Three-dimensional organoid culture unveils resistance to clinical therapies in adult and pediatric glioblastoma.

BACKGROUND: Glioblastoma (GBM) is the most common primary brain tumor with a dismal prognosis. The inherent cellular diversity and interactions within tumor microenvironments represent significant challenges to effective treatment. Traditional culture methods such as adherent or sphere cultures may mask such complexities whereas three-dimensional (3D) organoid culture systems derived from patient cancer stem cells (CSCs) can preserve cellular complexity and microenvironments. The objective of this study was to determine if GBM organoids may offer a platform, complimentary to traditional sphere culture methods, to recapitulate patterns of clinical drug resistance arising from 3D growth. METHODS: Adult and pediatric surgical specimens were collected and established as organoids. We created organoid microarrays and visualized bulk and spatial differences in cell proliferation using immunohistochemistry (IHC) staining, and cell cycle analysis by flow cytometry paired with 3D regional labeling. We tested the response of CSCs grown in each culture method to temozolomide, ibrutinib, lomustine, ruxolitinib, and radiotherapy. RESULTS: GBM organoids showed diverse and spatially distinct proliferative cell niches and include heterogeneous populations of CSCs/non-CSCs (marked by SOX2) and cycling/senescent cells. Organoid cultures display a comparatively blunted response to current standard-of-care therapy (combination temozolomide and radiotherapy) that reflects what is seen in practice. Treatment of organoids with clinically relevant drugs showed general therapeutic resistance with drug- and patient-specific antiproliferative, apoptotic, and senescent effects, differing from those of matched sphere cultures. CONCLUSIONS: Therapeutic resistance in organoids appears to be driven by altered biological mechanisms rather than physical limitations of therapeutic access. GBM organoids may therefore offer a key technological approach to discover and understand resistance mechanisms of human cancer cells.

Cuprizone-induced demyelination under physiological and post-stroke condition leads to decreased neurogenesis response in adult mouse brain.

Due to the limitation in treatment window of the rtPA (recombinant tissue plasminogen activator), the development of delayed treatment for stroke is needed. We previously reported that there is a difference in neurogenesis and neuroblast migration patterns in different mouse stroke models (proximal and distal middle cerebral artery occlusion models, pMCAo or dMCAo). Specifically, compared to robust neurogenesis and substantial migration of newly born neuroblasts in pMCAo model, dMCAo only illicit limited neurogenesis and migration of neuroblasts towards ischemic area. One potential reason for this difference is the relative location of ischemic area to white matter and the neurogenic niche (subventricular zone, SVZ). Specifically, white matter could serve as a physical barrier or inhibitory factor to neurogenesis and migration in the dMCAo model. Given that a major difference in human and rodent brains is the content of white matter in the brain, in this study, we further characterize these two models and test the important hypothesis that white matter is an important contributing inhibitory factor for the limited neurogenesis in the dMCAo model. We utilized a genetically inducible NSC-specific reporter mouse line (nestin-CreERT2-R26R-YFP) to label and track NSC proliferation, survival and differentiation in ischemic brain. To test whether myelin is inhibitory to neurogenesis in dMCAo model, we demyelinated mouse brains using cuprizone treatment after stroke and examined whether there is enhanced neurogenesis or migration of neuroblasts cells in stroke mice treated with cuprizone. Our data suggests that demyelination of the brain does not result in enhanced neurogenesis or migration of neuroblasts, supporting that myelin is not a major inhibitory factor for stroke-induced neurogenesis. In addition, our results suggest that in non-stroke mice, demyelination causes decreased neurogenesis in adult brain, indicating a potential positive role of myelin in maintenance of adult neural stem cell niche.

Sequential combined Treatment of Pifithrin-α and Posiphen Enhances Neurogenesis and Functional Recovery After Stroke.

OBJECTIVE: Although cerebral ischemia can activate endogenous reparative processes, such as proliferation of endogenous neural stem cells (NSCs) in the subventricular zone (SVZ) and subgranular zone (SGZ), the majority of these new cells die shortly after injury and do not appropriately differentiate into neurons, or migrate and functionally integrate into the brain. The purpose of this study was to examine a novel strategy for treatment of stroke after injury by optimizing the survival of ischemia-induced endogenous NSCs in the SVZ and SGZ. METHODS: Adult SVZ and SGZ NSCs were grown as neurospheres in culture and treated with a p53 inactivator, pifithrin-α (PFT-α), and an amyloid precursor protein (APP)-lowering drug, posiphen, and effects on neurosphere number, size and neuronal differentiation were evaluated. This combined sequential treatment approach was then evaluated in mice challenged with middle cerebral artery occlusion (MCAo). Locomotor behavior and cognition were evaluated at 4 weeks, and the number of new surviving neurons was quantified in nestin creERT2-YFP mice. RESULTS: PFT-α and posiphen enhanced the self-renewal, proliferation rate and neuronal differentiation of adult SVZ and SGZ NSCs in culture. Their sequential combination in mice challenged with MCAo-induced stroke mitigated locomotor and cognitive impairments and increased the survival of SVZ and SGZ NSCs cells. PFT-α and the combined posiphen+PFT-α treatment similarly improved locomotion behavior in stroke challenged mice. Notably, however, the combined treatment provided significantly more potent cognitive function enhancement in stroke mice, as compared with PFT-α single treatment. INTERPRETATION: Delayed combined sequential treatment with an inhibitor of p53 dependent apoptosis (PFT-α) and APP synthesis (posiphen) proved able to enhance stroke-induced endogenous neurogenesis and improve the functional recovery in stroke animals. Whereas the combined sequential treatment provided no further improvement in locomotor function, as compared with PFT-α alone treatment, suggesting a potential ceiling in the locomotion behavioral outcome in stroke animals, combined treatment more potently augmented cognitive function recovery after stroke.

