Subpopulation Of Reactive Astrocytes Research Articles

RTID-02. SILMET TRIAL: A RANDOMIZED, PLACEBO-CONTROLLED STUDY TO EVALUATE SILIBININ IN PREVENTING INTRACRANIAL RECURRENCE AFTER COMPLETE RESECTION OF A SINGLE BRAIN METASTASIS FROM NSCLC OR BREAST CANCER

Abstract BACKGROUND: A subpopulation of reactive astrocytes (RAs) with activated signal transducer and activator of transcription 3 (STAT3) signalling in peritumoral tissue of brain metastases (BM) has been shown to favor a pro-metastatic microenvironment. High-pSTAT3 expression in RAs of BM has been correlated with a shorter intracranial progression-free survival (i-PFS) as compared with low-pSTAT3 BM. Pharmacological inhibition of the STAT3 pathway with silibinin has been suggested to be active in preliminary cohort of patients with BM. Patients and methods. A new “prevention” trial has been initiated in June 2023 to investigate the efficacy of silibinin in comparison to placebo (PCB) to prevent a secondary intracranial relapse in a multicentric cohort of patients with gross-total resected BM from NSCLC and breast cancer. Inclusion criteria are as follows: histologically confirmed BM from NSCLC or BC; single BM who underwent a complete resection; systemic disease not requiring a change of antineoplastic therapy; pSTAT3 presence in RAs of peritumoral tissue confirmed by central assessment; ≥ 18 – 70 years of age; KPS ≥ 70. The primary endpoint is to demonstrate a superiority of silibinin in prolonging time to local recurrence as compared to PCB. The secondary endpoints are time to distant recurrence of BM in the brain, i-PFS, overall PFS (intracranial and/or systemic), overall survival, tolerability, quality of life. Collection of blood and CSF samples for the analysis of STAT3 downstream pathways is an exploratory objective. The sample size (70 patients) was calculated by assuming a median time to local recurrence of BM of 7.5 months for PCB (control arm) according to Mahajan [1] and ≥15 months for silibinin arm. Clinical trial information: NCT05689619 [1] Mahajan A et al. Post-operative stereotactic radiosurgery versus observation for completely resected brain metastases: a single-centre, randomised, controlled, phase 3 trial. Lancet Oncol. 2017

BSBM-03 A NOVEL COMBINED IMMUNOTHERAPY TO IMPROVE THERAPEUTIC RESPONSES IN SYMPTOMATIC BRAIN METASTASIS BASED ON TARGETING A SUBPOPULATION OF IMMUNOSUPPRESSIVE ASTROCYTES

Abstract The diagnosis of brain metastasis involves high morbidity and mortality. Recently, immune checkpoint blockade antibodies (ICB) have shown clinical benefits mainly when applied to asymptomatic brain metastasis patients. However, variability is broad and responses to this therapy drop considerably when treating clinically relevant disease. Although potentially involved in the lack of response to immunotherapy, corticoids do not seem to fully explain this situation. Thus, it is currently unknown how to effectively target symptomatic brain metastases with immunotherapy. We previously reported a clinically relevant protumoral program driven by STAT3 activation in a subpopulation of reactive astrocytes during advanced stages of the disease. By further exploiting astrocyte heterogeneity, we have found a potential strategy to improve the number of responders to immunotherapy in symptomatic brain metastasis. Specifically, we have developed a comprehensive strategy including genetic and pharmacologic approaches to define a novel immunosuppressive axis involving astrocyte-secreted TIMP1 signaling on CD63+ CD8+ T cells. Based on these findings, we developed a combined immunotherapy to boost the systemic activation of T cells with ICB while blocking local TIMP1-dependent immunosuppression in various preclinical models. Furthermore, the detection of TIMP1 in the CSF provides a biomarker to select patients who would benefit the most from the combined immunotherapy. Even more, our data using Patient Derived Organotypic Cultures from fresh brain metastasis neurosurgeries confirmed that our therapeutic strategy is valid for symptomatic brain metastases from any primary source. In conclusion, our study has identified a novel combined immunotherapy that could be especially relevant for symptomatic brain metastases. Our findings have emerged from a strong scientific rationale whereby a specific subpopulation of astrocytes is shown to activate the local immunosuppressive environment for brain-infiltrating CD8+ T cells.

