A metastasis map of human cancer cell lines.

Most deaths from cancer are explained by metastasis, and yet large-scale metastasis research has been impractical owing to the complexity of in vivo models. Here we introduce an in vivo barcoding strategy that is capable of determining the metastatic potential of human cancer cell lines in mouse xenografts at scale. We validated the robustness, scalability and reproducibility of the method and applied it to 500 cell lines1,2 spanning 21 types of solid tumour. We created a first-generation metastasis map (MetMap) that reveals organ-specific patterns of metastasis, enabling these patterns to be associated with clinical and genomic features. We demonstrate the utility of MetMap by investigating the molecular basis of breast cancers capable of metastasizing to the brain-a principal cause of death in patients with this type of cancer. Breast cancers capable of metastasizing to the brain showed evidence of altered lipid metabolism. Perturbation of lipid metabolism in these cells curbed brain metastasis development, suggesting a therapeutic strategy to combat the disease and demonstrating the utility of MetMap as a resource to support metastasis research.

Association of Lack of Speech Arrest During Cortical Stimulation With Interhemispheric Reorganization of the Functional Language Network in Patients With Brain Tumors.

BACKGROUND. Brain tumors induce language reorganization, which may influence the extent of resection in surgical planning. Direct cortical stimulation (DCS) allows definitive language mapping during awake surgery by locating areas of speech arrest (SA) surrounding the tumor. Although functional MRI (fMRI) combined with graph theory analysis can illustrate whole-brain network reorganization, few studies have corroborated these findings with DCS intraoperative mapping and clinical language performance. OBJECTIVE. We evaluated whether patients with low-grade gliomas (LGGs) without SA during DCS show increased right-hemispheric connections and better speech performance compared with patients with SA. METHODS. We retrospectively recruited 44 consecutive patients with left perisylvian LGG, preoperative language task-based fMRI, speech performance evaluation, and awake surgery with DCS. We generated language networks from ROIs corresponding to known language areas (i.e., language core) on fMRI using optimal percolation. Language core connectivity in the left and right hemispheres was quantified as fMRI laterality index (LI) and connectivity LI on the basis of fMRI activation maps and connectivity matrices. We compared fMRI LI and connectivity LI between patients with SA and without SA and used multivariable logistic regression (p < .05) to assess associations between DCS and connectivity LI, fMRI LI, tumor location, Broca area and Wernicke area involvement, prior treatments, age, handedness, sex, tumor size, and speech deficit before surgery, within 1 week after surgery, and 3-6 months after surgery. RESULTS. Patients with SA showed left-dominant connectivity; patients without SA lateralized more to the right hemisphere (p < .001). Between patients with SA and those without, fMRI LI was not significantly different. Patients without SA showed right-greater-than-left connectivity of Broca area and premotor area compared with patients with SA. Regression analysis showed significant association between no SA and right-lateralized connectivity LI (p < .001) and fewer speech deficits before (p < .001) and 1 week after (p = .02) surgery. CONCLUSION. Patients without SA had increased right-hemispheric connections and right translocation of the language core, suggesting language reorganization. Lack of interoperative SA was associated with fewer speech deficits both before and immediately after surgery. CLINICAL IMPACT. These findings support tumor-induced language plasticity as a compensatory mechanism, which may lead to fewer postsurgical deficits and allow extended resection.

Kendrick Mass Defect Variation to Decipher Isotopic Labeling in Brain Metastases Studied by Mass Spectrometry Imaging.

