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Glycocalyx bulkiness is fostered by enhanced mechanics and is associated with GBM aggression a, RNA from control (Scr) and mesenchymal shFAK cells was analysed by qPCR. Mean ± s.e.m. (n = 3 and 4 independent experiments for MUC1 and CD44, respectively). *P = 0.03 and 0.027 for MUC1 and CD44, respectively, by one-sided paired t-test. b, Hyaluronan (HA) was measured from human proneural (Pro), Scr and shFAK cells. Mean ± s.e.m. (n = 4 independent experiments). *P = 0.01 and 0.021 for proneural verus Scr and Scr versus shFAK, respectively, by two-sided paired t-test. c, qPCR of V737N and WT proneural xenografts. Mean ± s.e.m. (n = 5 mice per group). *P = 0.08 and 0.012 for MUC1 and CD44, respectively, by two-sided unpaired t-test. d, Alcian blue staining of WT and V737N xenograft, syngeneic and transgenic proneural tumour models. Scale bar, 250 µm (n = 3 mice per group). e, Mesenchymal cells were loaded with short (3 nm) or long (90 nm) glycopolymers, and glycocalyx height was measured by SAIM. Mean ± s.e.m.; each point represents measurement of glycocalyx height per frame (n = 24 and 23 measurements for short and long glycopolymers, respectively, pooled from two independent experiments). *P = 0.035 by two-sided paired t-test. f, Immunoblot assays were performed for pY397-FAK and HAS2 on soft and stiff hydrogels for long versus short glycopolymer decorated mesenchymal cells (n = 3 independent experiments). g, Cells loaded with long or short glycopolymers were injected into nude mice, and xenografts stained for TNC and by H&E. Insets show differential invasion and black broken lines indicate tumour boundary (n = 7 mice per group). Scale bar, 250 µm. h, Quantification of invasive edge was performed as for Fig. 3j. Mean ± s.e.m. (n = 5 and 6 distance measurements for WT and V737N tumours, respectively, for 4 tissue samples per group). *P = 0.047 by two-sided unpaired t-test. i, qPCR was performed for these tumours. Mean ± s.e.m. (n = 5 tissues per group). *P = 0.048 and 0.028 for TNC and MET, respectively, by one-sided unpaired t-test. j, Survival analysed by Kaplan–Meier test (n = 7 mice per group). P value calculated using two-sided logrank test. k, Schematic showing that enhanced mechanical signalling in tumour cells promotes the upregulation of mesenchymal and bulky glycocalyx (Glyx) genes, which results in the production of ECM proteins such as TNC and bulky glycocalyx components and modulators (CD44, hyaluronan (HA) and Gal-1. Together, these drive glioma aggressiveness in a tension-dependent feedback loop. Unprocessed blot: Supplementary Fig. 9.
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Glioblastoma multiforme (GBMs) are recurrent lethal brain tumours. Recurrent GBMs often exhibit mesenchymal, stem-like phenotypes that could explain their resistance to therapy. Analyses revealed that recurrent GBMs have increased tension and express high levels of glycoproteins that increase the bulkiness of the glycocalyx. Studies showed that a b...
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Cell migration is a force-dependent adaptive process mediated by integrin-dependent adhesion as well as other yet poorly defined interactions to the extracellular matrix. Using enzymatic multi-targeted digestion of sugar moieties on the surface of mesenchymal cells and leukocytes after interference with integrin function, we demonstrate that the su...
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... Our results suggest that TERT promoter mutant cells and EGFR amplified cells may favor this environment or perhaps contribute to its inception. The physical properties of ECM organization have also been shown to promote more mesenchymal phenotype of GBM cells (31). To unravel the full extent of the hidden structure within the ECM, future studies should include proteomic analyses to provide a more comprehensive understanding of the molecular composition of the ECM and cell types associated with these unique structures. ...
Tumor evolution is driven by genetic variation; however, it is the tumor microenvironment (TME) that provides the selective pressure contributing to evolution in cancer. Despite high histopathological heterogeneity within glioblastoma (GBM), the most aggressive brain tumor, the interactions between the genetically distinct GBM cells and the surrounding TME are not fully understood. To address this, we analyzed matched primary and recurrent GBM archival tumor tissues with imaging-based techniques aimed to simultaneously evaluate tumor tissues for the presence of hypoxic, angiogenic, and inflammatory niches, extracellular matrix (ECM) organization, TERT promoter mutational status, and several oncogenic amplifications on the same slide and location. We found that the relationships between genetic and TME diversity are different in primary and matched recurrent tumors. Interestingly, the texture of the ECM, identified by label-free reflectance imaging, was predictive of single-cell genetic traits present in the tissue. Moreover, reflectance of ECM revealed structured organization of the perivascular niche in recurrent GBM, enriched in immunosuppressive macrophages. Single-cell spatial transcriptomics further confirmed the presence of the niche-specific macrophage populations and identified interactions between endothelial cells, perivascular fibroblasts, and immunosuppressive macrophages. Our results underscore the importance of GBM tissue organization in tumor evolution and highlight genetic and spatial dependencies.
