Glioblastoma is the most common and aggressive primary brain tumour in adults. The development of anti-brain cancer agents are challenged by the blood-brain barrier and the resistance conferred by the local tumour microenvironment. Heptamethine cyanine dyes (HMCDs) are a class of near-infrared fluorescence compounds that have recently emerged as promising agents for drug delivery. We conjugated palbociclib, a cyclin-dependent kinase (CDK) 4/6 inhibitor, to an HMCD, MHI-148, and conducted drug activity analysis on primary patient-derived glioblastoma cell lines. In addition to the expected cytostatic activity, our in vitro studies revealed that palbociclib-MHI-148 conjugate resulted in an almost 100-fold increase in cytotoxicity compared to palbociclib alone. This shift of palbociclib from cytostatic to cytotoxic when conjugated to MHI-148 was due to increased DNA damage, as indicated by an increase in γH2AX foci, followed by an increased expression of key extrinsic apoptosis genes, including TP53, TNFR1, TRAIL, FADD and caspase 8. In addition, we observed a time-dependent increase in the cell surface expression of TNFR1, consistent with an observed increase in the secretion TNFα, followed by TNFR1 endocytosis at 48 h. The treatment of patient GBM cells with the palbociclib-MHI-148 conjugate prevented TNFα-induced NFκB translocation, suggesting conjugate-induced TNFR1 signalling favoured the TNFR1-mediated apoptotic response rather than the pro-inflammatory response pathway. Notably, pharmacological inhibition of endocytosis of TNFR1, and siRNA-knockdown of TNFR1 reversed the palbociclib-MHI-148-induced cell death. These results show a novel susceptibility of glioblastoma cells to TNFR1-dependent apoptosis, dependent on inhibition of canonical NFκB signalling using our previously reported palbociclib-HMCD conjugate.

When modeling disease in the laboratory, it is important to use clinically relevant models. Patient-derived human brain cells grown in vitro to study and test potential treatments provide such a model. Here, we present simple, highly reproducible coordinated procedures that can be used to routinely culture most cell types found in the human brain from single neurosurgically excised brain specimens. The cell types that can be cultured include dissociated cultures of neurons, astrocytes, microglia, pericytes and brain endothelial and neural precursor cells, as well as explant cultures of the leptomeninges, cortical slice cultures and brain tumor cells. The initial setup of cultures takes ~2 h, and the cells are ready for further experiments within days to weeks. The resulting cells can be studied as purified or mixed population cultures, slice cultures and explant-derived cultures. This protocol therefore enables the investigation of human brain cells to facilitate translation of neuroscience research to the clinic.

Abstract Background Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults. Whilst the role of the efflux transporters are well established in GBM, the expression and function of uptake transporters, such as the organic anion transporting polypeptide (OATP) family, are not well understood. OATPs possess broad substrate specificity that includes anti-cancer agents; therefore, we sought to investigate the expression of four OATP isoforms in human GBM cell types using patient tumor tissue. Methods We used fluorescent immunohistochemical labeling of paraffin-embedded surgically resected tissues and single-cell image analysis methods to explore the expression of the OATP isoforms in different tumor cell types through co-labeling with cell-type specific markers, such as IBA1 (pan-myeloid), GFAP (tumor cell), PDGFRβ (stromal cell), and UEA-1-lectin (endothelial). Results We found significant over-expression of all the OATP isoforms (OATP1A2, 2B1, 1C1 and 4A1) in GBM tumor sections when compared to non-neoplastic brain. A single-cell image analysis revealed that OATPs were significantly upregulated throughout the tumor parenchyma, with significantly higher expression found on lectin-positive blood vessels and IBA1-positive myeloid cells in GBM compared to non-tumor brain tissue. Qualitative analysis of the four OATP isoforms demonstrated greater expression of OATP4A1 in peri-necrotic regions of GBM tissue, which correlated with hypoxia-related markers within the Ivy GAP RNAseq dataset. Conclusion Here, we demonstrate, for the first time, the protein expression of four OATPs in human GBM tissue, including upregulation within the tumor microenvironment by myeloid cells and tumor vasculature, and isoform-specific upregulation within hypoxic niches.