Besides many other applications, isotopic labeling is commonly used to decipher the metabolism of living biological systems. By giving a stable isotopically labeled compound as a substrate, the biological system will use this labeled nutrient as it would with a regular substrate and incorporate stable heavy atoms into new metabolites. Utilizing mass spectrometry, by comparing heavy atom enriched isotopic profiles and naturally occurring ones, it is possible to identify these metabolites and deduce valuable information about metabolism and biochemical pathways. The coupling of this approach with mass spectrometry imaging (MSI) allows one then to obtain 2D maps of metabolisms used by living specimens. As metabolic networks are convoluted, a global overview of the isotopically labeled data set to detect unexpected metabolites is crucial. Unfortunately, due to the complexity of MSI spectra, such untargeted processing approaches are difficult to decipher. In this technical note, we demonstrate the potential of a variation around the Kendrick analysis concept to detect the incorporation of stable heavy atoms into metabolites. The Kendrick analysis uses as a base unit the difference between the mass of the most abundant isotope and the mass of the corresponding stable isotopic tracer (namely, 12C and 13C). The resulting Kendrick plot offers an alternative method to process the MSI data set with a new perspective allowing for the rapid detection of the 13C-enriched metabolites and separating unrelated compounds. This processing method of MS data could therefore be a useful tool to decipher isotopic labeling and study metabolic networks, especially as it does not require advanced computational capabilities.

Mechanisms of enhanced drug delivery in brain metastases with focused ultrasound-induced blood-tumor barrier disruption.

Blood-brain/blood-tumor barriers (BBB and BTB) and interstitial transport may constitute major obstacles to the transport of therapeutics in brain tumors. In this study, we examined the impact of focused ultrasound (FUS) in combination with microbubbles on the transport of two relevant chemotherapy-based anticancer agents in breast cancer brain metastases at cellular resolution: doxorubicin, a nontargeted chemotherapeutic, and ado-trastuzumab emtansine (T-DM1), an antibody-drug conjugate. Using an orthotopic xenograft model of HER2-positive breast cancer brain metastasis and quantitative microscopy, we demonstrate significant increases in the extravasation of both agents (sevenfold and twofold for doxorubicin and T-DM1, respectively), and we provide evidence of increased drug penetration (>100 vs. <20 µm and 42 ± 7 vs. 12 ± 4 µm for doxorubicin and T-DM1, respectively) after the application of FUS compared with control (non-FUS). Integration of experimental data with physiologically based pharmacokinetic (PBPK) modeling of drug transport reveals that FUS in combination with microbubbles alleviates vascular barriers and enhances interstitial convective transport via an increase in hydraulic conductivity. Experimental data demonstrate that FUS in combination with microbubbles enhances significantly the endothelial cell uptake of the small chemotherapeutic agent. Quantification with PBPK modeling reveals an increase in transmembrane transport by more than two orders of magnitude. PBPK modeling indicates a selective increase in transvascular transport of doxorubicin through small vessel wall pores with a narrow range of sizes (diameter, 10-50 nm). Our work provides a quantitative framework for the optimization of FUS-drug combinations to maximize intratumoral drug delivery and facilitate the development of strategies to treat brain metastases.

Targeting the cMET pathway augments radiation response without adverse effect on hearing in NF2 schwannoma models.

Neurofibromatosis type II (NF2) is a disease that needs new solutions. Vestibular schwannoma (VS) growth causes progressive hearing loss, and the standard treatment, including surgery and radiotherapy, can further damage the nerve. There is an urgent need to identify an adjunct therapy that, by enhancing the efficacy of radiation, can help lower the radiation dose and preserve hearing. The mechanisms underlying deafness in NF2 are still unclear. One of the major limitations in studying tumor-induced hearing loss is the lack of mouse models that allow hearing testing. Here, we developed a cerebellopontine angle (CPA) schwannoma model that faithfully recapitulates the tumor-induced hearing loss. Using this model, we discovered that cMET blockade by crizotinib (CRZ) enhanced schwannoma radiosensitivity by enhancing DNA damage, and CRZ treatment combined with low-dose radiation was as effective as high-dose radiation. CRZ treatment had no adverse effect on hearing; however, it did not affect tumor-induced hearing loss, presumably because cMET blockade did not change tumor hepatocyte growth factor (HGF) levels. This cMET gene knockdown study independently confirmed the role of the cMET pathway in mediating the effect of CRZ. Furthermore, we evaluated the translational potential of cMET blockade in human schwannomas. We found that human NF2-associated and sporadic VSs showed significantly elevated HGF expression and cMET activation compared with normal nerves, which correlated with tumor growth and cyst formation. Using organoid brain slice culture, cMET blockade inhibited the growth of patient-derived schwannomas. Our findings provide the rationale and necessary data for the clinical translation of combined cMET blockade with radiation therapy in patients with NF2.

