Decoding the epitranscriptional landscape from native RNA sequences.

Traditional epitranscriptomics relies on capturing a single RNA modification by antibody or chemical treatment, combined with short-read sequencing to identify its transcriptomic location. This approach is labor-intensive and may introduce experimental artifacts. Direct sequencing of native RNA using Oxford Nanopore Technologies (ONT) can allow for directly detecting the RNA base modifications, although these modifications might appear as sequencing errors. The percent Error of Specific Bases (%ESB) was higher for native RNA than unmodified RNA, which enabled the detection of ribonucleotide modification sites. Based on the %ESB differences, we developed a bioinformatic tool, epitranscriptional landscape inferring from glitches of ONT signals (ELIGOS), that is based on various types of synthetic modified RNA and applied to rRNA and mRNA. ELIGOS is able to accurately predict known classes of RNA methylation sites (AUC > 0.93) in rRNAs from Escherichiacoli, yeast, and human cells, using either unmodified in vitro transcription RNA or a background error model, which mimics the systematic error of direct RNA sequencing as the reference. The well-known DRACH/RRACH motif was localized and identified, consistent with previous studies, using differential analysis of ELIGOS to study the impact of RNA m6A methyltransferase by comparing wild type and knockouts in yeast and mouse cells. Lastly, the DRACH motif could also be identified in the mRNA of three human cell lines. The mRNA modification identified by ELIGOS is at the level of individual base resolution. In summary, we have developed a bioinformatic software package to uncover native RNA modifications.

Exosomal MicroRNA and Protein Profiles of Hepatitis B Virus-Related Hepatocellular Carcinoma Cells.

Infection with hepatitis B virus (HBV) is a main risk factor for hepatocellular carcinoma (HCC). Extracellular vesicles, such as exosomes, play an important role in tumor development and metastasis, including regulation of HBV-related HCC. In this study, we have characterized exosome microRNA and proteins released in vitro from hepatitis B virus (HBV)-related HCC cell lines SNU-423 and SNU-182 and immortalized normal hepatocyte cell lines (THLE2 and THLE3) using microRNA sequencing and mass spectrometry. Bioinformatics, including functional enrichment and network analysis, combined with survival analysis using data related to HCC in The Cancer Genome Atlas (TCGA) database, were applied to examine the prognostic significance of the results. More than 40 microRNAs and 200 proteins were significantly dysregulated (p < 0.05) in the exosomes released from HCC cells in comparison with the normal liver cells. The functional analysis of the differentially expressed exosomal miRNAs (i.e., mir-483, mir-133a, mir-34a, mir-155, mir-183, mir-182), their predicted targets, and exosomal differentially expressed proteins (i.e., POSTN, STAM, EXOC8, SNX9, COL1A2, IDH1, FN1) showed correlation with pathways associated with HBV, virus activity and invasion, exosome formation and adhesion, and exogenous protein binding. The results from this study may help in our understanding of the role of HBV infection in the development of HCC and in the development of new targets for treatment or non-invasive predictive biomarkers of HCC.

Liposome Formulation for Tumor-Targeted Drug Delivery Using Radiation Therapy.

Targeted delivery of drugs or other therapeutic agents through internal or external triggers has been used to control and accelerate the release from liposomal carriers in a number of studies, but relatively few utilize energy of therapeutic X-rays as a trigger. We have synthesized liposomes that are triggered by ionizing radiation (RTLs) to release their therapeutic payload. These liposomes are composed of natural egg phosphatidylethanolamine (PE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and 1,2-disteroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (DSPE-PEG-2000), and the mean size of the RTL was in the range of 114 to 133 nm, as measured by nanoparticle tracking analysis (NTA). The trigger mechanism is the organic halogen, chloral hydrate, which is known to generate free protons upon exposure to ionizing radiation. Once protons are liberated, a drop in internal pH of the liposome promotes destabilization of the lipid bilayer and escape of the liposomal contents. In proof of principle studies, we assessed RTL radiation-release of fluorescent tracers upon exposure to a low pH extracellular environment or exposure to X-ray irradiation. Biodistribution imaging before and after irradiation demonstrated a preferential uptake and release of the liposomes and their cargo at the site of local tumor irradiation. Finally, a potent metabolite of the commonly used chemotherapy irinotecan, SN-38, was loaded into RTL along with near infrared (NIR) fluorescent dyes for imaging studies and measuring tumor cell cytotoxicity alone or combined with radiation exposure, in vitro and in vivo. Fully loaded RTLs were found to increase tumor cell killing with radiation in vitro and enhance tumor growth delay in vivo after three IV injections combined with three, 5 Gy local tumor radiation exposures compared to either treatment modality alone.