Abstract 5105: TIMP1 mediates astrocyte-dependent local immunosuppression in brain metastasis

Abstract Brain metastasis is an unmet clinical need, affecting between 10-30% of cancer patients with 200000 to 400000 newly diagnosis per annum in the US. Recently, several clinical trials have reported benefits using immunotherapy to treat brain metastasis. However, variability of the responses is broad and high benefit is found mainly in asymptomatic brain metastasis, while the benefit is dramatically reduced in the clinically relevant stage. Thus, it is currently unknown how to effectively target symptomatic brain metastases with immunotherapy. We previously reported a clinically relevant protumoral program driven by STAT3 activation in a subpopulation of reactive astrocytes in these advanced stages of the disease. Our current study further exploited the heterogeneity within the metastasis-associated microenvironment as a resource to identify novel therapeutic vulnerabilities to improve the benefits of immunotherapies based on immune checkpoint blocking antibodies (ICB) in symptomatic brain metastasis. Our results demonstrate that reactive astrocytes are strong immunomodulatory cells in brain tumors. We have identified the molecular profile of disease-associated glial cells and defined its connection to modulatory activities on specific lymphocyte populations in experimental brain metastasis as well as human-derived samples. scRNASeq and high content multiplex immunofluorescence allowed us to report a novel local immunomodulatory axis dependent on TIMP1 (astrocytes)/CD63 (CD8 T cells), which is present in brain metastasis patients with high immunoscore and would imply an additional immunosuppressive signal for potential ICB responders in brain metastasis. Genetic and pharmacologic approaches targeting this STAT3-dependent local immunomodulatory axis have allowed us to define the rationale to combine immune checkpoint blockade with a STAT3 inhibitor, which we previously used in patients. We proved that such combined immunotherapy boost the systemic activation of T cells while also preventing the local blockade. Additionally, our comprehensive strategy includes the possibility to stratify patients that are best qualified to benefit from this therapy by measuring TIMP1 in liquid biopsies from CSF. Even more, our data using Patient Derived Organotypic Cultures (PDOC) from fresh brain metastasis neurosurgeries confirms that our therapeutic strategy might benefit brain metastases generated from any primary source. In conclusion, we describe an immunosuppressive mechanism in the brain microenvironment that could explain the lack of response to ICB in patients with advanced brain metastasis. Our finding provides the rationale to implement complementary approaches targeting local immunosuppression to increase the benefit of immunotherapy in symptomatic brain metastasis. Citation Format: Neibla Priego, Pedro García-Gómez, Ana de Pablos-Aragoneses, María Perea-García, Laura Álvaro-Espinosa, Carolina Hernández-Oliver, Elena Martínez-Saez, Ángel Pérez-Núñez, Aurelio Hernández-Laín, Rebeca Sanz-Pamplona, Marc Schmitz, Stephen J. Crocker, Diego Serrano, Asís Palazón, RENACER Red Nacional de Metástasis Cerebral, Manuel Valiente. TIMP1 mediates astrocyte-dependent local immunosuppression in brain metastasis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5105.

Abstract 2746: Stat3 labels a subpopulation of reactive astrocytes required for brain metastasis

