Glioblastoma (GBM) remains an incurable disease with a median survival of ~15-18 months post-diagnosis, despite ongoing efforts to improve treatment outcome [[1]Marenco-Hillembrand L. Wijesekera O. Suarez-Meade P. Mampre D. Jackson C. Peterson J. et al.Trends in glioblastoma: outcomes over time and type of intervention: a systematic evidence based analysis.J Neurooncol. 2020; (https://doi.org/)https://doi.org/10.1007/s11060-020-03451-6Crossref PubMed Scopus (27) Google Scholar]. Standard-of-care consists of maximum safe surgical resection followed by systemic temozolomide (TMZ) and local radiation. After initial treatment with this regimen, GBM universally recurs and subsequent lines of therapy have limited efficacy. As a result, there is a great need to understand the biology of acquired therapeutic resistance in this disease. One mechanism that has been proposed to confer GBM treatment unresponsiveness and recurrence is enrichment of cancer stem cells (CSCs), a population that is resistant to standard-of-care therapies and has the capacity to give rise to the recurrent tumor. In this issue, Ramakrishnan et al. report that GBM acquires a treatment-resistant CSC phenotype following radiation therapy by actively exporting miR-603 in extracellular vesicles (EV) [[2]Ramakrishnan V. Xu B. Akers J. Nguyen T. Ma J. Dhawan S. et al.Radiation-induced extracellular vesicle (EV) release of miR-603 promotes IGF1- mediated stem cell state in glioblastomas.EBioMedicine. 2020; (https://doi.org/)https://doi.org/10.1016/j.ebiom.2020.102736Summary Full Text Full Text PDF PubMed Scopus (12) Google Scholar]. Ramakrishnan et al. performed microRNA sequencing on paired patient GBM specimens obtained at initial diagnosis and recurrence, in order to identify microRNAs (miRNA/miR) potentially involved in therapeutic resistance. Through this unbiased approach they discovered that miR-603 was significantly lower in recurrent GBM specimens. A similar trend is observed in human GBM cell lines and freshly resected GBM tissue following irradiation, which was accompanied by an increase in the abundance of insulin growth factor 1 (IGF1), IGF1 receptor (IGF1R), and O6-methylguanine–DNA methyltransferase (MGMT), which are predicted targets of miR-603. The authors further established that loss of miR-603 led to a CSC phenotype of GBM cells, accompanied by therapeutic resistance to radiation and TMZ of cultured human GBM cell lines and mouse xenograft models. Acquired resistance to radiation due to loss of miR-603 was primarily mediated by de-repression of IGF1-IGF1R, while acquired resistance to TMZ was linked to de-repression of MGMT, a clinically used biomarker of TMZ response in GBM [[3]Hegi M.E. Diserens A.C. Gorlia T. Hamou M.F. de Tribolet N. Weller M. et al.MGMT gene silencing and benefit from temozolomide in glioblastoma.N Engl J Med. 2005; 352: 997-1003Crossref PubMed Scopus (4690) Google Scholar]. Accordingly, overexpression of miR-603 in an intracranial xenograft GBM model rescued the impaired radiation response and further synergized with radiation and TMZ combination therapy. This study went on to reveal that the decreased levels of miR-603 driving post-radiation therapeutic resistance of GBM was a result of en masse export of this miRNA in EV (summarized in Fig. 1). Earlier studies established that GBM cells secrete EV containing RNAs and proteins, which serve as communication vectors to transfer pro-tumorigenic signals to different cell types within the tumor microenvironment [[4]Skog J. Wurdinger T. van Rijn S. Meijer D.H. Gainche L. Sena-Esteves M. et al.Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers.Nat Cell Biol. 2008; 10: 1470-1476Crossref PubMed Scopus (3246) Google Scholar]. Since then, there has been great interest in interrogating EV-associated macromolecules, including miRNAs, in GBM pathogenesis, diagnosis, and treatment, albeit primarily focusing on the role of EV delivery to target cells. For example, transfer of specific miRNAs from mesenchymal stem cells in stroma via EV were shown to have a CSC-promoting or -restrictive role [[5]Figueroa J. Phillips L.M. Shahar T. Hossain A. Gumin J. Kim H. et al.Exosomes from glioma-associated mesenchymal stem cells increase the tumorigenicity of glioma stem-like cells via transfer of miR-1587.Cancer Res. 2017; 77: 5808-5819Crossref PubMed Scopus (103) Google Scholar,[6]Lang F.M. Hossain A. Gumin J. Momin E.N. Shimizu Y. Ledbetter D. et al.Mesenchymal stem cells as natural biofactories for exosomes carrying miR-124a in the treatment of gliomas.Neuro Oncol. 