Lymph Liquid Biopsy for Detection of Cancer Stem Cells.

Collection of a blood sample defined by the term "blood liquid biopsy" is commonly used to detect diagnostic, prognostic, and therapeutic decision-making markers of metastatic tumors including circulating tumor cells (CTCs). Many tumors also release CTCs and other markers into lymph fluid, but the utility of lymphatic markers largely remains unexplored. Here, we introduce lymph liquid biopsy through collection of peripheral (afferent) and central (thoracic duct [TD]) lymph samples and demonstrates its feasibility for detection of stem-like CTCs potentially responsible for metastasis development and tumor relapse. Stemness of lymphatic CTCs (L-CTCs) was determined by spheroid-forming assay in vitro. Simultaneously, we tested blood CTCs by conventional blood liquid biopsy, and monitored the primary tumor size, early metastasis in a sentinel lymph node (SLN) and distant metastasis in lungs. Using a mouse model at early melanoma stage with no distant metastasis, we identified stem-like L-CTCs in lymph samples from afferent lymphatic vessels. Since these vessels transport cells from the primary tumor to SLN, our finding emphasizes the significance of the lymphatic pathway in development of SLN metastasis. Surprisingly, in pre-metastatic disease, stem-like L-CTCs were detected in lymph samples from the TD, which directly empties lymph into blood circulation. This suggests a new contribution of the lymphatic system to initiation of distant metastasis. Integration of lymph and blood liquid biopsies demonstrated that all mice with early melanoma had stem-like CTCs in at least one of three samples (afferent lymph, TD lymph, and blood). At the stage of distant metastasis, spheroid-forming L-CTCs were detected in TD lymph, but not in afferent lymph. Altogether, our results demonstrated that lymph liquid biopsy and testing L-CTCs holds promise for diagnosis and prognosis of early metastasis. © 2020 International Society for Advancement of Cytometry.

Proteomic basis of modulation of postischemic fibrosis by MSC exosomes.

After an ischemic event, there is activation of fibroblasts leading to scar formation. It is critical to limit the profibrotic remodeling and activate the reparative remodeling phase to limit cardiac diastolic dysfunction. Mesenchymal stem cell (MSC) exosomes offer significant protection against ischemia-related systolic dysfunction. Here, we studied if MSC exosomes would offer protection against profibrotic events in mouse hearts subjected to acute ischemia [1 h left coronary artery (LCA) occlusion] or chronic ischemia (7 days LCA occlusion). After acute ischemia, there was activation of inflammatory signals, more in the peri-infarct than in the infarct area, in the saline (vehicle)-treated mice. At the same time, there was expression of cardiac remodeling signals (vimentin, collagens-1 and -3, and fibronectin), more in the infarct area. Treatment with MSC exosomes before LCA ligation suppressed inflammatory signals during acute and chronic ischemia. Furthermore, exosome treatment promoted pro-reparative cardiac extracellular matrix (ECM) remodeling in both infarct and peri-infarct areas by suppressing fibronectin secretion and by modulating collagen secretion to reduce fibrotic scar formation through altered cellular signaling pathways. Proteomics study revealed intense expression of IL-1ß and activation of profibrotic signals in the saline-treated hearts and their suppression in MSC exosome-treated hearts. To our knowledge, this is the first report on the infarct and peri-infarct area proteomics of ischemic mice hearts to explain MSC exosome-mediated suppression of scar formation in the ischemic mouse hearts.