Abstract The diagnosis of brain metastasis involves high morbidity and mortality and remains as an unmet clinical need in spite of being the most common tumor in the brain. Brain metastasis affects between 10-30% of cancer patients with 200.000 new cases yearly only in the US. Increasing evidences point out the relevance of the microenvironment to understand the biology of brain colonization by metastatic cells, however translation strategies targeting it are lacking. We have identified altered signaling pathways in pro-metastatic reactive astrocytes (RA), and translated that into a novel therapeutic application for brain metastasis. Specifically we have identified a subpopulation of RA characterized by activated STAT3 pathway (pSTAT3+). This subpopulation is located in the vicinity of metastatic lesions intermingled with pSTAT3- RA in several experimental models and in 89% of human brain metastases independently of the primary tumor source. The pro-metastatic behaviour of RA is regulated by the activation of the STAT3 pathway, which modulates the immune system locally promoting the survival of cancer cells. Specifically the secretome of pSTAT3+ RA decreases the anti-tumor activity of CD8+ T cells as well as promotes the expansion of the pro-tumor population of CD74+ microglia/macrophage, which infiltrates metastasis cores. Interestingly, the immunosuppressive nature of pSTAT3+ RA is linked to the acquisition of stem cell-like properties, which might reflect the misuse of brain responses to injury instigated by the presence of cancer cells. Genetic and pharmacologic approaches targeting STAT3 in RA impair the progression of brain metastasis, even at advanced stages of the disease. Moreover, a safe and orally bioavailable STAT3 inhibitor reduced brain metastasis in 75% of 18 stage IV lung adenocarcinoma patients with established brain metastases, improving the outcome of the disease by increasing patient survival from 4 to 15 months. Besides increasing therapeutic opportunities for patients with brain metastasis, we have described for the first time the role of reactive astrocytes as regulators of local immunosuppression in brain metastasis as well as the importance of uncovering the heterogeneity within the metastasis-associated microenvironment. Citation Format: Neibla Priego, Lucía Zhu, Catia Monteiro, Manon Mulders, David Wasilewski, Wendy Bindeman, Laura Doglio, Elena Martínez-Saez, Santiago Ramón y Cajal, Coral Fustero-Torre, Elena Piñeiro-Yáñez, Aurelio Hernández-Laín, Valeria Poli, Javier A. Menéndez, Ricardo Soffietti, Joaquim Bosch-Barrera, Manuel Valiente. Stat3 labels a subpopulation of reactive astrocytes required for brain metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2746.

Astroglial connexin43 contributes to neuronal suffering in a mouse model of Alzheimer's disease.

In Alzheimer's disease (AD), astrocyte properties are modified but their involvement in this pathology is only beginning to be appreciated. The expression of connexins, proteins forming gap junction channels and hemichannels, is increased in astrocytes contacting amyloid plaques in brains of AD patients and APP/PS1 mice. The consequences on their channel functions was investigated in a murine model of familial AD, the APPswe/PS1dE9 mice. Whereas gap junctional communication was not affected, we revealed that hemichannels were activated in astrocytes of acute hippocampal slices containing Aβ plaques. Such hemichannel activity was detected in all astrocytes, whatever their distance from amyloid plaques, but with an enhanced activity in the reactive astrocytes contacting amyloid plaques. Connexin43 was the main hemichannel contributor, however, a minor pannexin1 component was also identified in the subpopulation of reactive astrocytes in direct contact with plaques. Distinct regulatory pathways are involved in connexin and pannexin hemichannel activation. Inflammation triggered pannexin hemichannel activity, whereas connexin43 hemichannels were activated by the increase in resting calcium level of astrocytes. Importantly, hemichannel activation led to the release of ATP and glutamate that contributed to maintain a high calcium level in astrocytes placing them in the center of a vicious circle. The astroglial targeted connexin43 gene knocking-out in APPswe/PS1dE9 mice allowed to diminish gliotransmitter release and to alleviate neuronal damages, reducing oxidative stress and neuritic dystrophies in hippocampal neurons associated to plaques. Altogether, these data highlight the importance of astroglial hemichannels in AD and suggest that blocking astroglial hemichannel activity in astrocytes could represent an alternative therapeutic strategy in AD.

Adult Neural Stem Cells from the Subventricular Zone Give Rise to Reactive Astrocytes in the Cortex after Stroke

Reactive astrocytes (RAs) have been reported to convert to multipotent neural stem cells (NSCs) capable of neurosphere (NS) formation and multilineage differentiation in vitro. Using genetic tagging, we determined that subventricular zone (SVZ) NSCs give rise to NSs derived from the stroke-injured cortex. We demonstrate that these cells can be isolated from the cortex in two different models of stroke and from different stroke-lesioned cortical regions. Interestingly, SVZ NSCs give rise to a subpopulation of RAs in the cortex that contribute to astrogliosis and scar formation. Last, we show that these SVZ derived RAs can be converted to neurons in vivo by forced expression of Ascl1. Identifying the contribution of cells originating from the SVZ to injury repair has implications for neural regeneration strategies.

