Multiplexed spatial profiling of Hodgkin Reed-Sternberg cell neighborhoods in classic Hodgkin lymphoma.

PURPOSE: Classic Hodgkin lymphoma (cHL) is a B cell lymphoma that occurs primarily in young adults and, less frequently, in elderly individuals. A hallmark of cHL is the exceptional scarcity (1-5%) of the malignant Hodgkin Reed-Sternberg (HRS) cells within a network of non-malignant immune cells. Molecular determinants governing the relationship between HRS cells and their proximal microenvironment remain largely unknown. EXPERIMENTAL DESIGN: We performed spatially resolved multiplexed protein imaging and transcriptomic sequencing to characterize HRS cell states, cellular neighborhoods, and gene expression signatures of 23.6 million cells from 36 newly diagnosed Epstein-Barr virus (EBV) positive and EBV-negative cHL tumors. RESULTS: We show that MHC-I expression on HRS cells is associated with immune inflamed neighborhoods containing CD8+ T cells, MHC-II+ macrophages, and immune checkpoint expression (i.e., PD-1 and VISTA). We identified spatial clustering of HRS cells, consistent with the syncytial variant of cHL, and its association with T cell excluded neighborhoods in a subset of EBV-negative tumors. Finally, a subset of both EBV-positive and EBV-negative tumors contained regulatory T cells high neighborhoods harboring HRS cells with augmented proliferative capacity. CONCLUSIONS: Our study links HRS cell properties with distinct immunophenotypes and potential immune escape mechanisms in cHL.

Integrated clinical and genomic analysis identifies driver events and molecular evolution of colitis-associated cancers.

Inflammation has long been recognized to contribute to cancer development, particularly across the gastrointestinal tract. Patients with inflammatory bowel disease have an increased risk for bowel cancers, and it has been posited that a field of genetic changes may underlie this risk. Here, we define the clinical features, genomic landscape, and germline alterations in 174 patients with colitis-associated cancers and sequenced 29 synchronous or isolated dysplasia. TP53 alterations, an early and highly recurrent event in colitis-associated cancers, occur in half of dysplasia, largely as convergent evolution of independent events. Wnt pathway alterations are infrequent, and our data suggest transcriptional rewiring away from Wnt. Sequencing of multiple dysplasia/cancer lesions from mouse models and patients demonstrates rare shared alterations between lesions. These findings suggest neoplastic bowel lesions developing in a background of inflammation experience lineage plasticity away from Wnt activation early during tumorigenesis and largely occur as genetically independent events.

Noninvasive Imaging of CD4+ T Cells in Humanized Mice.

Antibody-based PET (immunoPET) with radiotracers that recognize specific cells of the immune system provides an opportunity to monitor immune cell trafficking at the organismal scale. We previously reported the visualization of human CD8+ T cells, including CD8+ tumor-infiltrating lymphocytes (TIL), in mice using a humanized CD8-targeted minibody. Given the important role of CD4+ T cells in adaptive immune responses of health and disease including infections, tumors, and autoimmunity, we explored immunoPET using an anti-human-CD4 minibody. We assessed the ability of [64Cu]Cu-NOTA-IAB41 to bind to various CD4+ T-cell subsets in vitro. We also determined the effect of the CD4-targeted minibody on CD4+ T-cell abundance, proliferation, and activation state in vitro. We subsequently evaluated the ability of the radiotracer to visualize CD4+ T cells in T-cell rich organs and orthotopic brain tumors in vivo. For the latter, we injected the [64Cu]Cu-NOTA-IAB41 radiotracer into humanized mice that harbored intracranial patient-derived glioblastoma (GBM) xenografts and performed in vivo PET, ex vivo autoradiography, and anti-CD4 IHC on serial brain sections. [64Cu]Cu-NOTA-IAB41 specifically detects human CD4+ T cells without impacting their abundance, proliferation, and activation. In humanized mice, [64Cu]Cu-NOTA-IAB41 can visualize various peripheral tissues in addition to orthotopically implanted GBM tumors. [64Cu]Cu-NOTA-IAB41 is able to visualize human CD4+ T cells in humanized mice and can provide noninvasive quantification of CD4+ T-cell distribution on the organismal scale.

Tumor MHC Class I Expression Associates with Intralesional IL2 Response in Melanoma.

