Cancer cell heterogeneity and plasticity: A paradigm shift in glioblastoma

Phenotypic plasticity has emerged as a major contributor to intra-tumoral heterogeneity and treatment resistance in cancer. Increasing evidence shows that glioblastoma (GBM) cells display prominent intrinsic plasticity and reversibly adapt to dynamic microenvironmental conditions. Limited genetic evolution at recurrence further suggests that resistance mechanisms also largely operate at the phenotypic level. Here we review recent literature underpinning the role of GBM plasticity in creating gradients of heterogeneous cells including those that carry cancer stem cell (CSC) properties. A historical perspective from the hierarchical to the nonhierarchical concept of CSCs towards the recent appreciation of GBM plasticity is provided. Cellular states interact dynamically with each other and with the surrounding brain to shape a flexible tumor ecosystem, which enables swift adaptation to external pressure including treatment. We present the key components regulating intra-tumoral phenotypic heterogeneity and the equilibrium of phenotypic states, including genetic, epigenetic, and microenvironmental factors. We further discuss plasticity in the context of intrinsic tumor resistance, where a variable balance between preexisting resistant cells and adaptive persisters leads to reversible adaptation upon treatment. Innovative efforts targeting regulators of plasticity and mechanisms of state transitions towards treatment-resistant states are needed to restrict the adaptive capacities of GBM.publishedVersio

Revealing and Harnessing Tumour-Associated Microglia/Macrophage Heterogeneity in Glioblastoma

Cancer heterogeneity and progression are subject to complex interactions between neoplastic cells and their microenvironment, including the immune system. Although glioblastomas (GBMs) are classified as ‘cold tumours’ with very little lymphocyte infiltration, they can contain up to 30–40% of tumour-associated macrophages, reported to contribute to a supportive microenvironment that facilitates tumour proliferation, survival and migration. In GBM, tumour-associated macrophages comprise either resident parenchymal microglia, perivascular macrophages or peripheral monocyte-derived cells. They are recruited by GBMs and in turn release growth factors and cytokines that affect the tumour. Notably, tumour-associated microglia/macrophages (TAMs) acquire different expression programs, which shape the tumour microenvironment and contribute to GBM molecular subtyping. Further, emerging evidence highlights that TAM programs may adapt to specific tumour features and landscapes. Here, we review key evidence describing TAM transcriptional and functional heterogeneity in GBM. We propose that unravelling the intricate complexity and diversity of the myeloid compartment as well as understanding how different TAM subsets may affect tumour progression will possibly pave the way to new immune therapeutic avenues for GBM patients.publishedVersio

Glioblastoma organoids: pre-clinical applications and challenges in the context of immunotherapy

Malignant brain tumors remain uniformly fatal, even with the best-to-date treatment. For Glioblastoma (GBM), the most severe form of brain cancer in adults, the median overall survival is roughly over a year. New therapeutic options are urgently needed, yet recent clinical trials in the field have been largely disappointing. This is partially due to inappropriate preclinical model systems, which do not reflect the complexity of patient tumors. Furthermore, clinically relevant patient-derived models recapitulating the immune compartment are lacking, which represents a bottleneck for adequate immunotherapy testing. Emerging 3D organoid cultures offer innovative possibilities for cancer modeling. Here, we review available GBM organoid models amenable to a large variety of pre-clinical applications including functional bioassays such as proliferation and invasion, drug screening, and the generation of patient-derived orthotopic xenografts (PDOX) for validation of biological responses in vivo. We emphasize advantages and technical challenges in establishing immunocompetent ex vivo models based on co-cultures of GBM organoids and human immune cells. The latter can be isolated either from the tumor or from patient or donor blood as peripheral blood mononuclear cells (PBMCs). We also discuss the challenges to generate GBM PDOXs based on humanized mouse models to validate efficacy of immunotherapies in vivo. A detailed characterization of such models at the cellular and molecular level is needed to understand the potential and limitations for various immune activating strategies. Increasing the availability of immunocompetent GBM models will improve research on emerging immune therapeutic approaches against aggressive brain cancer.publishedVersio