A subpopulation of reactive astrocytes at affected neuronal perikarya after hypophysectomy in adult rats

Intermediate filaments (IFs) of nestin and vimentin are expressed in immature astrocytes. In this study, we examined the re-expression of these early glial traits in rat reactive astrocytes in affected neuronal perikarya in supraoptic (SON) and paraventricular (PVN) nuclei induced by hypophysectomy. Double-labeling immunofluorescence confocal laser microscopy demonstrated that by 7 days post-lesion, both nestin and vimentin were present intensely in hypertrophied GFAP-IR reactive astrocytes in the area of hypophysectomized magnocellular neurons in SON and PVN, while nestin and vimentin are absent in the normal or sham-operated animals. As the gliotic reaction progressed, the morphology of nestin or vimentin-positive reactive astrocytes in SON but not PVN changed from stellate form at 7 days to thin and elongated shape, morphologically compatible with radial glia during development, at 14 days post-lesion. By 28 days post-lesion, while vimentin-IR persisted in reactive astrocytes in SON and PVN, nestin-IR could hardly be detected. The spatiotemporal pattern of nestin-IR and/or vimentin-IR in reactive astrocytes suggests astrocytes attempt to revert to a more primitive glia form indicated by changes in morphology and phenotype following hypophysectomy, which may contribute to neuronal trophism and plasticity in the lesioned HNS favoring neuronal maintenance and fiber outgrowth.

Proliferation of a subpopulation of reactive astrocytes following needle-insertion lesion in rat

It is well known that traumatic injuries of the CNS induce a gliotic reaction, characterized by the presence of reactive astrocytes. Reactive astrocytes exhibit enhanced expression of the astrocyte-specific intermediate filament, glial fibrillary acidic protein (GFAP), hypertrophy, and thickened processes. Recently, we have demonstrated that injuries of the CNS induce a re-expression of an embryonic intermediate filament-associated protein, IFAP-70/280kDa. Based on IFAP-70/280kDa immunolabeling, we have shown that reactive astrocytes, activated by stab-wound injury, can be divided into two major groups: 1. persistent IFAP+/GFAP+ cells which are close to the wound in the area of glial scar, and 2. transient IFAP-/GFAP+ cells which are farther from the wound. In this study, we use BrdU incorporation to examine proliferation in these two groups of reactive astrocytes induced by stab injury of the rat cerebrum. Triple/double-label immunofluorescence microscopy was performed using antibodies to IFAP-70/280kDa, GFAP, and BrdU. The results showed that BrdU+ reactive astrocytes (GFAP+) were always IFAB-70/280kDa+ as well. However, not all IFAP+ reactive astrocytes are BrdU+. BrdU+ signal was not observed in any IFAP- reactive astrocytes. At five days post-lesion, IFAP+ reactive astrocytes were increasing in the area of the wound (0-50 µm from the wound edge), but had reached a peak in the proximal area (50-800 µm away from the wound edge). At eight days post-lesion, IFAP+ reactive astrocytes achieved the highest percentage in the wound area. At the same time, BrdU-containing reactive astrocytes occupied an area closer to the wound. By 20 days post-lesion, following the formation of the gliotic scar at the stab-wound, a few IFAP+/GFAP+ cells still persisted. BrdU-containing reactive astrocytes were only observed in the scar. These results indicate that many IFAP+ reactive astrocytes close to the wound, in contrast to the IFAP- ones farther from the wound, appear to regain their proliferative potential to increase in number and participate in the formation of the gliotic scar.

Reactive astrocytes show enhanced inwardly rectifying K+ currents in situ.

Injury and diseases of the nervous system can induce astrocytes to form tenacious glial scars. We induced focal cortical freeze-lesions in neonatal rats and examined scars histologically and electrophysiologically in tissue slices isolated 2-3 weeks after lesioning. Lesions displayed marked gliosis, characterized by upregulation of GFAP labeling. Reactive astrocytes surrounding the scar showed marked hypertrophy, enlarged cell bodies and extended processes frequently terminating with endfeet-like structures on blood vessels. These reactive astrocytes showed enhanced expression of inwardly rectifying K+ (K(IR)) channels, widely believed to be an important pathway for astrocytic K+ buffering. These results suggest that a subpopulation of reactive astrocytes along a glial scar might be instrumental in buffering K+ away from the lesion.