Cancer immunotherapy can result in lasting tumor regression, but predictive biomarkers of treatment response remain ill-defined. Here, we performed single-cell proteomics, transcriptomics, and genomics on matched untreated and IL2 injected metastases from patients with melanoma. Lesions that completely regressed following intralesional IL2 harbored increased fractions and densities of nonproliferating CD8+ T cells lacking expression of PD-1, LAG-3, and TIM-3 (PD-1-LAG-3-TIM-3-). Untreated lesions from patients who subsequently responded with complete eradication of all tumor cells in all injected lesions (individuals referred to herein as "extreme responders") were characterized by proliferating CD8+ T cells with an exhausted phenotype (PD-1+LAG-3+TIM-3+), stromal B-cell aggregates, and expression of IFNγ and IL2 response genes. Loss of membranous MHC class I expression in tumor cells of untreated lesions was associated with resistance to IL2 therapy. We validated this finding in an independent cohort of metastatic melanoma patients treated with intralesional or systemic IL2. Our study suggests that intact tumor-cell antigen presentation is required for melanoma response to IL2 and describes a multidimensional and spatial approach to develop immuno-oncology biomarker hypotheses using routinely collected clinical biospecimens.

GCN2 kinase activation by ATP-competitive kinase inhibitors.

Small-molecule kinase inhibitors represent a major group of cancer therapeutics, but tumor responses are often incomplete. To identify pathways that modulate kinase inhibitor response, we conducted a genome-wide knockout (KO) screen in glioblastoma cells treated with the pan-ErbB inhibitor neratinib. Loss of general control nonderepressible 2 (GCN2) kinase rendered cells resistant to neratinib, whereas depletion of the GADD34 phosphatase increased neratinib sensitivity. Loss of GCN2 conferred neratinib resistance by preventing binding and activation of GCN2 by neratinib. Several other Food and Drug Administration (FDA)-approved inhibitors, such erlotinib and sunitinib, also bound and activated GCN2. Our results highlight the utility of genome-wide functional screens to uncover novel mechanisms of drug action and document the role of the integrated stress response (ISR) in modulating the response to inhibitors of oncogenic kinases.

Imaging Tumor-Infiltrating Lymphocytes in Brain Tumors with [64Cu]Cu-NOTA-anti-CD8 PET.

PURPOSE: Glioblastoma (GBM) is the most common malignant brain tumor in adults. Various immunotherapeutic approaches to improve patient survival are being developed, but the molecular mechanisms of immunotherapy resistance are currently unknown. Here, we explored the ability of a humanized radiolabeled CD8-targeted minibody to noninvasively quantify tumor-infiltrating CD8-positive (CD8+) T cells using PET. EXPERIMENTAL DESIGN: We generated a peripheral blood mononuclear cell (PBMC) humanized immune system (HIS) mouse model and quantified the absolute number of CD8+ T cells by flow cytometry relative to the [64Cu]Cu-NOTA-anti-CD8 PET signal. To evaluate a patient-derived orthotopic GBM HIS model, we intracranially injected cells into NOG mice, humanized cohorts with multiple HLA-matched PBMC donors, and quantified CD8+ tumor-infiltrating lymphocytes by IHC. To determine whether [64Cu]Cu-NOTA-anti-CD8 images brain parenchymal T-cell infiltrate in GBM tumors, we performed PET and autoradiography and subsequently stained serial sections of brain tumor tissue by IHC for CD8+ T cells. RESULTS: Nontumor-bearing NOG mice injected with human PBMCs showed prominent [64Cu]Cu-NOTA-anti-CD8 uptake in the spleen and minimal radiotracer localization to the normal brain. NOG mice harboring intracranial human GBMs yielded high-resolution PET images of tumor-infiltrating CD8+ T cells. Radiotracer retention correlated with CD8+ T-cell numbers in spleen and tumor tissue. Our study demonstrates the ability of [64Cu]Cu-NOTA-anti-CD8 PET to quantify peripheral and tumor-infiltrating CD8+ T cells in brain tumors. CONCLUSIONS: Human CD8+ T cells infiltrate an orthotopic GBM in a donor-dependent manner. Furthermore, [64Cu]Cu-NOTA-anti-CD8 quantitatively images both peripheral and brain parenchymal human CD8+ T cells.

Vorasidenib (AG-881): A First-in-Class, Brain-Penetrant Dual Inhibitor of Mutant IDH1 and 2 for Treatment of Glioma.