MT3-MMP Promotes Excitatory Synapse Formation by Promoting Nogo-66 Receptor Ectodomain Shedding.

Cell-surface molecules are dynamically regulated at the synapse to assemble and disassemble adhesive contacts that are important for synaptogenesis and for tuning synaptic transmission. Metalloproteinases dynamically regulate cellular behaviors through the processing of cell surface molecules. In the present study, we evaluated the role of membrane-type metalloproteinases (MT-MMPs) in excitatory synaptogenesis. We find that MT3-MMP and MT5-MMP are broadly expressed in the mouse cerebral cortex and that MT3-MMP loss-of-function interferes with excitatory synapse development in dissociated cortical neurons and in vivo We identify Nogo-66 receptor (NgR1) as an MT3-MMP substrate that is required for MT3-MMP-dependent synapse formation. Introduction of the shed ectodomain of NgR1 is sufficient to accelerate excitatory synapse formation in dissociated cortical neurons and in vivo Together, our findings support a role for MT3-MMP-dependent shedding of NgR1 in regulating excitatory synapse development.SIGNIFICANCE STATEMENT In this study, we identify MT3-MMP, a membrane-bound zinc protease, to be necessary for the development of excitatory synapses in cortical neurons. We identify Nogo-66 receptors (NgR1) as a downstream target of MT3-MMP proteolytic activity. Furthermore, processing of surface NgR1 by MT3-MMP generates a soluble ectodomain fragment that accelerates the formation of excitatory synapses. We propose that MT3-MMP activity and NgR1 shedding could stimulate circuitry remodeling in the adult brain and enhance functional connectivity after brain injury.

IgLON cell adhesion molecules are shed from the cell surface of cortical neurons to promote neuronal growth.

Matrix metalloproteinases and a disintegrin and metalloproteinases are members of the zinc endopeptidases, which cleave components of the extracellular matrix as well as cell surface proteins resulting in degradation or release of biologically active fragments. Surface ectodomain shedding affects numerous biological processes, including survival, axon outgrowth, axon guidance, and synaptogenesis. In this study, we evaluated the role of metalloproteinases in regulating cortical neurite growth. We found that treatment of mature cortical neurons with pan-metalloproteinase inhibitors or with tissue inhibitors of metalloproteinase-3 reduced neurite outgrowth. Through mass spectrometry, we characterized the metalloproteinase-sensitive cell surface proteome of mature cortical neurons. Members of the IgLON family of glycosylphosphatidylinositol-anchored neural cell adhesion molecules were identified and validated as proteins that were shed from the surface of mature cortical neurons in a metalloproteinase-dependent manner. Introduction of two members of the IgLON family, neurotrimin and NEGR1, in early embryonic neurons was sufficient to confer sensitivity to metalloproteinase inhibitors in neurite outgrowth assays. Outgrowth experiments on immobilized IgLON proteins revealed a role for all IgLON family members in promoting neurite extension from cortical neurons. Together, our findings support a role for metalloproteinase-dependent shedding of IgLON family members in regulating neurite outgrowth from mature cortical neurons.

Anergy in self-directed B lymphocytes: A statistical mechanics perspective.

Self-directed lymphocytes may evade clonal deletion at ontogenesis but still remain harmless due to a mechanism called clonal anergy. For B-lymphocytes, two major explanations for anergy developed over the last decades: according to Varela theory, anergy stems from a proper orchestration of the whole B-repertoire, such that self-reactive clones, due to intensive feed-back from other clones, display strong inertia when mounting a response. Conversely, according to the model of cognate response, self-reacting cells are not stimulated by helper lymphocytes and the absence of such signaling yields anergy. Through statistical mechanics we show that helpers do not prompt activation of a sub-group of B-cells: remarkably, the latter are just those broadly interacting in the idiotypic network. Hence Varela theory can finally be reabsorbed into the prevailing framework of the cognate response model. Further, we show how the B-repertoire architecture may emerge, where highly connected clones are self-directed as a natural consequence of ontogenetic learning.