2018; 20: 380-390Crossref PubMed Scopus (89) Google Scholar]. Ramakrishnan et al. assessed miRNA-mediated CSC regulation from a different angle, focusing on the EV-mediated export of a CSC-limiting miRNA as resistance mechanism. Still, their observation that miR-603 containing EV are taken up by microglia, the brain-resident innate immune cells, also opens the possibility of miR-603 delivery to non-tumor cells leading to effects beyond the therapeutic resistance observed in the microRNA-secreting GBM cells. Although, the functional consequence of this uptake remains to be elucidated, a recent study by Abels et al. demonstrated that transfer of another miRNA, miR-21, can alter the proliferation status of GBM-associated microglia [[7]Abels E.R. Maas S.L.N. Nieland L. Wei Z. Cheah P.S. Tai E. et al.Glioblastoma-associated microglia reprogramming is mediated by functional transfer of extracellular miR-21.Cell Rep. 2019; 28 (e7): 3105-3119Summary Full Text Full Text PDF PubMed Scopus (56) Google Scholar]. These observations support further investigation of CSC-independent roles of EV-associated miR-603 export in therapeutic resistance and its potential role in the repolarization of microglia into a tumor-supportive phenotype. Building on the authors' findings, reversal of therapeutic resistance by augmenting miR-603 is a strategy that warrants investigation in GBM, and perhaps even other tumors. To this point, systemic delivery of miR-603 containing liposomes significantly delayed the growth of the triple-negative breast tumors in mice by targeting cellular proliferation and migration [[8]Bayraktar R. Pichler M. Kanlikilicer P. Ivan C. Bayraktar E. Kahraman N. et al.MicroRNA 603 acts as a tumor suppressor and inhibits triple-negative breast cancer tumorigenesis by targeting elongation factor 2 kinase.Oncotarget. 2017; 8: 11641-11658Crossref PubMed Scopus (51) Google Scholar]. Regarding targeting of the downstream IGF1-IGF1R axis, previous studies in preclinical models showed that IGF1 blockade primes radiation response and reduces GBM tumor burden [[9]Osuka S. Sampetrean O. Shimizu T. Saga I. Onishi N. Sugihara E. et al.IGF1 receptor signaling regulates adaptive radioprotection in glioma stem cells.Stem Cells. 2013; 31: 627-640Crossref PubMed Scopus (85) Google Scholar]. However, clinical studies of IGF1R blockade in various cancers proved ineffective [[10]Ferrarotto R. William Jr., W.N. Tseng J.E. Marur S. Shin D.M. Murphy B. et al.Randomized phase II trial of cixutumumab alone or with cetuximab for refractory recurrent/metastatic head and neck squamous cell carcinoma.Oral Oncol. 2018; 82: 83-90Crossref PubMed Scopus (9) Google Scholar]. Ramakrishnan et al. describe a treatment-resistant phenotype driven by the export of miR-603, and resulting in de-repression of multiple downstream resistance genes, including TMZ resistance through concomitant regulation of MGMT expression. These observations provide a possible explanation as to why targeting IGF1 or IGF1R alone is insufficient to modulate the full spectrum of post-radiation resistance. Therefore, this study suggests that additional therapeutic targets could be identified by further exploring potential triggers of the novel mechanism of therapeutic resistance described, and by elucidating the upstream factors and signaling pathways leading to the packaging and expulsion of miR-603 EV. The authors declare no conflict of interest. We would like to thank Amanda Mendelsohn (Cleveland Clinic) for artwork. Radiation-induced extracellular vesicle (EV) release of miR-603 promotes IGF1-mediated stem cell state in glioblastomasProfiling of matched pre- and post-treatment glioblastoma specimens revealed altered homeostasis of select miRNAs in response to radiation. Radiation-induced EV export of miR-603 simultaneously promoted the CSC state and up-regulated DNA repair to promote acquired resistance. These effects were abolished by exogenous miR-603 expression, suggesting potential for clinical translation. Full-Text PDF Open Access
<p>Supplementary Fig. S1. Immune cell-dependent survival differences between males and females in murine glioblastoma models. Supplementary Fig. S2. Mouse syngeneic GBM cell lines do not contain a Y chromosome. Supplementary Fig. S3. Frequencies of tumor-infiltrating immune cells in SB28 model. Supplementary Fig. S4. Sex difference in survival in SB28-OVA model and GL261 model. Supplementary Fig. S5. No sex difference in T cells was observed at an earlier time point. Supplementary Fig. S6. Male T cells are more exhausted in the GL261 model. Supplementary Fig. S7. Phenotype of T cells in the periphery does not replicate the sex differences shown in tumor-infiltrating T cells. Supplementary Fig. S8. Frequencies of exhausted T cell subsets. Supplementary Fig. S9. PD1 blockade enhanced immune responses Supplementary Fig. S10. No significant difference in the immune cell composition of bone marrow chimera mice before tumor implantation. Supplementary Fig. S11. Male and female CD8+ T cells in mixed bone marrow chimera model. Supplementary Fig. S12. Phenotyping tumor-infiltrating CD8+ T cell from GBM patient tumors. Supplementary Fig. S13. scRNA-seq analysis of tumor-infiltrating CD8+ T cells from GBM patients. Supplementary Fig. S14. mRNA expression level of transcription factors in human in vitro exhausted T cells. Supplementary Fig. S15. Expression level of X chromosome inactivation (XCI) genes in exhausted T cells. Supplementary Fig. S16. Inhibition of UTX abrogates sex differences in T cell exhaustion.</p>