Inhibitors of mutant isocitrate dehydrogenase (mIDH) 1 and 2 cancer-associated enzymes prevent the accumulation of the oncometabolite d-2-hydroxyglutarate (2-HG) and are under clinical investigation for the treatment of several cancers harboring an IDH mutation. Herein, we describe the discovery of vorasidenib (AG-881), a potent, oral, brain-penetrant dual inhibitor of both mIDH1 and mIDH2. X-ray cocrystal structures allowed us to characterize the compound binding site, leading to an understanding of the dual mutant inhibition. Furthermore, vorasidenib penetrates the brain of several preclinical species and inhibits 2-HG production in glioma tissue by >97% in an orthotopic glioma mouse model. Vorasidenib represents a novel dual mIDH1/2 inhibitor and is currently in clinical development for the treatment of low-grade mIDH glioma.

Tracking tumour evolution in glioma through liquid biopsies of cerebrospinal fluid.

Diffuse gliomas are the most common malignant brain tumours in adults and include glioblastomas and World Health Organization (WHO) grade II and grade III tumours (sometimes referred to as lower-grade gliomas). Genetic tumour profiling is used to classify disease and guide therapy1,2, but involves brain surgery for tissue collection; repeated tumour biopsies may be necessary for accurate genotyping over the course of the disease3-10. While the detection of circulating tumour DNA (ctDNA) in the blood of patients with primary brain tumours remains challenging11,12, sequencing of ctDNA from the cerebrospinal fluid (CSF) may provide an alternative way to genotype gliomas with lower morbidity and cost13,14. We therefore evaluated the representation of the glioma genome in CSF from 85 patients with gliomas who underwent a lumbar puncture because they showed neurological signs or symptoms. Here we show that tumour-derived DNA was detected in CSF from 42 out of 85 patients (49.4%) and was associated with disease burden and adverse outcome. The genomic landscape of glioma in the CSF included a broad spectrum of genetic alterations and closely resembled the genomes of tumour biopsies. Alterations that occur early during tumorigenesis, such as co-deletion of chromosome arms 1p and 19q (1p/19q codeletion) and mutations in the metabolic genes isocitrate dehydrogenase 1 (IDH1) or IDH21,2, were shared in all matched ctDNA-positive CSF-tumour pairs, whereas growth factor receptor signalling pathways showed considerable evolution. The ability to monitor the evolution of the glioma genome through a minimally invasive technique could advance the clinical development and use of genotype-directed therapies for glioma, one of the most aggressive human cancers.

Phase 1b trial of an ibrutinib-based combination therapy in recurrent/refractory CNS lymphoma.

Ibrutinib is a first-in-class inhibitor of Bruton tyrosine kinase (BTK) and has shown single-agent activity in recurrent/refractory central nervous system (CNS) lymphoma. Clinical responses are often transient or incomplete, suggesting a need for a combination therapy approach. We conducted a phase 1b clinical trial to explore the sequential combination of ibrutinib (560 or 840 mg daily dosing) with high-dose methotrexate (HD-MTX) and rituximab in patients with CNS lymphoma (CNSL). HD-MTX was given at 3.5 g/m2 every 2 weeks for a total of 8 doses (4 cycles; 1 cycle = 28 days). Ibrutinib was held on days of HD-MTX infusion and resumed 5 days after HD-MTX infusion or after HD-MTX clearance. Single-agent daily ibrutinib was administered continuously after completion of induction therapy until disease progression, intolerable toxicity, or death. We also explored next-generation sequencing of circulating tumor DNA (ctDNA) in cerebrospinal fluid (CSF) before and during treatment. The combination of ibrutinib, HD-MTX, and rituximab was tolerated with an acceptable safety profile (no grade 5 events, 3 grade 4 events). No dose-limiting toxicity was observed. Eleven of 15 patients proceeded to maintenance ibrutinib after completing 4 cycles of the ibrutinib/HD-MTX/rituximab combination. Clinical responses occurred in 12 of 15 patients (80%). Sustained tumor responses were associated with clearance of ctDNA from the CSF. This trial was registered at www.clinicaltrials.gov as #NCT02315326.

EGFR feedback-inhibition by Ran-binding protein 6 is disrupted in cancer.