... We used the q value <0.25 recommended by broad GSEA software. The glycosylation gene signatures were taken from GSEA and (16,49) to calculate enrichment scores using GSVA package (50). ...
Brain metastatic breast cancer is particularly lethal largely due to therapeutic resistance. Almost half of the patients with metastatic HER2-positive breast cancer develop brain metastases, representing a major clinical challenge. We previously described that cancer-associated fibroblasts are an important source of resistance in primary tumors. Here, we report that breast cancer brain metastasis stromal cell interactions in 3D cocultures induce therapeutic resistance to HER2-targeting agents, particularly to the small molecule inhibitor of HER2/EGFR neratinib. We investigated the underlying mechanisms using a synthetic Notch reporter system enabling the sorting of cancer cells that directly interact with stromal cells. We identified mucins and bulky glycoprotein synthesis as top-up-regulated genes and pathways by comparing the gene expression and chromatin profiles of stroma-contact and no-contact cancer cells before and after neratinib treatment. Glycoprotein gene signatures were also enriched in human brain metastases compared to primary tumors. We confirmed increased glycocalyx surrounding cocultures by immunofluorescence and showed that mucinase treatment increased sensitivity to neratinib by enabling a more efficient inhibition of EGFR/HER2 signaling in cancer cells. Overexpression of truncated MUC1 lacking the intracellular domain as a model of increased glycocalyx-induced resistance to neratinib both in cell culture and in experimental brain metastases in immunodeficient mice. Our results highlight the importance of glycoproteins as a resistance mechanism to HER2-targeting therapies in breast cancer brain metastases.
... This reduced tumoricide is further seen in more complex TM teractions involving the tumor cells. GBM propagates in a soft matrix that does no mally promote upregulated FAK activity [92], which can result in reduced MRFT-A ization [93]. Abundant MRFT localization has been demonstrated to sensitize oth gressive cancers to the T cell response; therefore, a soft matrix such as the brain wou only reduce T cell activation but also remove a targetable receptor [94]. ...
... This reduced tumoricide is further seen in more complex TME interactions involving the tumor cells. GBM propagates in a soft matrix that does not normally promote upregulated FAK activity [92], which can result in reduced MRFT-A localization [93]. Abundant MRFT localization has been demonstrated to sensitize other aggressive cancers to the T cell response; therefore, a soft matrix such as the brain would not only reduce T cell activation but also remove a targetable receptor [94]. ...
GBM is the most aggressive and common form of primary brain cancer with a dismal prognosis. Current GBM treatments have not improved patient survival, due to the propensity for tumor cell adaptation and immune evasion, leading to a persistent progression of the disease. In recent years, the tumor microenvironment (TME) has been identified as a critical regulator of these pro-tumorigenic changes, providing a complex array of biomolecular and biophysical signals that facilitate evasion strategies by modulating tumor cells, stromal cells, and immune populations. Efforts to unravel these complex TME interactions are necessary to improve GBM therapy. Immunotherapy is a promising treatment strategy that utilizes a patient’s own immune system for tumor eradication and has exhibited exciting results in many cancer types; however, the highly immunosuppressive interactions between the immune cell populations and the GBM TME continue to present challenges. In order to elucidate these interactions, novel bioengineering models are being employed to decipher the mechanisms of immunologically “cold” GBMs. Additionally, these data are being leveraged to develop cell engineering strategies to bolster immunotherapy efficacy. This review presents an in-depth analysis of the biophysical interactions of the GBM TME and immune cell populations as well as the systems used to elucidate the underlying immunosuppressive mechanisms for improving current therapies.
... 6 Previous studies have reported that the mechanical modulus of the glioma tumor ECM increases with disease progression and have investigated how this increased stiffness may influence GBM cell behavior. [7][8][9][10] Increased production of both polysaccharide (e.g., hyaluronic acid [HA]) and protein (e.g., fibronectin) components of the ECM leads to elevated stiffness of the tumor ECM. 11,12 Simultaneously, tumor progression correlates with increased expression of ECM receptors, including various integrins and the CD44 receptor for HA, by GBM cells. ...