Discovery of the Potent and Selective ATR Inhibitor Camonsertib (RP-3500).

ATR is a key kinase in the DNA-damage response (DDR) that is synthetic lethal with several other DDR proteins, making it an attractive target for the treatment of genetically selected solid tumors. Herein we describe the discovery of a novel ATR inhibitor guided by a pharmacophore model to position a key hydrogen bond. Optimization was driven by potency and selectivity over the related kinase mTOR, resulting in the identification of camonsertib (RP-3500) with high potency and excellent ADME properties. Preclinical evaluation focused on the impact of camonsertib on myelosuppression, and an exploration of intermittent dosing schedules to allow recovery of the erythroid compartment and mitigate anemia. Camonsertib is currently undergoing clinical evaluation both as a single agent and in combination with talazoparib, olaparib, niraparib, lunresertib, or gemcitabine (NCT04497116, NCT04972110, NCT04855656). A preliminary recommended phase 2 dose for monotherapy was identified as 160 mg QD given 3 days/week.

Longitudinal Evaluation of Brain Plasticity in Low-Grade Gliomas: fMRI and Graph-Theory Provide Insights on Language Reorganization.

Language reorganization may represent an adaptive phenomenon to compensate tumor invasion of the dominant hemisphere. However, the functional changes over time underlying language plasticity remain unknown. We evaluated language function in patients with low-grade glioma (LGG), using task-based functional MRI (tb-fMRI), graph-theory and standardized language assessment. We hypothesized that functional networks obtained from tb-fMRI would show connectivity changes over time, with increased right-hemispheric participation. We recruited five right-handed patients (4M, mean age 47.6Y) with left-hemispheric LGG. Tb-fMRI and language assessment were conducted pre-operatively (pre-op), and post-operatively: post-op1 (4-8 months), post-op2 (10-14 months) and post-op3 (16-23 months). We computed the individual functional networks applying optimal percolation thresholding. Language dominance and hemispheric connectivity were quantified by laterality indices (LI) on fMRI maps and connectivity matrices. A fixed linear mixed model was used to assess the intra-patient correlation trend of LI values over time and their correlation with language performance. Individual networks showed increased inter-hemispheric and right-sided connectivity involving language areas homologues. Two patterns of language reorganization emerged: Three/five patients demonstrated a left-to-codominant shift from pre-op to post-op3 (type 1). Two/five patients started as atypical dominant at pre-op, and remained unchanged at post-op3 (type 2). LI obtained from tb-fMRI showed a significant left-to-right trend in all patients across timepoints. There were no significant changes in language performance over time. Type 1 language reorganization may be related to the treatment, while type 2 may be tumor-induced, since it was already present at pre-op. Increased inter-hemispheric and right-side connectivity may represent the initial step to develop functional plasticity.

Co-Targeting IL-6 and EGFR signaling for the treatment of schwannomatosis and associated pain.

Patients with Schwannomatosis (SWN) overwhelmingly present with intractable, debilitating chronic pain. There are no effective therapies to treat SWN. The drivers of pain response and tumor progression in SWN are not clear. The pain is not proportionally linked to tumor size and is not always relieved by tumor resection, suggesting that mechanisms other than mechanical nerve compression exist to cause pain. SWN research is limited by the lack of clinically-relevant models. Here, we established novel patient-derived xenograft (PDX) models, dorsal root ganglia (DRG) imaging model, and combined with single-cell resolution intravital imaging and RNASeq, we discovered: i) schwannomas on the peripheral nerve cause macrophage influx into the DRG, via secreting HMGB1 to directly stimulate DRG neurons to express CCL2, the key macrophage chemokine, ii) once recruited, macrophages cause pain response via overproduction of IL-6, iii) IL-6 blockade in a therapeutic setting significantly reduces pain but has modest efficacy on tumor growth, iv) EGF signaling is a potential driver of schwannoma growth and escape mechanism from anti-IL6 treatment, and v) combined IL-6 and EGFR blockade simultaneously controlled pain and tumor growth in SWN models. Our findings prompted the initiation of phase II clinical trial ( NCT05684692 ) for pain relief in patients with SWN.