Transport of macromolecules through the nuclear pore by importins and exportins plays a critical role in the spatial regulation of protein activity. How cancer cells co-opt this process to promote tumorigenesis remains unclear. The epidermal growth factor receptor (EGFR) plays a critical role in normal development and in human cancer. Here we describe a mechanism of EGFR regulation through the importin ß family member RAN-binding protein 6 (RanBP6), a protein of hitherto unknown functions. We show that RanBP6 silencing impairs nuclear translocation of signal transducer and activator of transcription 3 (STAT3), reduces STAT3 binding to the EGFR promoter, results in transcriptional derepression of EGFR, and increased EGFR pathway output. Focal deletions of the RanBP6 locus on chromosome 9p were found in a subset of glioblastoma (GBM) and silencing of RanBP6 promoted glioma growth in vivo. Our results provide an example of EGFR deregulation in cancer through silencing of components of the nuclear import pathway.

Ibrutinib Unmasks Critical Role of Bruton Tyrosine Kinase in Primary CNS Lymphoma.

Bruton tyrosine kinase (BTK) links the B-cell antigen receptor (BCR) and Toll-like receptors with NF-κB. The role of BTK in primary central nervous system (CNS) lymphoma (PCNSL) is unknown. We performed a phase I clinical trial with ibrutinib, the first-in-class BTK inhibitor, for patients with relapsed or refractory CNS lymphoma. Clinical responses to ibrutinib occurred in 10 of 13 (77%) patients with PCNSL, including five complete responses. The only PCNSL with complete ibrutinib resistance harbored a mutation within the coiled-coil domain of CARD11, a known ibrutinib resistance mechanism. Incomplete tumor responses were associated with mutations in the B-cell antigen receptor-associated protein CD79B. CD79B-mutant PCNSLs showed enrichment of mammalian target of rapamycin (mTOR)-related gene sets and increased staining with PI3K/mTOR activation markers. Inhibition of the PI3K isoforms p110α/p110δ or mTOR synergized with ibrutinib to induce cell death in CD79B-mutant PCNSL cells.Significance: Ibrutinib has substantial activity in patients with relapsed or refractory B-cell lymphoma of the CNS. Response rates in PCNSL were considerably higher than reported for diffuse large B-cell lymphoma outside the CNS, suggesting a divergent molecular pathogenesis. Combined inhibition of BTK and PI3K/mTOR may augment the ibrutinib response in CD79B-mutant human PCNSLs. Cancer Discov; 7(9); 1018-29. ©2017 AACR.See related commentary by Lakshmanan and Byrd, p. 940This article is highlighted in the In This Issue feature, p. 920.

Multiplexed immunofluorescence delineates proteomic cancer cell states associated with metabolism.

The phenotypic diversity of cancer results from genetic and nongenetic factors. Most studies of cancer heterogeneity have focused on DNA alterations, as technologies for proteomic measurements in clinical specimen are currently less advanced. Here, we used a multiplexed immunofluorescence staining platform to measure the expression of 27 proteins at the single-cell level in formalin-fixed and paraffin-embedded samples from treatment-naive stage II/III human breast cancer. Unsupervised clustering of protein expression data from 638,577 tumor cells in 26 breast cancers identified 8 clusters of protein coexpression. In about one-third of breast cancers, over 95% of all neoplastic cells expressed a single protein coexpression cluster. The remaining tumors harbored tumor cells representing multiple protein coexpression clusters, either in a regional distribution or intermingled throughout the tumor. Tumor uptake of the radiotracer 18F-fluorodeoxyglucose was associated with protein expression clusters characterized by hormone receptor loss, PTEN alteration, and HER2 gene amplification. Our study demonstrates an approach to generate cellular heterogeneity metrics in routinely collected solid tumor specimens and integrate them with in vivo cancer phenotypes.

Integration of 2-hydroxyglutarate-proton magnetic resonance spectroscopy into clinical practice for disease monitoring in isocitrate dehydrogenase-mutant glioma.

BACKGROUND: The majority of WHO grades II and III gliomas harbor a missense mutation in the metabolic gene isocitrate dehydrogenase (IDH) and accumulate the metabolite R-2-hydroxyglutarate (R-2HG). Prior studies showed that this metabolite can be detected in vivo using proton magnetic-resonance spectroscopy (MRS), but the sensitivity of this methodology and its clinical implications are unknown. METHODS: We developed an MR imaging protocol to integrate 2HG-MRS into routine clinical glioma imaging and examined its performance in 89 consecutive glioma patients. RESULTS: Detection of 2-hydroxyglutarate (2HG) in IDH-mutant gliomas was closely linked to tumor volume, with sensitivity ranging from 8% for small tumors (<3.4 mL) to 91% for larger tumors (>8 mL). In patients undergoing 2HG-MRS prior to surgery, tumor levels of 2HG corresponded with tumor cellularity but not with tumor grade or mitotic index. Cytoreductive therapy resulted in a gradual decrease in 2HG levels with kinetics that closely mirrored changes in tumor volume. CONCLUSIONS: Our study demonstrates that 2HG-MRS can be linked with routine MR imaging to provide quantitative measurements of 2HG in glioma and may be useful as an imaging biomarker to monitor the abundance of IDH-mutant tumor cells noninvasively during glioma therapy and disease monitoring.