... Tumor cells interact with their surrounding ECM through cell receptors, including RGDbinding integrins and HA-binding CD44, 4 both of which can transduce mechanical cues. 7,15 To investigate whether integrins or CD44 mediated the stiffness-induced metabolic shift of GBM cells cultured in soft hydrogels, we used the small-molecule inhibitors cilengitide (CRGD) and NSC668394. CRGD, a cyclic peptide containing RGD, is a competitive inhibitor of integrin-RGD binding in the hydrogel matrices. ...
... 4 Among features of the TME, mechanical stresses have been reported to impact tumor progression. 7,9,10 In this investigation, we used a 3D culture system to isolate the effects of matrix elasticity from other features of the TME, including ECM composition and density, to discover that ECM stiffness directly induces shifts in metabolism, which in turn influences GBM cell behavior. ...
The mechanical properties of solid tumors influence tumor cell phenotype and the ability to invade surrounding tissues. Using bioengineered scaffolds to provide a matrix microenvironment for patient-derived glioblastoma (GBM) spheroids, this study demonstrates that a soft, brain-like matrix induces GBM cells to shift to a glycolysis-weighted metabolic state, which supports invasive behavior. We first show that orthotopic murine GBM tumors are stiffer than peritumoral brain tissues, but tumor stiffness is heterogeneous where tumor edges are softer than the tumor core. We then developed 3D scaffolds with μ-compressive moduli resembling either stiffer tumor core or softer peritumoral brain tissue. We demonstrate that the softer matrix microenvironment induces a shift in GBM cell metabolism toward glycolysis, which manifests in lower proliferation rate and increased migration activities. Finally, we show that these mechanical cues are transduced from the matrix via CD44 and integrin receptors to induce metabolic and phenotypic changes in cancer cells.
... IHC of lung tissues for the 4T07 MUC1, HER2 mouse model IHC was performed as previously described 75 using antibodies specific to phospho-FAK Y397, cyclin D1 and phospho-(Ser/Thr)-Akt substrates (see Supplemental Table 5). Briefly, antigen retrieval was accomplished by boiling sections in 10 mM citrate buffer for 10 min. ...
Targeted protein degradation is an emerging strategy for the elimination of classically undruggable proteins. Here, to expand the landscape of targetable substrates, we designed degraders that achieve substrate selectivity via recognition of a discrete peptide and glycan motif and achieve cell-type selectivity via antigen-driven cell-surface binding. We applied this approach to mucins, O-glycosylated proteins that drive cancer progression through biophysical and immunological mechanisms. Engineering of a bacterial mucin-selective protease yielded a variant for fusion to a cancer antigen-binding nanobody. The resulting conjugate selectively degraded mucins on cancer cells, promoted cell death in culture models of mucin-driven growth and survival, and reduced tumor growth in mouse models of breast cancer progression. This work establishes a blueprint for the development of biologics that degrade specific protein glycoforms on target cells.
... The finding of myeloid subpopulations at the MVP region is particularly interesting to further study the interaction of tumor and immune cells that may promote MES transition and tumor progression. Given the role of the ECM to foster tumorigenesis and relapse [82][83][84], its component genes have been potential targets for cancer treatment [14,63,[85][86][87]. Therefore, it is essential to find key regulators for aberrant ECM production and determine the molecular features of key cell subpopulations responsible for ECM gene expression. ...
Glioblastoma (GBM) is the most frequent malignant brain tumor, the relapse of which is unavoidable following standard treatment. However, the effective treatment for recurrent GBM is lacking, necessitating the understanding of key mechanisms driving tumor recurrence and the identification of new targets for intervention. Here, we integrated single-cell RNA-sequencing data spanning 36 patient-matched primary and recurrent GBM (pGBM and rGBM) specimens, with 6 longitudinal GBM spatial transcriptomics to explore molecular alterations at recurrence, with each cell type characterized in parallel. Genes involved in extracellular matrix (ECM) organization are preferentially enriched in rGBM cells, and MAFK is highlighted as a potential regulator. Notably, we uncover a unique subpopulation of GBM cells that is much less detected in pGBM and highly expresses ECM and mesenchyme related genes, suggesting it may contribute to the molecular transition of rGBM. Further regulatory network analysis reveals that transcription factors, such as NFATC4 and activator protein 1 members, may function as hub regulators. All non-tumor cells alter their specific sets of genes as well and certain subgroups of myeloid cells appear to be physically associated with the mesenchyme-like GBM subpopulation. Altogether, our study provides new insights into the molecular understanding of GBM relapse and candidate targets for rGBM treatment.