The development of chemotherapies for glioblastoma is hindered by their limited bioavailability and toxicity on normal brain function. To overcome these limitations, we investigated the structure‐dependent activity of heptamethine cyanine dyes (HMCD), a group of tumour‐specific and BBB permeable near‐infrared fluorescent dyes, in both commercial (U87MG) and patient‐derived GBM cell lines. HMCD analogues with strongly ionisable sulphonic acid groups were not taken up by patient‐derived GBM cells, but were taken up by the U87MG cell line. HMCD uptake relies on a combination of transporter uptake through organic anion‐transporting polypeptides (OATPs) and endocytosis into GBM cells. The uptake of HMCDs was not affected by p‐glycoprotein efflux in GBM cells. Finally, we demonstrate structure‐dependent cytotoxic activity at high concentrations (EC50: 1–100 μM), likely due to mitochondrial damage‐induced apoptosis. An in vivo orthotopic glioblastoma model highlights tumour‐specific accumulation of our lead HMCD, MHI‐148, for up to 7 days following a single intraperitoneal injection. These studies suggest that strongly ionisable groups like sulphonic acids hamper the cellular uptake of HMCDs in patient‐derived GBM cell lines, highlighting cell line‐specific differences in HMCD uptake. We envisage these findings will help in the design and structural modifications of HMCDs for drug‐delivery applications for glioblastoma.

Amyotrophic lateral sclerosis (ALS) is a fatal movement disorder involving degeneration of motor neurons through dysfunction of the RNA-binding protein TDP-43. Pericytes, the perivascular cells of the blood-brain, blood-spinal cord, and blood-CSF barriers also degenerate in ALS. Indeed, pericytes are among the earliest cell types to show gene expression changes in pre-symptomatic animal models of ALS. This suggests that pericyte degeneration precedes neurodegeneration and may involve pericyte cell-autonomous TDP-43 dysfunction. Here we determined the effect of TDP-43 dysfunction in human brain pericytes on interleukin 6 (IL-6), a critical secreted inflammatory mediator reported to be regulated by TDP 43. Primary human brain pericytes were cultured from biopsy tissue from epilepsy surgeries and TDP-43 was silenced using siRNA. TDP-43 silencing of pericytes stimulated with pro-inflammatory cytokines, interleukin-1β or tumour necrosis factor alpha, robustly suppressed the induction of IL-6 transcript and protein. IL-6 regulation by TDP-43 did not involve the assembly of TDP-43 nuclear splicing bodies, and did not occur via altered splicing of IL6. Instead, transcriptome-wide analysis by RNA-Sequencing identified a poison exon in the IL6 destabilising factor HNRNPD (AUF1) as a splicing target of TDP-43. Our data support a model whereby TDP-43 silencing favours destabilisation of IL6 mRNA, via enhanced AU-rich element-mediated decay by HNRNP/AUF1. This suggests that cell-autonomous deficits in TDP-43 function in human brain pericytes would suppress their production of IL-6. Given the importance of the blood-brain and blood-spinal cord barriers in maintaining motor neuron health, TDP-43 in human brain pericytes may represent a cellular target for ALS therapeutics.

