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

Oudin, Anaïs; Baus, Virginie; Barthelemy, Vanessa; Fabian, Carina; Klein, Eliane; Dieterle, Monika; Wantz, May; Hau, Ann-Christin; Dording, Claire M.; Bernard, Amandine; Michelucci, Alessandro; Yabo, Yahaya A; Kanli, Georgia; Keunen, Olivier; Bjerkvig, Rolf; Niclou, Simone P.; Golebiewska, Anna

doi:10.1016/j.xpro.2021.100534

Cited by 18 publications

(33 citation statements)

References 15 publications

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“…We collected GBM tissues from the Centre Hospitalier de Luxembourg (CHL, Neurosurgical Department) or the Haukeland University Hospital (Bergen, Norway) from patients who had given their informed consent, and with approval from the local ethics committees (National Committee for Ethics in Research Luxembourg and local ethics committee Haukeland University Hospital in Bergen). PDOX derivation was previously described 17,18,61 ( Table S1 ). For this study, tumor organoids were implanted (6 per brain) into the right frontal cortex of nude mice (athymic nude mice, Charles River Laboratories, France).…”

Section: Methodsmentioning

confidence: 99%

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Glioblastoma-instructed microglia transition to heterogeneous phenotypic states with phagocytic and dendritic cell-like features in patient tumors and patient-derived orthotopic xenografts

Yabo

Moreno-Sanchez

Pires‐Afonso

et al. 2023

Preprint

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BackgroundGlioblastoma (GBM) evades the immune system by creating an immune-suppressive tumor microenvironment (TME), where GBM-associated myeloid cells are geared towards tumor-supportive roles. However, it is unclear whether recruited myeloid cells are phenotypically and functionally identical. Here, we aim to understand the TME heterogeneity in GBM patients recapitulated in patient-derived orthotopic xenografts (PDOXs) and systematically characterize myeloid cell type identities at the molecular and functional level.MethodsWe applied single-cell RNA-sequencing and spatial transcriptomics, multicolor flow cytometry, immunohistochemistry and functional assays to examine the heterogeneity of the TME in GBM. Various GBM PDOXs representing different tumor phenotypes were analyzed and compared to the patient tumors, normal brain and mouse GL261 glioma model.ResultsPDOX models recapitulate the major components of the TME detected in human GBM, where tumor cells reciprocally interact with host cells to create a GBM-specific TME. We detect the most prominent transcriptomic adaptations in myeloid cells, which are largely of microglial origin. We reveal intra-tumoral heterogeneity of microglia and identify diverse phenotypic states across distinct GBM landscapes and tumor niches. GBM-educated microglia acquire dendritic cell-like features, displaying increased migration and phagocytosis. We further find novel microglial states expressing astrocytic and endothelial markers. Lastly, we show that temozolomide (TMZ) treatment leads to transcriptomic plasticity of both GBM tumor cells and adjacent TME components.ConclusionsOur data provide insight into the phenotypic adaptation of the heterogeneous TME instructed by GBM. We uncover that GBM-educated microglia are represented by various concomitant states, both in patients and recapitulated in PDOXs, displaying different pro- or anti-tumoral properties that are modulated by anti-neoplastic treatments, such as TMZ.KEY POINTSGBM-educated tumor microenvironment is faithfully recapitulated and modulated in PDOX modelsMicroglia represent an essential myeloid cell population in the GBM microenvironmentGBM-educated microglia acquire heterogeneous transcriptomic states across distinct tumor nichesGBM-educated microglia subsets display phagocytic and dendritic cell-like gene expression programs, which are modulated upon TMZ treatmentIMPORTANCE OF THE STUDYThis manuscript addresses tumor-immune interactions in GBM, focusing on the molecular changes of the myeloid compartment. We find that myeloid cells, the most abundant immune cell population in brain tumors, undergo the most prominent transcriptional adaptation in the TME. Resident microglia represent the main myeloid cell population in the cellular tumor, while peripheral-derived myeloid cells appear to infiltrate the brain at sites of blood-brain barrier disruption. We identify reactive dendritic cell-like gene expression programs associated with enhanced phagocytic and antigen-presentation features in GBM-educated microglia subsets that might be harnessed for novel immunotherapeutic approaches. Overall, PDOX models faithfully recapitulate the major components of the GBM-educated TME and allow assessment of phenotypic changes in the GBM ecosystem upon treatment.

