The vexing difficulty in delineating brain tumor margins represents a major obstacle toward better outcome of brain tumor patients. 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 – surface enhanced Raman scattering (SERS) nanoparticle (MPR) can accurately help delineate the margins of brain tumors in living mice both pre- and intra-operatively. The MPRs were detected by all three modalities with at least picomolar sensitivity both in vitro and in living mice. Intravenous injection of MPRs into glioblastoma-bearing mice led to specific MPR accumulation and retention by the tumors, allowing for non-invasive tumor delineation by all three modalities through the intact skull. Raman imaging allowed guidance of intra-operative tumor resection, and histological correlation validated that Raman imaging is accurately delineating brain tumor margins. This novel triple-modality nanoparticle approach holds promise to enable more accurate brain tumor imaging and resection.

BAX is a pro-apoptotic protein of the BCL-2 family stationed in the cytosol until activated by a diversity of stress stimuli to induce cell death. Anti-apoptotic proteins such as BCL-2 counteract BAX-mediated cell death. Although an interaction site that confers survival functionality has been defined for anti-apoptotic proteins, an activation site has not been identified for BAX, rendering its explicit trigger mechanism unknown. We previously developed Stabilized Alpha-Helix of BCL-2 domains (SAHBs) that directly initiate BAX-mediated mitochondrial apoptosis. Here we demonstrate by NMR analysis that BIM SAHB binds BAX at an interaction site that is distinct from the canonical binding groove characterized for anti-apoptotic proteins. The specificity of the BIM SAHB-BAX interaction is highlighted by point mutagenesis that abrogates functional activity, confirming that BAX activation is initiated at this novel structural location. Thus, we have now defined a BAX interaction site for direct activation, establishing a new target for therapeutic modulation of apoptosis.

BAX is a multidomain proapoptotic BCL-2 family protein that resides in the cytosol until activated by an incompletely understood trigger mechanism, which facilitates BAX translocation to mitochondria and downstream death events. Whether BAX is activated by direct contact with select BH3-only members of the BCL-2 family is highly debated. Here we detect and quantify a direct binding interaction between BAX and a hydrocarbon-stapled BID BH3 domain, which triggers the functional activation of BAX at nanomolar doses in vitro. Chemical reinforcement of BID BH3 alpha helicity was required to reveal the direct BID BH3-BAX association. We confirm the specificity of this BH3 interaction by characterizing a stapled BAD BH3 peptide that interacts with antiapoptotic BCL-X(L) but does not bind or activate BAX. We further demonstrate that membrane targeting of stapled BID BH3 optimizes its ability to activate BAX, supporting a model in which BID directly engages BAX to trigger mitochondrial apoptosis.

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Summary Stem-like glioma cells reside within a perivascular niche and display hallmark radiation resistance. Understanding of the mechanisms underlying these properties will be vital for the development of effective therapies. Here we show that the stem cell marker CD44 promotes cancer stem cell phenotypes and radiation resistance. In a mouse model of glioma, Cd44−/− and Cd44+/− animals showed improved survival compared to controls. The CD44 ligand Osteopontin shared a perivascular expression pattern with CD44 and promoted glioma stem cell-like phenotypes. These effects were mediated via the γ-secretase regulated intracellular domain of CD44, which promoted aggressive glioma growth in vivo and stem cell-like phenotypes via CBP/p300-dependent enhancement of HIF-2α activity. In human glioblastoma multiforme, expression of CD44 correlated with hypoxia-induced gene signatures and poor survival. Together, these data suggest that in the glioma perivascular niche, Osteopontin promotes stem cell-like properties and radiation resistance in adjacent tumor cells via activation of CD44 signaling.

The inability to visualize the true extent of cancers represents a significant challenge in many areas of oncology. The margins of most cancer types are not well demarcated because the cancer diffusely infiltrates the surrounding tissues. Furthermore, cancers may be multifocal and characterized by the presence of microscopic satellite lesions. Such microscopic foci represent a major reason for persistence of cancer, local recurrences, and metastatic spread and are usually impossible to visualize with currently available imaging technologies. An imaging method to reveal the tumor extent is desired clinically and surgically. Here we show the precise visualization of tumor margins, microscopic tumor invasion, and multifocal loco-regional tumor spread using a new generation of surface-enhanced resonance Raman scattering (SERRS) nanoparticles, which are termed here SERRS-nanostars. The SERRS-nanostars feature a star-shaped gold core, a Raman reporter resonant in the near-infrared spectrum, and a primer-free silication method. In mouse models of pancreatic cancer, breast cancer, prostate cancer, and sarcoma, SERRS-nanostars enabled accurate detection of macroscopic malignant lesions as well as microscopic disease, without the need for a targeting moiety. Moreover, the sensitivity (1.5 femtomolar limit of detection under in vivo Raman imaging conditions) of SERRS-nanostars allowed imaging of premalignant lesions of pancreatic and prostatic neoplasias. High sensitivity and broad applicability, in conjunction with their inert gold-silica composition, render SERRS-nanostars a promising imaging agent for more precise cancer imaging and resection.

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 18F-fluorodeoxyglucose (18F-FDG). However, 18F-FDG is ineffective in evaluating gliomas due to 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 analogue 4-18F-(2S,4R)-fluoroglutamine (18F-FGln) shows high uptake in gliomas but low background brain uptake, facilitating clear tumor delineation. Chemo/radiation therapy reduced 18F-FGln-tumor avidity, corresponding with decreased tumor burden. 18F-FGln uptake was not observed in animals with a permeable blood-brain barrier or neuroinflammation. We translated these findings to human subjects, where 18F-FGln showed high tumor/background ratios with minimal uptake in the surrounding brain in human glioma patients with progressive disease. These data suggest that 18F-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.

Highlights d Lung cancer progression is accompanied by a stereotypic expansion of heterogeneity d Cell state heterogeneity arises largely independently of genetic variation d State transitions occur via an HPCS harboring high differentiation and growth capacity d The HPCS is drug resistant and portends poor patient survival across all cancers