The study of microglia isolated from adult human brain tissue provides unique insight into the physiology of these brain immune cells and their role in adult human brain disorders. Reports of microglia in post-mortem adult human brain tissue show regional differences in microglial populations, however, these differences have not been fully explored in living microglia. In this study biopsy tissue was obtained from epileptic patients undergoing surgery and consisted of both cortical areas and neurogenic ventricular and hippocampal (Hp) areas. Microglia were concurrently isolated from both regions and compared by immunochemistry. Our initial observation was that a greater number of microglia resulted from isolation and culture of ventricular/Hp tissue than cortical tissue. This was found to be due to a greater proliferative capacity of microglia from ventricular/Hp regions compared to the cortex. Additionally, ventricular/Hp microglia had a greater proliferative response to the microglial mitogen Macrophage Colony-Stimulating Factor (M-CSF). This enhanced response was found to be associated with higher M-CSF receptor expression and higher expression of proteins involved in M-CSF signalling DAP12 and C/EBPβ. Microglia from the ventricular/Hp region also displayed higher expression of the receptor for Insulin-like Growth Factor-1, a molecule with some functional similarity to M-CSF. Compared to microglia isolated from the cortex, ventricular/Hp microglia showed increased HLA-DP, DQ, DR antigen presentation protein expression and a rounded morphology. These findings show that microglia from adult human brain neurogenic regions are more proliferative than cortical microglia and have a distinct protein expression profile. The data present a case for differential microglial phenotype and function in different regions of the adult human brain and suggest that microglia in adult neurogenic regions are "primed" to an activated state by their unique tissue environment.

Cancer cells show higher energy and biomolecules requirements than healthy cells, making the targeting of cancer metabolic dysfunctions an appealing therapeutic approach.Unfortunately, tackling the tumor metabolism is a daunting task, due to the ability of cancer cells to re-organize their metabolism switching between glycolysis and oxidative phosphorylation.Cancer metabolic plasticity and re-programming is partially supported by altered ionic channels expression and activity, our laboratory has described Chloride Intracellular Channel 1 (CLIC1) as a marker of malignancy in tumor cells and a possible therapeutic target.CLIC1 is a metamorphic protein able to switch between cytoplasmic and transmembrane forms (tmCLIC1), the latter characterized by chloride conductance.TmCLIC1 was found to be overexpressed in several solid tumors where supports cancer proliferation and growth, while it is mainly absent in healthy cells.Recently, it has been demonstrated that tmCLIC1 activity is affected by biguanide compounds, in particular metformin.Metformin was recently repurposed as an anticancer drug with unclear mechanisms.We demonstrated that in hypoglycemia, metformin displays increased toxic effect on cancer cells, inducing apoptosis.Metformin inhibits Cancerous Inhibitor of Protein Phosphatase 2A (CIP2A), and hypoglycemia upregulates PP2A subunit B56δ targeting the Glycogen Synthase Kinase 3 Beta (GSK3b).Metformin treatment in combination with hypoglycemia leads to de-phosphorylation of GSK3b protein resulting in degradation of induced myeloid leukemia cell differentiation protein (MCL-1), a pro-survival protein, and cell death.In primary glioblastoma cell lines enriched in tumor-initiating-cells (GSCs), metformin has proven to be effective in reducing cell growth without affecting viability.Here, we are going to show studies trying to establish whether CLIC1 is a component of the established PP2A-mediated signaling network.Our work aims to clarify metformin molecular mechanism of action and to assess a new potential therapeutic approach for solid tumors in which tmCLIC1 is found to be overexpressed.

Platelet-derived growth factor-BB (PDGF-BB):PDGF receptor-β (PDGFRβ) signalling in brain pericytes is critical to the development, maintenance and function of a healthy blood-brain barrier (BBB). Furthermore, BBB impairment and pericyte loss in Alzheimer’s disease (AD) is well documented. We found that PDGF-BB:PDGFRβ signalling components were altered in human AD brains, with a marked reduction in vascular PDGFB . We hypothesised that reduced PDGF-BB:PDGFRβ signalling in pericytes may impact on the BBB. We therefore tested the effects of PDGF-BB on primary human brain pericytes in vitro to define pathways related to BBB function. Using pharmacological inhibitors, we dissected distinct aspects of the PDGF-BB response that are controlled by extracellular signal-regulated kinase (ERK) and Akt pathways. PDGF-BB promotes the proliferation of pericytes and protection from apoptosis through ERK signalling. In contrast, PDGF-BB:PDGFRβ signalling through Akt augments pericyte-derived inflammatory secretions. It may therefore be possible to supplement PDGF-BB signalling to stabilise the cerebrovasculature in AD.