Tumor-endothelial cell interaction in an experimental model of human hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a densely vascularized tumor that is highly dependent on angiogenic pathways to direct arterial blood flow to the growing neoplasm, though little is known about how the interaction of tumor and endothelial cells drives these processes and the degree of clinical importance. To this end, we examined the intercellular cross-talk between HepG2 (human HCC) and human endothelial progenitor cells (EPC) in a co-culture system that mimics some aspects of initial tumor parenchyma and stroma interactions. The results showed that the remote cell-to-cell (paracrine) interactions between HepG2 cells and EPC play a critical role in the differentiation and angiogenic activity of endothelial cells, possibly through intercellular signaling function of the exosomes released in the medium by HepG2 cells. The tumor-cell activated phenotype of EPC was associated with increased migration and elevated expression of ephrin-B2, and Delta-like 4 ligand (DLL4). Furthermore, ephrin-B2 was found to be overexpressed in HCC and cholangiocarcinoma tissue samples taken from humans. Overall, our results demonstrate that ephrin-B2 and Dll4 mediated co-dependence of HCC and EPC intercellular crosstalk in the initial stages of HCC establishment and development, a promising target for new clinical strategies.

Modulation of dietary methionine intake elicits potent, yet distinct, anticancer effects on primary versus metastatic tumors.

Methionine dependency describes the characteristic rapid in vitro death of most tumor cells in the absence of methionine. Combining chemotherapy with dietary methionine deprivation [methionine-deficient diet (MDD)] at tolerable levels has vast potential in tumor treatment; however, it is limited by MDD-induced toxicity during extended deprivation. Recent advances in imaging and irradiation delivery have created the field of stereotactic body radiotherapy (SBRT), where fewer large-dose fractions delivered in less time result in increased local-tumor control, which could be maximally synergistic with an MDD short course. Identification of the lowest effective methionine dietary intake not associated with toxicity will further enhance the cancer therapy potential. In this study, we investigated the effects of MDD and methionine-restricted diet (MRD) in primary and metastatic melanoma models in combination with radiotherapy (RT). In vitro, MDD dose-dependently sensitized mouse and human melanoma cell lines to RT. In vivo in mice, MDD substantially potentiated the effects of RT by a significant delay in tumor growth, in comparison with administering MDD or RT alone. The antitumor effects of an MDD/RT approach were due to effects on one-carbon metabolism, resulting in impaired methionine biotransformation via downregulation of Mat2a, which encodes methionine adenosyltransferase 2A. Furthermore, and probably most importantly, MDD and MRD substantially diminished metastatic potential; the antitumor MRD effects were not associated with toxicity to normal tissue. Our findings suggest that modulation of methionine intake holds substantial promise for use with short-course SBRT for cancer treatment.

Glutamine drives glutathione synthesis and contributes to radiation sensitivity of A549 and H460 lung cancer cell lines.

BACKGROUND: Increased glutamine uptake is known to drive cancer cell proliferation, making tumor cells glutamine-dependent. Glutamine provides additional carbon and nitrogen sources for cell growth. The first step in glutamine utilization is its conversion to glutamate by glutaminase (GLS). Glutamate is a precursor for glutathione synthesis, and we investigated the hypothesis that glutamine drives glutathione synthesis and thereby contributes to cellular defense systems. METHODS: The importance of glutamine for glutathione synthesis was studied in H460 and A549 lung cancer cell lines using glutamine-free medium and bis-2-(5-phenyl-acetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES) a GLS inhibitor. Metabolic activities were determined by targeted mass spectrometry. RESULTS: A significant correlation between glutamine consumption and glutathione excretion was demonstrated in H460 and A549 tumor cells. Culturing in the presence of [(13)C5]glutamine demonstrated that by 12h >50% of excreted glutathione was derived from glutamine. Culturing in glutamine-free medium or treatment with BPTES, a GLS-specific inhibitor, reduced cell proliferation and viability and abolished glutathione excretion. Treatment with glutathione-ester prevented BPTES-induced cytotoxicity. Inhibition of GLS markedly radiosensitized the lung tumor cell lines, suggesting an important role of glutamine-derived glutathione in determining radiation sensitivity. CONCLUSIONS: We demonstrate here for the first time that a significant amount of extracellular glutathione is directly derived from glutamine. This finding adds yet another important function to the already known glutamine dependence of tumor cells and probably tumors as well. GENERAL SIGNIFICANCE: Glutamine is essential for synthesis and excretion of glutathione to promote cell growth and viability.