Membrane-type matrix metalloproteinase-3 regulates neuronal responsiveness to myelin through Nogo-66 receptor 1 cleavage.

Nogo-66 receptor 1 (NgR1) is a glycosylphosphatidylinositol-anchored receptor for myelin-associated inhibitors that restricts plasticity and axonal regrowth in the CNS. NgR1 is cleaved from the cell surface of SH-SY5Y neuroblastoma cells in a metalloproteinase-dependent manner; however, the mechanism and physiological consequence of NgR1 shedding have not been explored. We now demonstrate that NgR1 is shed from multiple populations of primary neurons. Through a loss-of-function approach, we found that membrane-type matrix metalloproteinase-3 (MT3-MMP) regulates endogenous NgR1 shedding in primary neurons. Neuronal knockdown of MT3-MMP resulted in the accumulation of NgR1 at the cell surface and reduced the accumulation of the NgR1 cleavage fragment in medium conditioned by cortical neurons. Recombinant MT1-, MT2-, MT3-, and MT5-MMPs promoted NgR1 shedding from the surface of primary neurons, and this treatment rendered neurons resistant to myelin-associated inhibitors. Introduction of a cleavage-resistant form of NgR1 reconstitutes the neuronal response to these inhibitors, demonstrating that specific metalloproteinases attenuate neuronal responses to myelin in an NgR1-dependent manner.

14-3-3 proteins regulate protein kinase a activity to modulate growth cone turning responses.

Growth cones regulate the speed and direction of neuronal outgrowth during development and regeneration. How the growth cone spatially and temporally regulates signals from guidance cues is poorly understood. Through a proteomic analysis of purified growth cones we identified isoforms of the 14-3-3 family of adaptor proteins as major constituents of the growth cone. Disruption of 14-3-3 via the R18 antagonist or knockdown of individual 14-3-3 isoforms switches nerve growth factor- and myelin-associated glycoprotein-dependent repulsion to attraction in embryonic day 13 chick and postnatal day 5 rat DRG neurons. These effects are reminiscent of switching responses observed in response to elevated cAMP. Intriguingly, R18-dependent switching is blocked by inhibitors of protein kinase A (PKA), suggesting that 14-3-3 proteins regulate PKA. Consistently, 14-3-3 proteins interact with PKA and R18 activates PKA by dissociating its regulatory and catalytic subunits. Thus, 14-3-3 heterodimers regulate the PKA holoenzyme and this activity plays a critical role in modulating neuronal responses to repellent cues.

LGI1 is a Nogo receptor 1 ligand that antagonizes myelin-based growth inhibition.

Mutations in leucine-rich glioma inactivated (LGI1) are a genetic cause of autosomal dominant temporal lobe epilepsy with auditory features. LGI1 is a secreted protein that shares homology with members of the SLIT family, ligands that direct axonal repulsion and growth cone collapse, and we therefore considered the possibility that LGI1 may regulate neuronal process extension or growth cone collapse. Here we report that LGI1 does not affect growth directly but instead enhances neuronal growth on myelin-based inhibitory substrates and antagonizes myelin-induced growth cone collapse. We show that LGI1 mediates this effect by functioning as a specific Nogo receptor 1 (NgR1) ligand that antagonizes the action of myelin-based inhibitory cues. Finally, we demonstrate that NgR1 and ADAM22 physically associate to form a receptor complex in which NgR1 facilitates LGI1 binding to ADAM22.

Monolingual and bilingual language networks in healthy subjects using functional MRI and graph theory.