Immunohistochemical Study of the β-Chemokine Receptors CCR3 and CCR5 and Their Ligands in Normal and Alzheimer's Disease Brains

Chemokines belong to an expanding family of cytokines the primary function of which is recruitment of leukocytes to inflammatory sites. Recent evidence has shown their presence in the central nervous system. Because inflammatory responses have been implicated in the pathogenesis of Alzheimer's disease (AD), we studied the expression of CCR3, CCR5, and their ligands in normal and AD brains by immunohistochemistry. CCR3 and CCR5 are present on microglia of both control and AD brains, with increased expression on some reactive microglia in AD. Immunohistochemistry for MIP-1beta, MIP-1alpha, RANTES, eotaxin, and MCP-3 (ligands for CCR5 and/or CCR3) revealed the presence of MIP-1beta predominantly in a subpopulation of reactive astrocytes, which were more widespread in AD than control brains, and MIP-1alpha predominantly in neurons and weakly in some microglia in both AD and controls. Many of the CCR3+ or CCR5+ reactive microglia and MIP-1beta+ reactive astrocytes were found associated with amyloid deposits. Immunoreactivity for eotaxin, RANTES, and MCP-3 were not detected. Detection of these beta-chemokine receptors on microglia and some of their ligands in reactive astrocytes and neurons as well as microglia suggests a role for this system in glial-glial and glial-neuronal interactions, potentially influencing the progression of AD.

Qualitative analysis of membrane currents in glial cells from normal and gliotic tissue in situ: down-regulation of Na + current and lack of P 2 purinergic responses

To date, the electrophysiological properties of glial cells located in reactive scar tissue are unknown. To address this issue two subtypes of hippocampal glial cells, located in thin vital slices of normal or gliotic brain tissue, were analysed for their voltage controlled ion channels using the patch-clamp technique. Reactive gliosis was induced in adult rats by a single peritoneal injection of kainic acid. The intensity of the following seizures was rated ascending from 1 to 6. Rats which exhibited seizures of level 3 or higher showed, within three days, a marked loss of pyramidal cells (>60% in CA1 and CA3) and an increase in the density of glial fibrillary acidic protein immunostaining, representing an apparent increase in the number and size of astrocytes in all layers of the hippocampal CA1 subfield. Reactive and normal astrocytes of one subtype, electrophysiologically characterized by time-independent potassium currents, did not significantly differ in membrane potential and potassium conductivity. Glutamine synthetase-positive, but mostly glial fibrillary acidic protein-negative, glial cells (presumably representing immature astrocytes) were also included in this study. This subtype of glial cells showed several voltage- and time-dependent potassium currents and, under control conditions, tetrodotoxin-sensitive voltage-gated Na + channels, which were almost completely lost after reactive gliosis. Another part of this study focuses on the sensitivity of reactive and control glial cells for extracellular ATP. Several in vitro studies suggest that P 2 purinergic receptors on glial cells could trigger the induction of reactive gliosis. In contrast to results described on cultured astrocytes, we found in situ that hippocampal glial cells were not sensitive to ATP or stable P 2 receptor agonists in control or in gliotic brain slices. In summary, the presence of at least two different subtypes of hippocampal astrocytes was demonstrated for control as well as for gliotic brain tissue. A dramatic down-regulation of tetrodotoxin-sensitive sodium channels in one subpopulation of reactive astrocytes was shown. This result supports the hypothesis that the presence of active neurons could be required to maintain glial voltage-gated sodium channels. Furthermore, we conclude that there is no longtime expression of P2 purinoceptors on hippocampal astrocytes in situ, and therefore the involvement of astrocytic ATP receptors in the genesis of reactive gliosis is unlikely.