Acquired Radiation Resistance Induces Thiol-dependent Cisplatin Cross-resistance.

Resistance to radiation remains a significant clinical challenge in non-small cell lung carcinoma (NSCLC). It is therefore important to identify the underlying molecular and cellular features that drive acquired resistance. We generated genetically matched NSCLC cell lines to investigate characteristics of acquired resistance. Murine Lewis lung carcinoma (LLC) and human A549 cells acquired an approximate 1.5-2.5-fold increase in radiation resistance as compared to their parental match, which each had unique intrinsic radio-sensitivities. The radiation resistance (RR) was reflected in higher levels of DNA damage and repair marker γH2AX and reduced apoptosis induction after radiation. Morphologically, we found that radiation resistance A549 (A549-RR) cells exhibited a greater nucleus-to-cytosol (N/C) ratio as compared to its parental counterpart. Since the N/C ratio is linked to the differentiation state, we next investigated the epithelial-to-mesenchymal transition (EMT) phenotype and cellular plasticity. We found that A549 cells had a greater radiation-induced plasticity, as measured by E-cadherin, vimentin and double-positive (DP) modulation, as compared to LLC. Additionally, migration was suppressed in A549-RR cells, as compared to A549 cells. Subsequently, we confirmed in vivo that the LLC-RR and A549-RR cells are also more resistance to radiation than their isogenic-matched counterpart. Moreover, we found that the acquired radiation resistance also induced resistance to cisplatin, but not carboplatin or oxaliplatin. This cross-resistance was attributed to induced elevation of thiol levels. Gamma-glutamylcysteine synthetase inhibitor buthionine sulfoximine (BSO) sensitized the resistant cells to cisplatin by decreasing the amount of thiols to levels prior to obtaining acquired radiation resistance. By generating radiation-resistance genetically matched NSCLC we were able to identify and overcome cisplatin cross-resistance. This is an important finding arguing for combinatorial treatment regimens including glutathione pathway disruptors in patients with the potential of improving clinical outcomes in the future.

Exploiting nanopore sequencing for characterization and grading of IDH-mutant gliomas.

The 2021 WHO Classification of Central Nervous System Tumors recommended evaluation of cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B) deletion in addition to codeletion of 1p/19q to characterize IDH-mutant gliomas. Here, we demonstrated the use of a nanopore-based copy-number variation sequencing (nCNV-seq) approach to simultaneously identify deletions of CDKN2A/B and 1p/19q. The nCNV-seq approach was initially evaluated on three distinct glioma cell lines and then applied to 19 IDH-mutant gliomas (8 astrocytomas and 11 oligodendrogliomas) from patients. The whole-arm 1p/19q codeletion was detected in all oligodendrogliomas with high concordance among nCNV-seq, FISH, DNA methylation profiling, and whole-genome sequencing. For the CDKN2A/B deletion, nCNV-seq detected the loss in both astrocytoma and oligodendroglioma, with strong correlation with the CNV profiles derived from whole-genome sequencing (Pearson correlation (r) = 0.95, P < 2.2 × 10-16 to r = 0.99, P < 2.2 × 10-16 ) and methylome profiling. Furthermore, nCNV-seq can differentiate between homozygous and hemizygous deletions of CDKN2A/B. Taken together, nCNV-seq holds promise as a new, alternative approach for a rapid and simultaneous detection of the molecular signatures of IDH-mutant gliomas without capital expenditure for a sequencer.