Transcriptional and epigenetic mechanisms underlying astrocyte identity

Astrocytes play a significant role in coordinating neural development and provide critical support for the function of the CNS. They possess important adaptation capacities that range from their transition towards reactive astrocytes to their ability to undergo reprogramming, thereby revealing their potential to retain latent features of neural progenitor cells. We propose that the mechanisms underlying reactive astrogliosis or astrocyte reprogramming provide an opportunity for initiating neuronal regeneration, a process that is notably reduced in the mammalian nervous system throughout evolution. Conversely, this plasticity may also affect normal astrocytic functions resulting in pathologies ranging from neurodevelopmental disorders to neurodegenerative diseases and brain tumors. We postulate that epigenetic mechanisms linking extrinsic cues and intrinsic transcriptional programs are key factors to maintain astrocyte identity and function, and critically, to control the balance of regenerative and degenerative activity. Here, we will review the main evidences supporting this concept. We propose that unravelling the epigenetic and transcriptional mechanisms underlying the acquisition of astrocyte identity and plasticity, as well as understanding how these processes are modulated by the local microenvironment under specific threatening or pathological conditions, may pave the way to new therapeutic avenues for several neurological disorders including neurodegenerative diseases and brain tumors of astrocytic lineage.publishedVersio

Elucidating tumour-associated microglia/macrophage diversity along glioblastoma progression and under ACOD1 deficiency

In glioblastoma (GBM), tumour-associated microglia/macrophages (TAMs) represent the major cell type of the stromal compartment and contribute to tumour immune escape mechanisms. Thus, targeting TAMs is emerging as a promising strategy for immunotherapy. However, TAM heterogeneity and metabolic adaptation along GBM progression represent critical features for the design of effective TAM-targeted therapies. Here, we comprehensively study the cellular and molecular changes of TAMs in the GL261 GBM mouse model, combining single-cell RNA-sequencing with flow cytometry and immunohistological analyses along GBM progression and in the absence of Acod1 (also known as Irg1), a key gene involved in the metabolic reprogramming of macrophages towards an anti-inflammatory phenotype. Similarly to patients, we identify distinct TAM profiles, mainly based on their ontogeny, that reiterate the idea that microglia- and macrophage-like cells show key transcriptional differences and dynamically adapt along GBM stages. Notably, we uncover decreased antigen-presenting cell features and immune reactivity in TAMs along tumour progression that are instead enhanced in Acod1-deficient mice. Overall, our results provide insight into TAM heterogeneity and highlight a novel role for Acod1 in TAM adaptation during GBM progression.publishedVersio

Protocol for derivation of organoids and patient-derived orthotopic xenografts from glioma patient tumors

Tumor organoids and patient-derived orthotopic xenografts (PDOXs) are some of the most valuable pre-clinical tools in cancer research. In this protocol, we describe efficient derivation of organoids and PDOX models from glioma patient tumors. We provide detailed steps for organoid culture, intracranial implantation, and detection of tumors in the brain. We further present technical adjustments for standardized functional assays and drug testing.publishedVersio

XAB2 promotes Ku eviction from single-ended DNA double-strand breaks independently of the ATM kinase

Replication-associated single-ended DNA double-strand breaks (seDSBs) are repaired predominantly through RAD51-mediated homologous recombination (HR). Removal of the non-homologous end-joining (NHEJ) factor Ku from resected seDSB ends is crucial for HR. The coordinated actions of MRE11-CtIP nuclease activities orchestrated by ATM define one pathway for Ku eviction. Here, we identify the pre-mRNA splicing protein XAB2 as a factor required for resistance to seDSBs induced by the chemotherapeutic alkylator temozolomide. Moreover, we show that XAB2 prevents Ku retention and abortive HR at seDSBs induced by temozolomide and camptothecin, via a pathway that operates in parallel to the ATM-CtIP-MRE11 axis. Although XAB2 depletion preserved RAD51 focus formation, the resulting RAD51-ssDNA associations were unproductive, leading to increased NHEJ engagement in S/G2 and genetic instability. Overexpression of RAD51 or RAD52 rescued the XAB2 defects and XAB2 loss was synthetically lethal with RAD52 inhibition, providing potential perspectives in cancer therapy.publishedVersio

Mutant IDH1 Differently Affects Redox State and Metabolism in Glial Cells of Normal and Tumor Origin

