excitability after spinal cord injury (SCI) with the local anesthetic lidocaine (micro-injected by means of a lumbar puncture) blocks these adverse cellular effects. In contrast, treatment with an analgesic dose of morphine had no effect. Contused rats that received nociceptive stimulation soon after injury exhibited poor locomotor recovery, less weight gain, and greater tissue loss at the site of injury. Prophylactic application of lidocaine blocked the adverse effect of nociceptive stimulation on behavioral recovery and reduced tissue loss from secondary injury. The results suggest that quieting neural excitability using lidocaine can reduce the adverse effect of pain input (from polytrauma or surgery) after SCI.