Pancreatic Tumor-Derived Extracellular Vesicles Stimulate Schwann Cell Phenotype Indicative of Perineural Invasion via IL-8 Signaling.

Pancreatic cancer remains a pre-eminent cause of cancer-related deaths with late-stage diagnoses leading to an 11% five-year survival rate. Moreover, perineural invasion (PNI), in which cancer cells migrate into adjacent nerves, occurs in an overwhelming majority of patients, further enhancing tumor metastasis. PNI has only recently been recognized as a key contributor to cancer progression; thus, there are insufficient treatment options for the disease. Attention has been focused on glial Schwann cells (SC) for their mediation of pancreatic PNI. Under stress, SCs dedifferentiate from their mature state to facilitate the repair of peripheral nerves; however, this signaling can also re-direct cancer cells to accelerate PNI. Limited research has explored the mechanism that causes this shift in SC phenotype in cancer. Tumor-derived extracellular vesicles (TEV) have been implicated in other avenues of cancer development, such as pre-metastatic niche formation in secondary locations, yet how TEVs contribute to PNI has not been fully explored. In this study, we highlight TEVs as initiators of SC activation into a PNI-associated phenotype. Proteomic and pathway assessments of TEVs revealed an elevation in interleukin-8 (IL-8) signaling and nuclear factor kappa B (NFκB) over healthy cell-derived EVs. TEV-treated SCs exhibited higher levels of activation markers, which were successfully neutralized with IL-8 inhibition. Additionally, TEVs increased NFκB subunit p65 nuclear translocation, which may lead to increased secretion of cytokines and proteases indicative of SC activation and PNI. These findings present a novel mechanism that may be targeted for the treatment of pancreatic cancer PNI. Statement of Significance: Identifying pancreatic tumor extracellular vesicles as key players in Schwann cell activation and perineural invasion by way of IL-8 will educate for more specialized and effective targets for an under-valued disease.

Proteomic analysis of transcription factors involved in the alteration of ischemic mouse heart as modulated by MSC exosomes.

Mesenchymal stem cell (MSC) exosomes have been found to attenuate cardiac systolic and diastolic dysfunction in animal models of ischemia. Exosomes carry a plethora of active and inactive proteins as their cargo, which are readily available to the recipient cell for use in intracellular signaling pathways-depending on the stresses, such as ischemia or hypoxia. Among the exosomal proteins are the often-overlooked cargo of transcriptional regulators. These transcriptional regulators influence the transcriptome and subsequently the proteome of recipient cell. Here, we report the transcriptional factors and regulators differentially modulated and their potential role in modulating cardiac function in MSC exosome treated ischemic mice hearts. Our analysis shows ischemic stress modulating transcriptional regulators and factors such as HSF1 and HIF1A in the infarct and peri-infarct areas of ischemic hearts which is mitigated by MSC exosomes. Similarly, STAT3 and SMAD3 are also modulated by MSC exosomes. Interestingly, NOTCH1 and ß-catenin were detected in the ischemic hearts. The differential expression of these regulators and factors drives changes in various biological process governed in the ischemic cardiac cells. We believe these studies will advance our understanding of cardiac dysfunction occurring in the ischemic hearts and lay the groundwork for further studies on the modulation of cardiac function during ischemia by MSC exosomes.

Morphological Effects and In Vitro Biological Mechanisms of Radiation-Induced Cell Killing by Gold Nanomaterials.