Glutamine-based PET imaging facilitates enhanced metabolic evaluation of gliomas in vivo.

Glucose and glutamine are the two principal nutrients that cancer cells use to proliferate and survive. Many cancers show altered glucose metabolism, which constitutes the basis for in vivo positron emission tomography (PET) imaging with (18)F-fluorodeoxyglucose ((18)F-FDG). However, (18)F-FDG is ineffective in evaluating gliomas because of high background uptake in the brain. Glutamine metabolism is also altered in many cancers, and we demonstrate that PET imaging in vivo with the glutamine analog 4-(18)F-(2S,4R)-fluoroglutamine ((18)F-FGln) shows high uptake in gliomas but low background brain uptake, facilitating clear tumor delineation. Chemo/radiation therapy reduced (18)F-FGln tumor avidity, corresponding with decreased tumor burden. (18)F-FGln uptake was not observed in animals with a permeable blood-brain barrier or neuroinflammation. We translated these findings to human subjects, where (18)F-FGln showed high tumor/background ratios with minimal uptake in the surrounding brain in human glioma patients with progressive disease. These data suggest that (18)F-FGln is avidly taken up by gliomas, can be used to assess metabolic nutrient uptake in gliomas in vivo, and may serve as a valuable tool in the clinical management of gliomas.

A kinase-independent function of AKT promotes cancer cell survival.

The serine-threonine kinase AKT regulates proliferation and survival by phosphorylating a network of protein substrates. In this study, we describe a kinase-independent function of AKT. In cancer cells harboring gain-of-function alterations in MET, HER2, or Phosphatidyl-Inositol-3-Kinase (PI3K), catalytically inactive AKT (K179M) protected from drug induced cell death in a PH-domain dependent manner. An AKT kinase domain mutant found in human melanoma (G161V) lacked enzymatic activity in vitro and in AKT1/AKT2 double knockout cells, but promoted growth factor independent survival of primary human melanocytes. ATP-competitive AKT inhibitors failed to block the kinase-independent function of AKT, a liability that limits their effectiveness compared to allosteric AKT inhibitors. Our results broaden the current view of AKT function and have important implications for the development of AKT inhibitors for cancer.

mTOR complex 2 controls glycolytic metabolism in glioblastoma through FoxO acetylation and upregulation of c-Myc.

Aerobic glycolysis (the Warburg effect) is a core hallmark of cancer, but the molecular mechanisms underlying it remain unclear. Here, we identify an unexpected central role for mTORC2 in cancer metabolic reprogramming where it controls glycolytic metabolism by ultimately regulating the cellular level of c-Myc. We show that mTORC2 promotes inactivating phosphorylation of class IIa histone deacetylases, which leads to the acetylation of FoxO1 and FoxO3, and this in turn releases c-Myc from a suppressive miR-34c-dependent network. These central features of activated mTORC2 signaling, acetylated FoxO, and c-Myc levels are highly intercorrelated in clinical samples and with shorter survival of GBM patients. These results identify a specific, Akt-independent role for mTORC2 in regulating glycolytic metabolism in cancer.

An inhibitor of mutant IDH1 delays growth and promotes differentiation of glioma cells.

The recent discovery of mutations in metabolic enzymes has rekindled interest in harnessing the altered metabolism of cancer cells for cancer therapy. One potential drug target is isocitrate dehydrogenase 1 (IDH1), which is mutated in multiple human cancers. Here, we examine the role of mutant IDH1 in fully transformed cells with endogenous IDH1 mutations. A selective R132H-IDH1 inhibitor (AGI-5198) identified through a high-throughput screen blocked, in a dose-dependent manner, the ability of the mutant enzyme (mIDH1) to produce R-2-hydroxyglutarate (R-2HG). Under conditions of near-complete R-2HG inhibition, the mIDH1 inhibitor induced demethylation of histone H3K9me3 and expression of genes associated with gliogenic differentiation. Blockade of mIDH1 impaired the growth of IDH1-mutant--but not IDH1-wild-type--glioma cells without appreciable changes in genome-wide DNA methylation. These data suggest that mIDH1 may promote glioma growth through mechanisms beyond its well-characterized epigenetic effects.