Bilingualism requires control of multiple language systems, and may lead to architectural differences in language networks obtained from clinical fMRI tasks. Emerging connectivity metrics such as k-core may capture these differences, highlighting crucial network components based on resiliency. We investigated the influence of bilingualism on clinical fMRI language tasks and characterized bilingual networks using connectivity metrics to provide a patient care benchmark. Sixteen right-handed subjects (mean age 42-years; nine males) without neurological history were included: eight native English-speaking monolinguals and eight native Spanish-speaking (L1) bilinguals with acquired English (L2). All subjects underwent fMRI with gold-standard clinical language tasks. Starting from active clusters on fMRI, we inferred the persistent functional network across subjects and ran centrality measures to characterize differences. Our results demonstrated a persistent network "core" consisting of Broca's area, the pre-supplementary motor area, and the premotor area. K-core analysis showed that Wernicke's area was engaged by the "core" with weaker connection in L2 than L1.

Targeting Treg cells with GITR activation alleviates resistance to immunotherapy in murine glioblastomas.

Immune checkpoint blockers (ICBs) have failed in all phase III glioblastoma (GBM) trials. Here, we show that regulatory T (Treg) cells play a key role in GBM resistance to ICBs in experimental gliomas. Targeting glucocorticoid-induced TNFR-related receptor (GITR) in Treg cells using an agonistic antibody (αGITR) promotes CD4 Treg cell differentiation into CD4 effector T cells, alleviates Treg cell-mediated suppression of anti-tumor immune response, and induces potent anti-tumor effector cells in GBM. The reprogrammed GBM-infiltrating Treg cells express genes associated with a Th1 response signature, produce IFNγ, and acquire cytotoxic activity against GBM tumor cells while losing their suppressive function. αGITR and αPD1 antibodies increase survival benefit in three experimental GBM models, with a fraction of cohorts exhibiting complete tumor eradication and immune memory upon tumor re-challenge. Moreover, αGITR and αPD1 synergize with the standard of care treatment for newly-diagnosed GBM, enhancing the cure rates in these GBM models.

FATTY ACID SYNTHESIS IS REQUIRED FOR BREAST CANCER BRAIN METASTASIS.

Brain metastases are refractory to therapies that control systemic disease in patients with human epidermal growth factor receptor 2 (HER2+) breast cancer, and the brain microenvironment contributes to this therapy resistance. Nutrient availability can vary across tissues, therefore metabolic adaptations required for brain metastatic breast cancer growth may introduce liabilities that can be exploited for therapy. Here, we assessed how metabolism differs between breast tumors in brain versus extracranial sites and found that fatty acid synthesis is elevated in breast tumors growing in brain. We determine that this phenotype is an adaptation to decreased lipid availability in brain relative to other tissues, resulting in a site-specific dependency on fatty acid synthesis for breast tumors growing at this site. Genetic or pharmacological inhibition of fatty acid synthase (FASN) reduces HER2+ breast tumor growth in the brain, demonstrating that differences in nutrient availability across metastatic sites can result in targetable metabolic dependencies.

The blood-brain barrier and blood-tumour barrier in brain tumours and metastases.

For a blood-borne cancer therapeutic agent to be effective, it must cross the blood vessel wall to reach cancer cells in adequate quantities, and it must overcome the resistance conferred by the local microenvironment around cancer cells. The brain microenvironment can thwart the effectiveness of drugs against primary brain tumours as well as brain metastases. In this Review, we highlight the cellular and molecular components of the blood-brain barrier (BBB), a specialized neurovascular unit evolved to maintain brain homeostasis. Tumours are known to compromise the integrity of the BBB, resulting in a vasculature known as the blood-tumour barrier (BTB), which is highly heterogeneous and characterized by numerous distinct features, including non-uniform permeability and active efflux of molecules. We discuss the challenges posed by the BBB and BTB for drug delivery, how multiple cell types dictate BBB function and the role of the BTB in disease progression and treatment. Finally, we highlight emerging molecular, cellular and physical strategies to improve drug delivery across the BBB and BTB and discuss their impact on improving conventional as well as emerging treatments, such as immune checkpoint inhibitors and engineered T cells. A deeper understanding of the BBB and BTB through the application of single-cell sequencing and imaging techniques, and the development of biomarkers of BBB integrity along with systems biology approaches, should enable new personalized treatment strategies for primary brain malignancies and brain metastases.