... Tumor cells interact with their surrounding ECM through cell receptors, including such RGD-binding integrins and HA-binding CD44 3 , both of which can transduce mechanical cues 6,14 . To investigate whether integrins or CD44 mediated the stiffness-induced metabolic shift of GBM cells cultured in soft hydrogels, we used the small molecule inhibitors Cilengitide TM (CRGD) and NSC668394. ...
... ; https://doi.org/10.1101/2023.07.03.547558 doi: bioRxiv preprint partly attributed to the ECM content and stiffness. The CD44 receptor for the HA polysaccharide and integrin receptors for various ECM proteins have each been reported to relay mechanical cues from the matrix to cells 6,14 . Here, we showed that both CD44 and integrins on GBM cells can transduce mechanical cues that mediate downstream metabolic shifts, but the extent of their influence depends on the patient- was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. ...
The mechanical properties of solid tumors influence tumor cell phenotype and ability to invade into surrounding tissues. Using bioengineered scaffolds to provide a matrix microenvironment for patient-derived glioblastoma (GBM) spheroids, this study demonstrates that a soft, brain-like matrix induces GBM cells to shift to a glycolysisweighted metabolic state which supports invasive behavior. We first show that orthotopic murine GBM tumors are stiffer than peri-tumoral brain tissues, but tumor stiffness is heterogenous where tumor edges are softer than the tumor core. Then, we developed three-dimensional scaffolds with μ-compressive moduli resembling either stiffer, tumor core or softer, peri-tumoral brain tissue. We demonstrate that the softer matrix microenvironment induces a shift in GBM cell metabolism toward glycolysis which manifests in lower proliferation rate and increased migration activities. Finally, we show that these mechanical cues are transduced from the matrix via CD44 and integrin receptors to induce metabolic and phenotypic changes in cancer cells.
... Specifically, FN1 (fibronectin), a key gene in ECM, is a direct target of CEBPD. The significance of EGFR/PI3K/AKT pathway on GBM has been extensively studied [16,17], and the importance of ECM on GBM is being increasingly recognized in recent years [18][19][20]. Our results uncovered the intrinsic roles of CEBPD on HRGs and provided novel links between hypoxia, ECM and EGFR/PI3K/AKT pathway. ...
Hypoxia contributes to the initiation and progression of glioblastoma by regulating a cohort of genes called hypoxia-regulated genes (HRGs) which form a complex molecular interacting network (HRG-MINW). Transcription factors (TFs) often play central roles for MINW. The key TFs for hypoxia induced reactions were explored using proteomic analysis to identify a set of hypoxia-regulated proteins (HRPs) in GBM cells. Next, systematic TF analysis identified CEBPD as a top TF that regulates the greatest number of HRPs and HRGs. Clinical sample and public database analysis revealed that CEBPD is significantly up-regulated in GBM, high levels of CEBPD predict poor prognosis. In addition, CEBPD is highly expressed in hypoxic condition both in GBM tissue and cell lines. For molecular mechanisms, HIF1α and HIF2α can activate the CEBPD promotor. In vitro and in vivo experiments demonstrated that CEBPD knockdown impaired the invasion and growth capacity of GBM cells, especially in hypoxia condition. Next, proteomic analysis identified that CEBPD target proteins are mainly involved in the EGFR/PI3K pathway and extracellular matrix (ECM) functions. WB assays revealed that CEBPD significantly positively regulated EGFR/PI3K pathway. Chromatin immunoprecipitation (ChIP) qPCR/Seq analysis and Luciferase reporter assay demonstrated that CEBPD binds and activates the promotor of a key ECM protein FN1 (fibronectin). In addition, the interactions of FN1 and its integrin receptors are necessary for CEBPD-induced EGFR/PI3K activation by promoting EGFR phosphorylation. Furthermore, GBM sample analysis in the database corroborated that CEBPD is positively correlated with the pathway activities of EGFR/PI3K and HIF1α, especially in highly hypoxic samples. At last, HRPs are also enriched in ECM proteins, indicating that ECM activities are important components of hypoxia induced responses in GBM. In conclusion, CEPBD plays important regulatory roles in the GBM HRG-MINW as a key TF, which activates the EGFR/PI3K pathway through ECM, especially FN1, mediated EGFR phosphorylation.