A potently immunosuppressive tumor microenvironment facilitates progression of glioblastoma (GBM). Immunotherapies have had variable success in improving the outcome of GBM patients, suggesting that there is a need to gain insight into the mechanisms of immunosuppression. Our findings indicated that proliferating monocytic MDSCs (mMDSCs) accumulate in tumors of male mice and patients, while female tumor-bearing mice had an increase in circulating granulocytic MDSC (gMDSC) frequency, and a high gMDSC gene signature correlated with worse outcome of female patients.
Methods
To investigate the basis and prognostic value of sex differences in MDSC profile, we analyzed the role of sex hormones, determined gene expression signatures of MDSCs and preclinically tested the therapeutic benefit of candidate drugs predicted to be effective against individual MDSC subsets.
Results
In line with the differential MDSC accumulation pattern, targeting the systemic gMDSCs with the anti-Ly6G neutralizing antibody extended the lifespan of female mice without affecting males. These differences were not driven by sex steroids, as castration or ovariectomy failed to alter MDSC subset accumulation patterns in GBM-bearing mice. Drug-prediction algorithms using the differential MDSC gene expression profiles predicted IL-1 inhibitors are effective against gMDSCs. Correspondingly, IL-1β was highly expressed in female but not male gMDSCs. Single-cell sequencing revealed that circulating but not tumor-infiltrating gMDSCs were the primary source of IL-1β and that its neutralization provided a female-specific survival advantage by reducing circulating gMDSCs. This was accompanied by declines in tumor infiltration of microglia, microglia activation status and tumor cell proliferation. In vitro, IL-1β inhibition reduced viability and expression of activation markers by primary microglia.
Conclusions
These findings highlight a novel peripheral gMDSC-microglia IL-1β mediated communication axis in female GBM and indicate expression differences in MDSC subsets can be leveraged for improved immunotherapy efficacy in a sex-specific, precision medicine strategy.
The Swiss cheese model is used to assess risks and explain accidents in a variety of industries. This model can be applied to dissect the homeostatic mechanisms whose cumulative dysregulation contributes to disease states, including cancer. Using glioblastoma (GBM) as an exemplar, we discuss how specific protumorigenic mechanisms collectively drive disease by affecting genomic integrity, epigenetic regulation, metabolic homeostasis, and antitumor immunity. We further highlight how host factors, such as hormonal differences and aging, impact this process, and the interplay between these 'system failures' that enable tumor progression and foster therapeutic resistance. Finally, we examine therapies that consider the interactions between these elements, which may comprise more effective approaches given the multifaceted protumorigenic mechanisms that drive GBM.
ABSTRACT The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell‐released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV‐associated functional activities. Finally, a checklist is provided with summaries of key points.
Abstract Extracellular vesicles (EVs) large‐scale production is a crucial point for the translation of EVs from discovery to application of EV‐based products. In October 2021, the International Society for Extracellular Vesicles (ISEV), along with support by the FET‐OPEN projects, “The Extracellular Vesicle Foundry” (evFOUNDRY) and “Extracellular vesicles from a natural source for tailor‐made nanomaterials” (VES4US), organized a workshop entitled “massivEVs” to discuss the potential challenges for translation of EV‐based products. This report gives an overview of the topics discussed during “massivEVs”, the most important points raised, and the points of consensus reached after discussion among academia and industry representatives. Overall, the review of the existing EV manufacturing, upscaling challenges and directions for their resolution highlighted in the workshop painted an optimistic future for the expanding EV field.