Neuroinflammation is a key component of virtually all neurodegenerative diseases; preceding neuronal loss and associating directly with cognitive impairment. Neuroinflammatory signals can originate and be amplified at barrier tissues such as brain vasculature, surrounding meninges and the choroid plexus. We designed a high-throughput screening system to target inflammation in cells of the blood-brain barrier (primary human pericytes and endothelia) and microglia enabling us to target human disease-specific inflammatory modifiers. Screening an FDA-approved drug library we identified digoxin and lanatoside C, members of the cardiac glycoside family as inflammatory modulating drugs that work in blood-brain barrier cells. A novel ex vivo assay of leptomeningeal and choroid plexus explants further confirmed that these drugs maintain their function in 3D cultures of brain border tissues. While current therapeutic strategies for the treatment of neurodegenerative diseases are missing the mark in terms of targets, efficacy and translatability, our innovative approach using in vitro and ex vivo human barrier cells and tissues to target neuroinflammatory pathways is a step forward in drug development and testing, and brings us closer to translatable treatments for human neurodegenerative disease. One Sentence Summary We have identified cardiac glycosides as powerful regulators of neuroinflammatory pathways in brain-barrier tissues such as vasculature, meninges and choroid plexus.

Cytoprotective agents are mainly used to protect the gastrointestinal tract linings and in the treatment of gastric ulcers. These agents are devoid of appreciable cytotoxic or cytostatic effects, and medicinal chemistry efforts to modify them into anticancer agents are rare. A drug repurposing campaign initiated in our laboratory with the primary focus of discovering brain cancer drugs resulted in drug-dye conjugate 1, a combination of the cytoprotective agent troxipide and heptamethine cyanine dye MHI 148. The drug-dye conjugate 1 was evaluated in three different patient-derived adult glioblastoma cell lines, commercially available U87 glioblastoma, and one paediatric glioblastoma cell line. In all cases, the conjugate 1 showed potent cytotoxic activity with nanomolar potency (EC50: 267 nM). Interestingly, troxipide alone does not show any cytotoxic and cytostatic activity in the above cell lines. We also observe a synergistic effect of 1 with temozolomide (TMZ), the standard drug used for glioblastoma treatment, even though the cell lines we used in this study were resistant to TMZ treatment. Herein we disclose the synthesis and in vitro activity of drug-dye conjugate 1 for treatment of difficult-to-treat brain cancers such as glioblastoma.

BACKGROUND:Microglia and tumor-associated macrophages (TAMs) constitute up to half of the total tumor mass of glioblastomas. Despite these myeloid populations being ontogenetically distinct, they have been largely conflated. Recent single-cell transcriptomic studies have identified genes that distinguish microglia from TAMs. Here we investigated whether the translated proteins of genes enriched in microglial or TAM populations can be used to differentiate these myeloid cells in immunohistochemically stained human glioblastoma tissue.METHODS:Tissue sections from resected low-grade, meningioma, and glioblastoma (grade IV) tumors and epilepsy tissues were immunofluorescently triple-labeled for Iba1 (pan-myeloid marker), CD14 or CD163 (preferential TAM markers), and either P2RY12 or TMEM119 (microglial-specific markers). Using a single-cell-based image analysis pipeline, we quantified the abundance of each marker within single myeloid cells, allowing the identification and analysis of myeloid populations.RESULTS:P2RY12 and TMEM119 successfully discriminated microglia from TAMs in glioblastoma. In contrast, CD14 and CD163 expression were not restricted to invading TAMs and were upregulated by tumor microglia. Notably, a higher ratio of microglia to TAMs significantly correlated with increased patient survival.CONCLUSIONS:We demonstrate the validity of previously defined microglial-specific genes P2RY12 and TMEM119 as robust discriminators of microglia and TAMs at the protein level in human tissue. Moreover, our data suggest that a higher proportion of microglia may be beneficial for patient survival in glioblastoma. Accordingly, this tissue-based method for myeloid population differentiation could serve as a useful prognostic tool.