Poststroke Sonic Hedgehog Agonist Treatment Improves Functional Recovery by Enhancing Neurogenesis and Angiogenesis.

BACKGROUND AND PURPOSE: Because of the limitation in treatment window of the r-tPA (recombinant tissue-type plasminogen activator), the development of delayed treatment for stroke is needed. In this study, we examined the efficacy of delayed poststroke treatment (post 3-8 days) of the sonic hedgehog pathway agonist on functional recovery and the underlying mechanisms. METHODS: We evaluated functional recovery at 1 month after stroke using locomotion analysis and Barnes maze test for cognitive function. We used a genetically inducible neural stem cell-specific reporter mouse line (nestin-CreERT2-R26R-YFP) to label and track their proliferation, survival, and differentiation in ischemic brain. Brain tissue damage, angiogenesis, and cerebral blood flow recovery was evaluated using magnetic resonance imaging techniques and immunostaining. RESULTS: Our results show that delayed treatment of sonic hedgehog pathway agonist in stroke mice results in enhanced functional recovery both in locomotor function and in cognitive function at 1 month after stroke. Furthermore, using the Nestincre-ERT2-YFP mice, we showed that poststroke sonic hedgehog pathway agonist treatment increased surviving newly born cells derived from both subventricular zone and subgranular zone neural stem cells, total surviving DCX+ (Doublecortin) neuroblast cells, and neurons (NeuN+/YFP+) in the ischemic brain. Sonic hedgehog pathway agonist treatment also improved the brain tissue repair in ischemic region supported by our T2-weighted magnetic resonance imaging, cerebral blood flow map by arterial spin labeling, and immunohistochemistry (α-smooth muscle actin and CD31 immunostaining). CONCLUSIONS: These data confirm an important role for the hedgehog pathway in poststroke brain repair and functional recovery, suggesting a prolonged treatment window for potential treatment strategy to modulate sonic hedgehog pathway after stroke.

Dopaminergic neuron-specific deletion of p53 gene is neuroprotective in an experimental Parkinson's disease model.

p53, a stress response gene, is involved in diverse cell death pathways and its activation has been implicated in the pathogenesis of Parkinson's disease (PD). However, whether the neuronal p53 protein plays a direct role in regulating dopaminergic (DA) neuronal cell death is unknown. In this study, in contrast to the global inhibition of p53 function by pharmacological inhibitors and in traditional p53 knock-out (KO) mice, we examined the effect of DA specific p53 gene deletion in DAT-p53KO mice. These DAT-p53KO mice did not exhibit apparent changes in the general structure and neuronal density of DA neurons during late development and in aging. However, in DA-p53KO mice treated with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), we found that the induction of Bax and p53 up-regulated modulator of apoptosis (PUMA) mRNA and protein levels by MPTP were diminished in both striatum and substantia nigra of these mice. Notably, deletion of the p53 gene in DA neurons significantly reduced dopaminergic neuronal loss in substantia nigra, dopaminergic neuronal terminal loss at striatum and, additionally, decreased motor deficits in mice challenged with MPTP. In contrast, there was no difference in astrogliosis between WT and DAT-p53KO mice in response to MPTP treatment. These findings demonstrate a specific contribution of p53 activation in DA neuronal cell death by MPTP challenge. Our results further support the role of programmed cell death mediated by p53 in this animal model of PD and identify Bax, BAD and PUMA genes as downstream targets of p53 in modulating DA neuronal death in the in vivo MPTP-induced PD model. We deleted p53 gene in dopaminergic neurons in late developmental stages and found that DA specific p53 deletion is protective in acute MPTP animal model possibly through blocking MPTP-induced BAX and PUMA up-regulation. Astrocyte activation measured by GFAP positive cells and GFAP gene up-regulation in the striatum shows no difference between wt and DA-p53 ko mice.

The shh signaling pathway is upregulated in multiple cell types in cortical ischemia and influences the outcome of stroke in an animal model.

Recently the sonic hedgehog (shh) signaling pathway has been shown to play an important role in regulating repair and regenerative responses after brain injury, including ischemia. However, the precise cellular components that express and upregulate the shh gene and the cellular components that respond to shh signaling remain to be identified. In this study, using a distal MCA occlusion model, our data show that the shh signal is upregulated both at the cortical area near the injury site and in the adjacent striatum. Multiple cell types upregulate shh signaling in ischemic brain, including neurons, reactive astrocytes and nestin-expressing cells. The shh signaling pathway genes are also expressed in the neural stem cells (NSCs) niche in the subventricular zone (SVZ). Conditional deletion of the shh gene in nestin-expressing cells both at the SVZ niche and at the ischemic site lead to significantly more severe behavioral deficits in these shh iKO mice after cortical stroke, measured using an automated open field locomotion apparatus (Student's t-test, p<0.05). In contrast, animals given post-stroke treatment with the shh signaling agonist (SAG) demonstrated less deficits in behavioral function, compared to vehicle-treated mice. At 7 days after stroke, SAG-treated mice showed higher values in multiple horizontal movement parameters compared to vehicle treated mice (Student's t-test, p<0.05) whereas there were no differences in pre-stroke measurements, (Student's t-test, p>0.05). In summary, our data demonstrate that shh signaling plays critical and ongoing roles in response to ischemic injury and modulation of shh signaling in vivo alters the functional outcome after cortical ischemic injury.