<div>Abstract<p>In multiple types of cancer, an increased frequency in myeloid-derived suppressor cells (MDSC) is associated with worse outcomes and poor therapeutic response. In the glioblastoma (GBM) microenvironment, monocytic (m) MDSCs represent the predominant subset. However, the molecular basis of mMDSC enrichment in the tumor microenvironment compared with granulocytic (g) MDSCs has yet to be determined. Here we performed the first broad epigenetic profiling of MDSC subsets to define underlying cell-intrinsic differences in behavior and found that enhanced gene accessibility of cell adhesion programs in mMDSCs is linked to their tumor-accelerating ability in GBM models upon adoptive transfer. Mouse and human mMDSCs expressed higher levels of integrin β1 and dipeptidyl peptidase-4 (DPP-4) compared with gMDSCs as part of an enhanced cell adhesion signature. Integrin β1 blockade abrogated the tumor-promoting phenotype of mMDSCs and altered the immune profile in the tumor microenvironment, whereas treatment with a DPP-4 inhibitor extended survival in preclinical GBM models. Targeting DPP-4 in mMDSCs reduced pERK signaling and their migration towards tumor cells. These findings uncover a fundamental difference in the molecular basis of MDSC subsets and suggest that integrin β1 and DPP-4 represent putative immunotherapy targets to attenuate myeloid cell-driven immune suppression in GBM.</p>Significance:<p>Epigenetic profiling uncovers cell adhesion programming as a regulator of the tumor-promoting functions of monocytic myeloid-derived suppressor cells in glioblastoma, identifying therapeutic targets that modulate the immune response and suppress tumor growth.</p></div>
The transfer of intact mitochondria between heterogeneous cell types has been confirmed in various settings, including cancer. However, the functional implications of mitochondria transfer on tumor biology are poorly understood. Here we show that mitochondria transfer is a prevalent phenomenon in glioblastoma (GBM), the most frequent and malignant primary brain tumor. We identified horizontal mitochondria transfer from astrocytes as a mechanism that enhances tumorigenesis in GBM. This transfer is dependent on network-forming intercellular connections between GBM cells and astrocytes, which are facilitated by growth-associated protein 43 (GAP43), a protein involved in neuron axon regeneration and astrocyte reactivity. The acquisition of astrocyte mitochondria drives an increase in mitochondrial respiration and upregulation of metabolic pathways linked to proliferation and tumorigenicity. Functionally, uptake of astrocyte mitochondria promotes cell cycle progression to proliferative G2/M phases and enhances self-renewal and tumorigenicity of GBM. Collectively, our findings reveal a host-tumor interaction that drives proliferation and self-renewal of cancer cells, providing opportunities for therapeutic development.
The common γ-chain cytokine interleukin-15 (IL-15) plays a significant role in regulating innate and adaptive lymphocyte homeostasis and can stimulate anti-tumor activity of leukocytes. We have previously shown that the circulating IL-15 in the plasma is the heterodimeric form (hetIL-15), produced upon co-expression of IL-15 and IL-15 Receptor alpha (IL-15Rα) polypeptides in the same cell, heterodimerization of the two chains and secretion. We investigated the pharmacokinetic and pharmacodynamic profile and toxicity of purified human hetIL-15 cytokine upon injection in rhesus macaques. We compared the effects of repeated hetIL-15 administration during a two-week dosing cycle, using different subcutaneous dosing schemata, i.e. fixed doses of 0.5, 5 and 50 µg/kg or a doubling step-dose scheme ranging from 2 to 64 µg/kg. Following a fixed-dose regimen, dose-dependent peak plasma IL-15 levels decreased significantly between the first and last injection. The trough plasma IL-15 levels measured at 48 h after injections were significantly higher after the first dose, compared to subsequent doses. In contrast, following the step-dose regimen, the systemic exposure increased by more than 1 log between the first injection given at 2 µg/kg and the last injection given at 64 µg/kg, and the trough levels were comparable after each injection. Blood lymphocyte cell count, proliferation, and plasma IL-18 levels peaked at day 8 when hetIL-15 was provided at fixed doses, and at the end of the cycle following a step-dose regimen, suggesting that sustained expansion of target cells requires increasing doses of cytokine. Macaques treated with a 50 µg/kg dose showed moderate and transient toxicity, including fever, signs of capillary leak syndrome and renal dysfunction. In contrast, these effects were mild or absent using the step-dose regimen. The results provide a new method of optimal administration of this homeostatic cytokine and may have applications for the delivery of other cytokines.