The human brain shows remarkable complexity in its cellular makeup and function, which are distinct from nonhuman species, signifying the need for human-based research platforms for the study of human cellular neurophysiology and neuropathology. However, the use of adult human brain tissue for research purposes is hampered by technical, methodological, and accessibility challenges. One of the major problems is the limited number of in vitro systems that, in contrast, are readily available from rodent brain tissue. With recent advances in the optimization of protocols for adult human brain preparations, there is a significant opportunity for neuroscientists to validate their findings in human-based systems. This review addresses the methodological aspects, advantages, and disadvantages of human neuron in vitro systems, focusing on the unique properties of human neurons and synapses in neocortical microcircuits. These in vitro models provide the incomparable advantage of being a direct representation of the neurons that have formed part of the human brain until the point of recording, which cannot be replicated by animal models nor human stem-cell systems. Important distinct cellular mechanisms are observed in human neurons that may underlie the higher order cognitive abilities of the human brain. The use of human brain tissue in neuroscience research also raises important ethical, diversity, and control tissue limitations that need to be considered. Undoubtedly however, these human neuron systems provide critical information to increase the potential of translation of treatments from the laboratory to the clinic in a way animal models are failing to provide.

We describe the synthesis and in vitro activity of drug-dye conjugate 1, which is a combination of the PARP inhibitor rucaparib and heptamethine cyanine dye IR-786. The drug-dye conjugate 1 was evaluated in three different patient-derived glioblastoma cell lines and showed strong cytotoxic activity with nanomolar potency (EC50: 128 nM), which was a 780 fold improvement over rucaparib itself. We also observe a synergistic effect of 1 with temozolomide (TMZ), the standard drug for treatment for glioblastoma even though these cell lines were resistant to TMZ treatment. We envisage such conjugates to be worth exploring for their utility in the treatment of various brain cancers.

The ability to characterize and study primary neurons isolated directly from the adult human brain would greatly advance neuroscience research. However, significant challenges such as accessibility of human brain tissue and the lack of a robust neuronal cell culture protocol have hampered its progress. Here, we describe a simple and reproducible method for the isolation and culture of functional adult human neurons from neurosurgical brain specimens. In vitro, adult human neurons form a dense network and express a plethora of mature neuronal and synaptic markers. Most importantly, for the first time, we demonstrate the re-establishment of mature neurophysiological properties in vitro, such as repetitive fast-spiking action potentials, and spontaneous and evoked synaptic activity. Together, our dissociated and slice culture systems enable studies of adult human neurophysiology and gene expression under normal and pathological conditions and provide a high-throughput platform for drug testing on brain cells directly isolated from the adult human brain.

We describe the synthesis of drug-dye conjugate 1 between anaplastic lymphoma kinase inhibitor Crizotinib and heptamethine cyanine dye IR-786. The drug-dye conjugate 1 was evaluated in three different patient-derived glioblastoma cell lines and showed potent cytotoxic activity with nanomolar potency (EC50: 50.9 nM). We also demonstrate evidence for antiproliferative activity of 1 with single digit nanomolar potency (IC50: 4.7 nM). Furthermore, the cytotoxic effects conveyed a dramatic, 110-fold improvement over Crizotinib. This improvement was even more pronounced (492-fold) when 1 was combined with Temozolomide, the standard drug for treatment for glioblastoma. This work lays the foundation for future exploration of similar tyrosine kinase inhibitor drug-dye conjugates for the treatment of glioblastoma.