IDH1R132H (isocitrate dehydrogenase 1) mutations play a key role in the development of low-grade gliomas. IDH1wt converts isocitrate to α-ketoglutarate while reducing nicotinamide adenine dinucleotide phosphate (NADP+), whereas IDH1R132H uses α-ketoglutarate and NADPH to generate the oncometabolite 2-hydroxyglutarate (2-HG). While the effects of 2-HG have been the subject of intense research, the 2-HG independent effects of IDH1R132H are still ambiguous. The present study demonstrates that IDH1R132H expression but not 2-HG alone leads to significantly decreased tricarboxylic acid (TCA) cycle metabolites, reduced proliferation, and enhanced sensitivity to irradiation in both glioblastoma cells and astrocytes in vitro. Glioblastoma cells, but not astrocytes, showed decreased NADPH and NAD+ levels upon IDH1R132H transduction. However, in astrocytes IDH1R132H led to elevated expression of the NAD-synthesizing enzyme nicotinamide phosphoribosyltransferase (NAMPT). These effects were not 2-HG mediated. This suggests that IDH1R132H cells utilize NAD+ to restore NADP pools, which only astrocytes could compensate via induction of NAMPT. We found that the expression of NAMPT is lower in patient-derived IDH1-mutant glioma cells and xenografts compared to IDH1-wildtype models. The Cancer Genome Atlas (TCGA) data analysis confirmed lower NAMPT expression in IDH1-mutant versus IDH1-wildtype gliomas. We show that the IDH1 mutation directly affects the energy homeostasis and redox state in a cell-type dependent manner. Targeting the impairments in metabolism and redox state might open up new avenues for treating IDH1-mutant gliomas.publishedVersio

Stem cell-associated heterogeneity in Glioblastoma results from intrinsic tumor plasticity shaped by the microenvironment

The identity and unique capacity of cancer stem cells (CSC) to drive tumor growth and resistance have been challenged in brain tumors. Here we report that cells expressing CSC-associated cell membrane markers in Glioblastoma (GBM) do not represent a clonal entity defined by distinct functional properties and transcriptomic profiles, but rather a plastic state that most cancer cells can adopt. We show that phenotypic heterogeneity arises from non-hierarchical, reversible state transitions, instructed by the microenvironment and is predictable by mathematical modeling. Although functional stem cell properties were similar in vitro, accelerated reconstitution of heterogeneity provides a growth advantage in vivo, suggesting that tumorigenic potential is linked to intrinsic plasticity rather than CSC multipotency. The capacity of any given cancer cell to reconstitute tumor heterogeneity cautions against therapies targeting CSC-associated membrane epitopes. Instead inherent cancer cell plasticity emerges as a novel relevant target for treatment.publishedVersio

Glioblastoma organoids: pre-clinical applications and challenges in the context of immunotherapy

Malignant brain tumors remain uniformly fatal, even with the best-to-date treatment. For Glioblastoma (GBM), the most severe form of brain cancer in adults, the median overall survival is roughly over a year. New therapeutic options are urgently needed, yet recent clinical trials in the field have been largely disappointing. This is partially due to inappropriate preclinical model systems, which do not reflect the complexity of patient tumors. Furthermore, clinically relevant patient-derived models recapitulating the immune compartment are lacking, which represents a bottleneck for adequate immunotherapy testing. Emerging 3D organoid cultures offer innovative possibilities for cancer modeling. Here, we review available GBM organoid models amenable to a large variety of pre-clinical applications including functional bioassays such as proliferation and invasion, drug screening, and the generation of patient-derived orthotopic xenografts (PDOX) for validation of biological responses in vivo. We emphasize advantages and technical challenges in establishing immunocompetent ex vivo models based on co-cultures of GBM organoids and human immune cells. The latter can be isolated either from the tumor or from patient or donor blood as peripheral blood mononuclear cells (PBMCs). We also discuss the challenges to generate GBM PDOXs based on humanized mouse models to validate efficacy of immunotherapies in vivo. A detailed characterization of such models at the cellular and molecular level is needed to understand the potential and limitations for various immune activating strategies. Increasing the availability of immunocompetent GBM models will improve research on emerging immune therapeutic approaches against aggressive brain cancer