In Vivo and Ex Vivo Patient-Derived Tumor Xenograft Models of Lymphoma for Drug Discovery.

In the hemato-oncology field, remarkable scientific progress has been achieved, primarily propelled by the discovery of new technologies, improvement in genomics, and novel in vitro and in vivo models. The establishment of multiple cell line collections and the development of instrumental mouse models enhanced our ability to discover effective therapeutics. However, cancer models that faithfully mimic individual cancers are still imperfect. Patient-derived tumor xenografts (PDTXs) have emerged as a powerful tool for identifying the mechanisms which drive tumorigenesis and for testing potential therapeutic interventions. The recognition that PDTXs can maintain many of the donor samples' properties enabled the development of new strategies for discovering and implementing therapies. Described in this article are protocols for the generation and characterization of lymphoma PDTXs that may be used as the basis of shared procedures. Universal protocols will foster the model utilization, enable the integration of public and private repositories, and aid in the development of shared platforms. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Tissue handling and cryopreservation of primary and PDTX samples Basic Protocol 2: Performing tumor implant in immunocompromised mice PDTX models Alternate Protocol 1: Intra-medullary femoral injection Alternate Protocol 2: Intravenous injection Alternate Protocol 3: Intraperitoneal injection Support Protocol 1: Phenotypical characterization of PDTXs by flow cytometry Support Protocol 2: Biological and molecular characterization of PDTX tumors by PCR detection of IGK, IGH, and TCR rearrangements Basic Protocol 3: Harvesting PDTX-derived tumor cells for ex vivo experiments Basic Protocol 4: In vivo testing of multiple compounds in a PDTX mouse model.

Repeated anodal trans-spinal direct current stimulation results in long-term reduction of spasticity in mice with spinal cord injury.

KEY POINTS: Spasticity is a disorder of muscle tone that is associated with lesions of the motor system. This condition involves an overactive spinal reflex loop that resists the passive lengthening of muscles. Previously, we established that application of anodal trans-spinal direct current stimulation (a-tsDCS) for short periods of time to anaesthetized mice sustaining a spinal cord injury leads to an instantaneous reduction of spasticity. However, the long-term effects of repeated a-tsDCS and its mechanism of action remained unknown. In the present study, a-tsDCS was performed for 7 days and this was found to cause long-term reduction in spasticity, increased rate-dependent depression in spinal reflexes, and improved ground and skill locomotion. Pharmacological, molecular and cellular evidence further suggest that a novel mechanism involving Na-K-Cl cotransporter isoform 1 mediates the observed long-term effects of repeated a-tsDCS. ABSTRACT: Spasticity can cause pain, fatigue and sleep disturbances; restrict daily activities such as walking, sitting and bathing; and complicate rehabilitation efforts. Thus, spasticity negatively influences an individual's quality of life and novel therapeutic interventions are needed. We previously demonstrated in anaesthetized mice that a short period of trans-spinal subthreshold direct current stimulation (tsDCS) reduces spasticity. In the present study, the long-term effects of repeated tsDCS to attenuate abnormal muscle tone in awake female mice with spinal cord injuries were investigated. A motorized system was used to test velocity-dependent ankle resistance and associated electromyographical activity. Analysis of ground and skill locomotion was also performed, with electrophysiological, molecular and cellular studies being conducted to reveal a potential underlying mechanism of action. A 4 week reduction in spasticity was associated with an increase in rate-dependent depression of spinal reflexes, and ground and skill locomotion were improved following 7 days of anodal-tsDCS (a-tsDCS). Secondary molecular, cellular and pharmacological experiments further demonstrated that the expression of K-Cl co-transporter isoform 2 (KCC2) was not changed in animals with spasticity. However, Na-K-Cl cotransporter isoform 1 (NKCC1) was significantly up-regulated in mice that exhibited spasticity. When mice were treated with a-tsDCS, down regulation of NKCC1 was detected, and this level did not significantly differ from that in the non-injured control mice. Thus, long lasting reduction of spasticity by a-tsDCS via downregulation of NKCC1 may constitute a novel therapy for spasticity following spinal cord injury.

Transspinal direct current stimulation modulates migration and proliferation of adult newly born spinal cells in mice.

Direct current electrical fields have been shown to be a major factor in the regulation of cell proliferation, differentiation, migration, and survival, as well as in the maturation of dividing cells during development. During adulthood, spinal cord cells are continuously produced in both animals and humans, and they hold great potential for neural restoration following spinal cord injury. While the effects of direct current electrical fields on adult-born spinal cells cultured ex vivo have recently been reported, the effects of direct current electrical fields on adult-born spinal cells in vivo have not been characterized. Here, we provide convincing findings that a therapeutic form of transspinal direct current stimulation (tsDCS) affects the migration and proliferation of adult-born spinal cells in mice. Specifically, cathodal tsDCS attracted the adult-born spinal cells, while anodal tsDCS repulsed them. In addition, both tsDCS polarities caused a significant increase in cell number. Regarding the potential mechanisms involved, both cathodal and anodal tsDCS caused significant increases in expression of brain-derived neurotrophic factor, while expression of nerve growth factor increased and decreased, respectively. In the spinal cord, both anodal and cathodal tsDCS increased blood flow. Since blood flow and angiogenesis are associated with the proliferation of neural stem cells, increased blood flow may represent a major factor in the modulation of newly born spinal cells by tsDCS. Consequently, we propose that the method and novel findings presented in the current study have the potential to facilitate cellular, molecular, and/or bioengineering strategies to repair injured spinal cords.NEW & NOTEWORTHY Our results indicate that transspinal direct current stimulation (tsDCS) affects the migratory pattern and proliferation of adult newly born spinal cells, a cell population which has been implicated in learning and memory. In addition, our results suggest a potential mechanism of action regarding the functional effects of applying direct current. Thus tsDCS may represent a novel method by which to manipulate the migration and cell number of adult newly born cells and restore functions following brain or spinal cord injury.