Abstract BACKGROUND Glioblastoma Multiforme (GBM) is the most aggressive, fatal, yet most common form of brain malignancy in adults. Despite advances in immune-based treatments for other modes of cancer, GBM remains a challenge due to its ability to dampen immune responses via mechanisms not yet fully understood. With a median survival time of only 15 months following diagnosis, there is a strong push to find new targets for therapy. The microenvironment comprises a mixture of malignant tumour cells, stroma, blood vessels and infiltrating inflammatory cells. Despite advances in understanding the contribution of these cells in establishing an anti-inflammatory microenvironment, the contribution of pericytes, an important neurovascular mural cell that forms the blood-brain barrier, has been inadequately studied. Therefore, we investigated the differences in immune profile between patient-matched non-neoplastic brain- and GBM-derived pericytes under basal and induced conditions. MATERIAL AND METHODS Primary patient-matched non-neoplastic brain and GBM tumour derived pericytes were isolated from specimens excised from consenting patients undergoing GBM surgical resection at Auckland City Hospital. Pericytes were treated with inflammatory cytokines including IL-1β, IFN-γ, TNFα and TGFβ for up to 24 hours. Inflammatory profile changes were probed for using fluorescent immunocytochemistry, qRT-PCR and spectral flow cytometry. Media was also collected for secretome analysis via cytometric bead array. RESULTS GBM pericytes show decreased expression of CX3CL1, both basally and following IL-1β treatment, via qRT-PCR and CBA. In contrast, increased gene expression and secretion of IL-6 and IL-8 by GBM pericytes were observed. GBM pericytes also basally express CD90 and anti-inflammatory molecule PD-L1 compared to their normal counterparts. In terms of activated pathways, basal SMAD2/3 activation is increased in GBM pericytes, while also showing greater activation following treatment with IL-1β, IFN-γ but not TNFα. C/EBPδ is activated and translocated following inflammatory stimulation; however, shows localised expression within the cytoplasm only observed in GBM pericytes. CONCLUSION This immunological screen of GBM pericytes highlights them as key players in the establishment of the tumour microenvironment. With data suggesting the activation of pathways such as the SMAD2/3 pathway in an unconventional manner, it suggests the potential for pericytes to manipulate pathways towards a more immunosuppressive outcome. Further immune characterisation of such cells is required to fully understand how they might contribute to the immunosuppressive nature of GBM.

BACKGROUND:Pericytes and endothelial cells are critical cellular components of the blood-brain barrier (BBB) and play an important role in neuroinflammation. To date, the majority of inflammation-related studies in endothelia and pericytes have been carried out using immortalised cell lines or non-human-derived cells. Whether these are representative of primary human cells is unclear and systematic comparisons of the inflammatory responses of primary human brain-derived pericytes and endothelia has yet to be performed.METHODS:To study the effects of neuroinflammation at the BBB, primary brain endothelial cells and pericytes were isolated from human biopsy tissue. Culture purity was examined using qPCR and immunocytochemistry. Electrical cell-substrate impedance sensing (ECIS) was used to determine the barrier properties of endothelial and pericyte cultures. Using immunocytochemistry, cytometric bead array, and ECIS, we compared the responses of endothelia and pericytes to a panel of inflammatory stimuli (IL-1β, TNFα, LPS, IFN-γ, TGF-β1, IL-6, and IL-4). Secretome analysis was performed to identify unique secretions of endothelia and pericytes in response to IL-1β.RESULTS:Endothelial cells were pure, moderately proliferative, retained the expression of BBB-related junctional proteins and transporters, and generated robust TEER. Both endothelia and pericytes have the same pattern of transcription factor activation in response to inflammatory stimuli but respond differently at the secretion level. Secretome analysis confirmed that endothelia and pericytes have overlapping but distinct secretome profiles in response to IL-1β. We identified several cell-type specific responses, including G-CSF and GM-CSF (endothelial-specific), and IGFBP2 and IGFBP3 (pericyte-specific). Finally, we demonstrated that direct addition of IL-1β, TNFα, LPS, and IL-4 contributed to the loss of endothelial barrier integrity in vitro.CONCLUSIONS:Here, we identify important cell-type differences in the inflammatory response of brain pericytes and endothelia and provide, for the first time, a comprehensive profile of the secretions of primary human brain endothelia and pericytes which has implications for understanding how inflammation affects the cerebrovasculature.