Imaging tumor burden in the brain with 89Zr-transferrin.

UNLABELLED: A noninvasive technology that indiscriminately detects tumor tissue in the brain could substantially enhance the management of primary or metastatic brain tumors. Although the documented molecular heterogeneity of diseases that initiate or eventually deposit in the brain may preclude identifying a single smoking-gun molecular biomarker, many classes of brain tumors are generally avid for transferrin. Therefore, we reasoned that applying a radiolabeled derivative of transferrin ((89)Zr-labeled transferrin) may be an effective strategy to more thoroughly identify tumor tissue in the brain, regardless of the tumor's genetic background. METHODS: Transferrin was radiolabeled with (89)Zr, and its properties with respect to human models of glioblastoma multiforme were studied in vivo. RESULTS: In this report, we show proof of concept that (89)Zr-labeled transferrin ((89)Zr-transferrin) localizes to genetically diverse models of glioblastoma multiforme in vivo. Moreover, we demonstrate that (89)Zr-transferrin can detect an orthotopic lesion with exceptional contrast. Finally, the tumor-to-brain contrast conferred by (89)Zr-transferrin vastly exceeded that observed with (18)F-FDG, currently the most widely used radiotracer to assess tumor burden in the brain. CONCLUSION: The results from this study suggest that (89)Zr-transferrin could be a broadly applicable tool for identifying and monitoring tumors in the brain, with realistic potential for near-term clinical translation.

Differential sensitivity of glioma- versus lung cancer-specific EGFR mutations to EGFR kinase inhibitors.

UNLABELLED: Activation of the epidermal growth factor receptor (EGFR) in glioblastoma (GBM) occurs through mutations or deletions in the extracellular (EC) domain. Unlike lung cancers with EGFR kinase domain (KD) mutations, GBMs respond poorly to the EGFR inhibitor erlotinib. Using RNAi, we show that GBM cells carrying EGFR EC mutations display EGFR addiction. In contrast to KD mutants found in lung cancer, glioma-specific EGFR EC mutants are poorly inhibited by EGFR inhibitors that target the active kinase conformation (e.g., erlotinib). Inhibitors that bind to the inactive EGFR conformation, however, potently inhibit EGFR EC mutants and induce cell death in EGFR-mutant GBM cells. Our results provide first evidence for single kinase addiction in GBM and suggest that the disappointing clinical activity of first-generation EGFR inhibitors in GBM versus lung cancer may be attributed to the different conformational requirements of mutant EGFR in these 2 cancer types. SIGNIFICANCE: Approximately 40% of human glioblastomas harbor oncogenic EGFR alterations, but attempts to therapeutically target EGFR with first-generation EGFR kinase inhibitors have failed. Here, we demonstrate selective sensitivity of glioma-specific EGFR mutants to ATP-site competitive EGFR kinase inhibitors that target the inactive conformation of the catalytic domain.

A brain tumor molecular imaging strategy using a new triple-modality MRI-photoacoustic-Raman nanoparticle.

The difficulty in delineating brain tumor margins is a major obstacle in the path toward better outcomes for patients with brain tumors. Current imaging methods are often limited by inadequate sensitivity, specificity and spatial resolution. Here we show that a unique triple-modality magnetic resonance imaging-photoacoustic imaging-Raman imaging nanoparticle (termed here MPR nanoparticle) can accurately help delineate the margins of brain tumors in living mice both preoperatively and intraoperatively. The MPRs were detected by all three modalities with at least a picomolar sensitivity both in vitro and in living mice. Intravenous injection of MPRs into glioblastoma-bearing mice led to MPR accumulation and retention by the tumors, with no MPR accumulation in the surrounding healthy tissue, allowing for a noninvasive tumor delineation using all three modalities through the intact skull. Raman imaging allowed for guidance of intraoperative tumor resection, and a histological correlation validated that Raman imaging was accurately delineating the brain tumor margins. This new triple-modality-nanoparticle approach has promise for enabling more accurate brain tumor imaging and resection.