A Subpopulation of Reactive Astrocytes at the Immediate Site of Cerebral Cortical Injury

We have identified an early-appearing intermediate filament-associated protein (IFAP-70/280 kDa) in radial glia and their immediate derivatives. This IFAP is absent in the adult CNS. In this study, we examined the reexpression of this early glial differentiation trait in rat reactive astrocytes induced by stab injury of the cerebrum. Double-label immunofluorescence microscopy demonstrated that by 36 h postlesion, IFAP-70/280 kDa was present in a few GFAP-positive astrocytes in the area adjacent to the wound. As the gliotic reaction progressed, the number of IFAP-positive reactive astrocytes increased and by 5–6 days postlesion, IFAP-70/280 kDa was present in most of the hypertrophied astrocytes in tissue immediately adjacent to the wound. By 8 days postlesion, while the number of IFAP-negative reactive astrocytes away from the wound diminished, the IFAP-containing reactive astrocytes close to the wound persisted. Concurrently, they began to change from a stellate form to an elongated shape, with their longitudinal axes radiating from the wound. The immunoreactivity of this IFAP started to diminish at 20 days postlesion, and by 30 days postlesion, it was not observed in the remaining gliotic cells. These results demonstrate that reactive astrocytes induced by stab-wound injury can be divided into two subtypes: persistent IFAP-70/280 kDa-containing cells which are close to the wound in the area of the glial scar and transient IFAP-70/280 kDa-negative cells which are farther from the wound. The reappearance of IFAP-70/280 kDa also suggests that some reactive astrocytes have the capacity to recapitulate early developmental stages.

Reactive gliosis and monoamine oxidase B.

A double-staining method was applied to cryosections of human spinal cord from patients who died with amyotrophic lateral sclerosis (ALS) and corresponding controls in order to investigate cellular content of monoamine oxidase B (MAO-B). 3H-L-Deprenyl emulsion autoradiography was used in combination with histochemical methods for the detection of astrocytes and monocytes/microglia. In the ALS spinal cords an increased number of astrocytes as well as an increased content of MAO-B in reactive species of astrocytes was demonstrated. No significant 3H-L-deprenyl binding was observed in cells derived from the mesoderm, e.g. monocytes or microglia. Furthermore, a sub-population of reactive astrocytes that contained low levels of MAO-B was observed in spinal sections. These findings were further substantiated by studies performed on primary astrocyte cultures.

Acidic FGF expression in the surroundings of senile plaques.

Immunohistochemical examination of postmortem brain tissue of Alzheimer's disease revealed that acidic fibroblast growth factor (aFGF) was specifically expressed in a subpopulation of reactive astrocytes which were congregated at the margin of the senile plaque. Double immunostaining indicated that such upregulation of aFGF expression might be related to the presence of reactive microglia rather than beta-amyloid protein deposits. Although, on the other hand, immunohistochemical staining for fibroblast growth factor receptor-1 occurred in some cortical neurons of Alzheimer's disease, the staining pattern did not differ from that in age-matched controls. Possible significance of aFGF-positive astrocytes in the surroundings of the senile plaque will be discussed in relation to receptor mediated or non-mediated mechanisms.

Monoclonal antibody 14E recognizes an antigen common to human oligodendrocytes, Schwann cells, Bergmann glia, and a subpopulation of reactive glia.

The monoclonal antibody 14E immunocytochemically stains the nuclear membrane of oligodendrocytes but not myelin in tissue sections of adult normal human white matter. The nuclear membranes of Schwann cells in human peripheral nerve and cerebellar Bergmann glia were also visualized with this antibody. In actively demyelinating multiple sclerosis plaques the 14E antibody stained increased numbers of oligodendrocytes and the nuclei, perikarya and cell processes of hypertrophic glia, which were often multinucleate. Scattered small groups of these hypertrophic glia were present in areas of dense astrogliosis in acute plaques. The 14E-positive hypertrophic cells could be either a subpopulation of reactive astrocytes or bipotential glial precursors.

Monoclonal antibody 14E identifies the oligodendrocyte cell body in normal adult human and rat white matter

A monoclonal antibody, 14E, which detects immunocytochemically the perikaya of interfascicular oligodendrocytes in sections of normal adult human and rat white matter, was obtained using isolated normal human oligodendrocytes as the immunogen. 14E is an IgM antibody that reacts with a polypeptide of approximate molecular weight 23 000 on immunoblots of the particulate fraction of normal white matter. In sections of active multiple sclerosis plaques and recent infarcts, 14E immunostained the cell bodies and processes of only a small number of hypertrophic glial cells which could be oligodendrocytes undergoing pathological changes or a subpopulation of reactive astrocytes.