... Fibrotic elements of the TME are other environmental variables in the modulation of quiescent states and tumour cell fitness across different tumour types [118][119][120][121]. Mechanosignalling pathways can modulate all the elements of a cell's fitness: growth, survival, invasion and treatment resistance [118,[122][123][124]. ...
... Fibrotic elements of the TME are other environmental variables in the modulation of quiescent states and tumour cell fitness across different tumour types [118][119][120][121]. Mechanosignalling pathways can modulate all the elements of a cell's fitness: growth, survival, invasion and treatment resistance [118,[122][123][124]. For GBM in particular, it is suggested that the mesenchymal, stem-like phenotype observed in recurrent GBM could be induced via tension-mediated glycocalyx-integrin feedback, suggesting novel in situ modulation of quiescent states and treatment strategies aiming to reduce TME tension [119]. Furthermore, collagen also can mediate immune activity (see a discussion in [125]), creating an immunosuppressive microenvironment for gliomas [126]. ...
... The fourth level of organisation is ecosystems, where the focus is on the feedback between the cellular (cell types) and the physicochemical elements of the TME (e.g., collagen, pH, oxygen). At this level, different cell types are affected, and affect, the abiotic components TME [119]. For example, Yin et al. [126] suggested various collagen-associated genes (COL1A1, COL1A2, COL3A1, COL4A1, COL4A2, and COL5A2) that could regulate the immunosuppressive microenvironment of gliomas. ...
Quiescence is a state of cell cycle arrest, allowing cancer cells to evade anti-proliferative cancer therapies. Quiescent cancer stem cells are thought to be responsible for treatment resistance in glioblastoma, an aggressive brain cancer with poor patient outcomes. However, the regulation of quiescence in glioblastoma cells involves a myriad of intrinsic and extrinsic mechanisms that are not fully understood. In this review, we synthesise the literature on quiescence regulatory mechanisms in the context of glioblastoma and propose an ecological perspective to stemness-like phenotypes anchored to the contemporary concepts of niche theory. From this perspective, the cell cycle regulation is multiscale and multidimensional, where the niche dimensions extend to extrinsic variables in the tumour microenvironment that shape cell fate. Within this conceptual framework and powered by ecological niche modelling, the discovery of microenvironmental variables related to hypoxia and mechanosignalling that modulate proliferative plasticity and intratumor immune activity may open new avenues for therapeutic targeting of emerging biological vulnerabilities in glioblastoma that cannot be anticipated from genomics alone.
... [nm]) glycocalyx layer [3][4][5][6] . This glycocalyx imposes a physical separation between the cancer cell and its surroundings that is much wider than the typical length of adhesion proteins such as integrins (~20 nm). ...
Many types of cancer overexpress bulky glycoproteins to form a thick glycocalyx layer. The glycocalyx physically separates the cell from its surroundings, but recent work has shown that the glycocalyx can paradoxically increase adhesion to soft tissues and therefore promote the metastasis of cancer cells. This surprising phenomenon occurs because the glycocalyx forces adhesion molecules (called integrins) on the cell’s surface into clusters. These integrin clusters have cooperative effects that allow them to form stronger adhesions to surrounding tissues than would be possible with equivalent numbers of un-clustered integrins. These cooperative mechanisms have been intensely scrutinized in recent years; a more nuanced understanding of the biophysical underpinnings of glycocalyx-mediated adhesion could uncover therapeutic targets, deepen our general understanding of cancer metastasis, and elucidate general biophysical processes that extend far beyond the realm of cancer research.
This work examines the hypothesis that the glycocalyx has the additional effect of increasing mechanical tension experienced by clustered integrins. Integrins function as mechanosensors that undergo catch bonding – meaning the application of moderate tension increases integrin bond lifetime relative to the lifetime of integrins experiencing low tension. In this work, a three-state chemomechanical catch bond model of integrin tension is used to investigate catch bonding in the presence of a bulky glycocalyx.
This modeling suggests that a bulky glycocalyx can lightly trigger catch bonding, increasing the bond lifetime of integrins at adhesion edges by up to 100%. The total number of integrin-ligand bonds within an adhesion is predicted to increase by up to ~ 60% for certain adhesion geometries. Catch bonding is predicted to decrease the activation energy of adhesion formation by ~ 1–4 kBT, which translates to a ~ 3–50× increase in the kinetic rate of adhesion nucleation. This work reveals that integrin mechanic and clustering likely both contribute to glycocalyx-mediated metastasis.