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Section: Methodsmentioning

confidence: 99%

“…Treatment was administered by oral gavage 5x per week with a total of 8 doses, where the last treatment dose was given shortly before the endpoint ( Figure S9A ). Tumor growth was followed by MRI at day 37 and 42 and quantified as described in 61 . Statistical difference was assessed with two-tailed Student’s t-test.…”

Section: Methodsmentioning

confidence: 99%

Glioblastoma-instructed microglia transition to heterogeneous phenotypic states with phagocytic and dendritic cell-like features in patient tumors and patient-derived orthotopic xenografts

Yabo

Moreno-Sanchez

Pires‐Afonso

et al. 2023

Preprint

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“…To assess the invasion ability of the NCH601 cells in vivo , the cells (sh Scramble or MTHFD1L KD) were injected intracranially into the right frontal part of the brain according to the protocol published in Oudin A. et al, (Oudin et al, 2021) . Briefly, NU/NU Nude female mice (n=16) were anesthetized and 3 x 10 5 NCH601 cells were implanted into the right hemisphere of the brain.…”

Section: Methodsmentioning

confidence: 99%

Formate Promotes Invasion and Metastasis by Activating Fatty Acid Synthesis and Matrix Metalloproteinases

Delbrouck

Kiweler

Pozdeev

et al. 2023

Preprint

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Metabolic rewiring is essential to enable cancer onset and progression. One important metabolic pathway that is often hijacked by cancer cells is the one-carbon cycle, in which the third carbon of serine is oxidized to formate. We have previously shown that formate production in cancer cells often exceeds the anabolic demand, resulting in formate overflow. Furthermore, we observed that high extracellular formate promotes the in vitro invasiveness of glioblastoma (GBM) cells. However, additional data supporting this in vitro observation and mechanistic details remained elusive so far. In the present study, we now demonstrate that inhibition of formate overflow results in a decreased invasiveness of GBM cells ex vivo and in vivo. Additionally, we observed that exposure to exogeneous formate can induce a transiently stable pro-invasive phenotype that results in increased metastasis formation in vivo. All in all, these results suggest that a local formate increase within the tumor microenvironment may be one factor that can promote cancer cell motility and dissemination. Mechanistically, we uncover a previously undescribed interplay where formate acts as a trigger to alter fatty acid metabolism and matrix metalloproteinase (MMP) activity which in turn impacts cancer cell invasiveness. We thus highlight the role of formate as a pro-invasive metabolite. Gaining a deeper understanding of formate overflow and how it promotes invasion in cancer, may open new therapeutic opportunities to prevent cancer cell dissmination.

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“…17 After AAI induction, all mice underwent surgery for intracranial implantation of GL261 tumor cells as previously described. 18 MRI was used to measure the tumor volume.…”

Section: Allergic Airway Inflammation and Orthotopic Glioma Modelmentioning

confidence: 99%

Allergic airway inflammation delays glioblastoma progression and reinvigorates systemic and local immunity in mice

Poli

Oudin

Muller

et al. 2022

Allergy

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Background: Numerous patient-based studies have highlighted the protective role of immunoglobulin E-mediated allergic diseases on glioblastoma (GBM) susceptibility and prognosis. However, the mechanisms behind this observation remain elusive. Our objective was to establish a preclinical model able to recapitulate this phenomenon and investigate the role of immunity underlying such protection. Methods: An immunocompetent mouse model of allergic airway inflammation (AAI) was initiated before intracranial implantation of mouse GBM cells (GL261). RAG1-KO mice served to assess tumor growth in a model deficient for adaptive immunity. Tumor development was monitored by MRI. Microglia were isolated for functional analyses and RNA-sequencing. Peripheral as well as tumor-associated immune cells were characterized by flow cytometry. The impact of allergy-related microglial genes on patient survival was analyzed by Cox regression using publicly available datasets. Results: We found that allergy establishment in mice delayed tumor engraftment in the brain and reduced tumor growth resulting in increased mouse survival. AAI induced a transcriptional reprogramming of microglia towards a pro-inflammatory-like state, uncovering a microglia gene signature, which correlated with limited local immunosuppression in glioma patients. AAI increased effector memory T-cells in the circulation as well as tumor-infiltrating CD4 + T-cells. The survival benefit conferred by AAI was lost in mice devoid of adaptive immunity. Conclusion:Our results demonstrate that AAI limits both tumor take and progression in mice, providing a preclinical model to study the impact of allergy on GBM susceptibility and prognosis, respectively. We identify a potentiation of local and adaptive systemic immunity, suggesting a reciprocal crosstalk that orchestrates allergy-induced immune protection against GBM.

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

Cited by 18 publications

References 15 publications

Glioblastoma-instructed microglia transition to heterogeneous phenotypic states with phagocytic and dendritic cell-like features in patient tumors and patient-derived orthotopic xenografts

Glioblastoma-instructed microglia transition to heterogeneous phenotypic states with phagocytic and dendritic cell-like features in patient tumors and patient-derived orthotopic xenografts

Formate Promotes Invasion and Metastasis by Activating Fatty Acid Synthesis and Matrix Metalloproteinases

Allergic airway inflammation delays glioblastoma progression and reinvigorates systemic and local immunity in mice

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