Gold nanomaterials have been shown to augment radiation therapy both in vitro and in vivo. However, studies on these materials are mostly phenomenological due to nanoparticle heterogeneity and the complexity of biological systems. Even accurate quantification of the particle dose still results in bulk average biases; the effect on individual cells is not measured but rather the effect on the overall population. To perform quantitative nanobiology, we coated glass coverslips uniformly at varying densities with Au nanoparticle preparations with different morphologies (45 nm cages, 25 nm spheres, and 30 nm rods). Consequently, the effect of a specific number of particles per unit area in contact with breast cancer cells growing on the coated surfaces was ascertained. Gold nanocages showed the highest degree of radiosensitization on a per particle basis, followed by gold nanospheres and gold nanorods, respectively. All three materials showed little cytotoxic effect at 0 Gy, but clonogenic survival decreased proportionally with the radiation dose and particle coverage density. A similar trend was seen in vivo in the combined treatment antitumor response in 4T1 tumor-bearing animals. The presence of gold affected the type and quantity of reactive oxygen species generated, specifically superoxide and hydroxyl radicals, and the concentration of nanocages correlated with the development of more numerous double-stranded DNA breaks and increased protein oxidation as measured by carbonylation. This work demonstrates the dependence on morphology and concentration of radiation enhancement by gold nanomaterials and may lead to a novel method to differentiate intra- and extracellular functionalities of gold nanomedicine treatment strategies. It further provides insights that can guide the rational development of gold nanomaterial-based radiosensitizers for clinical use.

Photothermal Response Induced by Nanocage-Coated Artificial Extracellular Matrix Promotes Neural Stem Cell Differentiation.

Strategies to increase the proportion of neural stem cells that differentiate into neurons are vital for therapy of neurodegenerative disorders. In vitro, the extracellular matrix composition and topography have been found to be important factors in stem cell differentiation. We have developed a novel artificial extracellular matrix (aECM) formed by attaching gold nanocages (AuNCs) to glass coverslips. After culturing rat neural stem cells (rNSCs) on these gold nanocage-coated surfaces (AuNC-aECMs), we observed that 44.6% of rNSCs differentiated into neurons compared to only 27.9% for cells grown on laminin-coated glass coverslips. We applied laser irradiation to the AuNC-aECMs to introduce precise amounts of photothermally induced heat shock in cells. Our results showed that laser-induced thermal stimulation of AuNC-aECMs further enhanced neuronal differentiation (56%) depending on the laser intensity used. Response to these photothermal effects increased the expression of heat shock protein 27, 70, and 90α in rNSCs. Analysis of dendritic complexity showed that this thermal stimulation promoted neuronal maturation by increasing dendrite length as thermal dose was increased. In addition, we found that cells growing on AuNC-aECMs post laser irradiation exhibited action potentials and increased the expression of voltage-gated Na+ channels compared to laminin-coated glass coverslips. These results indicate that the photothermal response induced in cells growing on AuNC-aECMs can be used to produce large quantities of functional neurons, with improved electrochemical properties, that can potentially be transplanted into a damaged central nervous system to provide replacement neurons and restore lost function.

Mild temperature hyperthermia and radiation therapy: role of tumour vascular thermotolerance and relevant physiological factors.

Here we review the significance of changes in vascular thermotolerance on tumour physiology and the effects of multiple clinically relevant mild temperature hyperthermia (MTH) treatments on tumour oxygenation and corresponding radiation response. Thus far vascular thermotolerance referred to the observation of significantly greater blood flow response by the tumour to a second hyperthermia exposure than in response to a single thermal dose, even at temperatures that would normally cause vascular damage. New information suggests that although hyperthermia is a powerful modifier of tumour blood flow and oxygenation, sequencing and frequency are central parameters in the success of MTH enhancement of radiation therapy. We hypothesise that heat treatments every 2 to 3 days combined with traditional or accelerated radiation fractionation may be maximally effective in exploiting the improved perfusion and oxygenation induced by typical thermal doses given in the clinic.

In Vivo Lymphatic Circulating Tumor Cells and Progression of Metastatic Disease.

