ABSTRACT
Breast cancer therapies using checkpoints alone have not been highly effective. Based on previous experiences using the ConvitVax, an autologous tumor cells/bacillus Calmette-Guérin (BCG)/formalin-based vaccine, in breast cancer and the potential success of combined therapies, we sought to ascertain whether the ConvitVax combined with anti-PD-1 enhances the antitumor effect in a 4T1 breast cancer model. Animals received four weekly injections of either PBS (G1), ConvitVax (200 µg cell homogenate, 0.0625 mg BCG, 0.02% formalin) (G2), 50 µg anti-PD-1 (G3), or ConvitVax plus anti-PD-1 (200 µg cell homogenate, 0.0625 mg BCG, 0.02% formalin, 50 µg anti-PD-1) (G4). Five weeks post tumor induction all mice were euthanized, tumors extracted and evaluated pathologically and by immunohistochemistry. The combination group (G4) showed 10% more tumor necrosis, greater infiltration of PD-1+ cells and lower infiltration of TAMs, evidencing that the combination of ConvitVax and anti-PD-1 can improve the antitumor effect of the vaccine. Using a higher anti-PD-1 dose and administering each treatment at different times could further potentiate the effect of our therapy. Given the vaccine's low cost and simple preparation, its use in combination with checkpoints or other target-specific compounds may lead to a highly effective personalized breast cancer immunotherapy.
ABSTRACT
Collapsin Response Mediator Protein 2 (CRMP2) is an intracellular protein involved in axon and dendrite growth and specification. In this study, CRMP2 was identified in a conditioned media derived from degenerated sciatic nerves (CM). On cultured rat hippocampal neurons, acute extracellular application of CM or partially purified recombinant CRMP2 produced an increase in cytoplasmic calcium. The increase in cytoplasmic calcium was mostly mediated through NMDA receptors, with a minor contribution of N-type VDCC, and it was maintained as long as CM was present. By using live-labeling of CRMP2, Ca2+ channel binding domain 3 (CBD3) peptide derived from CRMP2, and recombinant CRMP2, we demonstrated that that this effect was mediated by an action on the extracellular side of the NMDA receptor. This is the first report of an extracellular action of CRMP2. Prolonged exposure to extracellular CRMP2, may contribute to neuronal calcium dysregulation and neuronal damage.
Subject(s)
Calcium/metabolism , Central Nervous System Agents/administration & dosage , Cytoplasm/drug effects , Intercellular Signaling Peptides and Proteins/administration & dosage , Nerve Tissue Proteins/administration & dosage , Receptors, N-Methyl-D-Aspartate/metabolism , Animals , Cations, Divalent/metabolism , Cells, Cultured , Central Nervous System Agents/isolation & purification , Culture Media, Conditioned , Cytoplasm/metabolism , Extracellular Space , Hippocampus/drug effects , Hippocampus/metabolism , Male , Mice , Molecular Docking Simulation , Nerve Tissue Proteins/isolation & purification , Nerve Tissue Proteins/metabolism , Neurons/drug effects , Neurons/metabolism , Optic Nerve/metabolism , Rats, Sprague-Dawley , Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors , Recombinant Proteins/administration & dosage , Sciatic Nerve/metabolismABSTRACT
Pharmacological treatment in animal models of cerebral disease imposes the problem of repeated injection protocols that may induce stress in animals and result in impermanent tissue levels of the drug. Additionally, drug delivery to the brain is delicate due to the blood brain barrier (BBB), thus significantly reducing intracerebral concentrations of selective drugs after systemic administration. Therefore, a system that allows both constant drug delivery without peak levels and circumvention of the BBB is in order to achieve sufficiently high intracerebral concentrations of drugs that are impermeable to the BBB. In this context, miniosmotic pumps represent an ideal system for constant drug delivery at a fixed known rate that eludes the problem of daily injection stress in animals and that may also be used for direct brain delivery of drugs. Here, we describe a method for miniosmotic pump implantation and post operatory care that should be given to animals in order to successfully apply this technique. We embed the aforementioned experimental paradigm in standard procedures that are used for studying neuroplasticity within the brain of C57BL6 mice. Thus, we exposed animals to 30 min brain infarct and implanted with miniosmotic pumps connected to the skull via a cannula in order to deliver a pro-plasticity drug. Behavioral testing was done during 30 days of treatment. After removal the animals received injections of anterograde tract tracers to analyze neuronal plasticity in the chronic phase of recovery. Results indicated that neuroprotection by the delivered drug was accompanied with increase in motor fibers crossing the midline of the brain at target structures. The results affirm the value of these techniques for drug administration and brain plasticity studies in modern neuroscience.