The dissemination of circulating tumor cells (CTCs) by lymph fluid is one of the key events in the development of tumor metastasis. However, little progress has been made in studying lymphatic CTCs (L-CTCs). Here, we demonstrate the detection of L-CTCs in preclinical mouse models of melanoma and breast cancer using in vivo high-sensitivity photoacoustic and fluorescent flow cytometry. We discovered that L-CTCs are be detected in pre-metastatic disease stage. The smallest primary tumor that shed L-CTCs was measured as 0.094mm×0.094mm, its volume was calculated as 0.0004 mm3; and its productivity was estimated as 1 L-CTC per 30 minutes. As the disease progressed, primary tumors continued releasing L-CTCs with certain individual dynamics. The integrated assessment of lymph and blood underlined the parallel dissemination of CTCs at all disease stages. However, the analysis of links between L-CTC counts, blood CTC (B-CTC) counts, primary tumor size and metastasis did not reveal statistically significant correlations, likely due to L-CTC heterogeneity. Altogether, our results showed the feasibility of our diagnostic platform using photoacoustic flow cytometry for preclinical L-CTC research with translational potential. Our findings also demonstrated new insights into lymphatic system involvement in CTC dissemination. They help to lay the scientific foundation for the consideration of L-CTCs as prognostic markers of metastasis and to emphasize the integrative assessment of lymph and blood.

Enhanced Photothermal Treatment Efficacy and Normal Tissue Protection via Vascular Targeted Gold Nanocages.

A major challenge in photothermal treatment is generating sufficient heat to eradicate diseased tissue while sparing normal tissue. Au nanomaterials have shown promise as a means to achieve highly localized photothermal treatment. Toward that end, the synthetic peptide anginex was conjugated to Au nanocages. Anginex binds to galectin-1, which is highly expressed in dividing endothelial cells found primarily in the tumor vasculature. The skin surface temperature during a 10 min laser exposure of subcutaneous murine breast tumors did not exceed 43°C and no normal tissue damage was observed, yet a significant anti-tumor effect was observed when laser was applied 24 h post-injection of targeted nanocages. Untargeted particles showed little effect in immunocompetent, tumor-bearing mice under these conditions. Photoacoustic, photothermal, and ICP-MS mapping of harvested tissue showed distribution of particles near the vasculature throughout the tumor. This uptake pattern within the tumor combined with a minimal overall temperature rise were nonetheless sufficient to induce marked photothermal efficacy and evidence of tumor control. Importantly, this evidence suggests that bulk tumor temperature during treatment does not correlate with treatment outcome, which implies that targeted nanomedicine can be highly effective when closely bound/distributed in and around the tumor endothelium and extensive amounts of direct tumor cell binding may not be a prerequisite of effective photothermal approaches.

Bioinspired magnetic nanoparticles as multimodal photoacoustic, photothermal and photomechanical contrast agents.

Nanoparticles from magnetotactic bacteria have been used in conventional imaging, drug delivery, and magnetic manipulations. Here, we show that these natural nanoparticles and their bioinspired hybrids with near-infrared gold nanorods and folic acid can serve as molecular high-contrast photoacoustic probes for single-cell diagnostics and as photothermal agents for single-cell therapy using laser-induced vapor nanobubbles and magnetic field as significant signal and therapy amplifiers. These theranostics agents enable the detection and photomechanical killing of triple negative breast cancer cells that are resistant to conventional chemotherapy, with just one or a few low-energy laser pulses. In studies in vivo, we discovered that circulating tumor cells labeled with the nanohybrids generate transient ultrasharp photoacoustic resonances directly in the bloodstream as the basis for new super-resolution photoacoustic flow cytometry in vivo. These properties make natural and bioinspired magnetic nanoparticles promising biocompatible, multimodal, high-contrast, and clinically relevant cellular probes for many in vitro and in vivo biomedical applications.

Glutaminase inhibitor CB-839 increases radiation sensitivity of lung tumor cells and human lung tumor xenografts in mice.

PURPOSE: The purpose of this study was to translate our in vitro therapy approach to an in vivo model. Increased glutamine uptake is known to drive cancer cell proliferation, making tumor cells glutamine-dependent. Studying lymph-node aspirates containing malignant lung tumor cells showed a strong correlation between glutamine consumption and glutathione (GSH) excretion. Subsequent experiments with A549 and H460 lung tumor cell lines provided additional evidence for glutamine's role in driving synthesis and excretion of GSH. Using stable-isotope-labeled glutamine as a tracer metabolite, we demonstrated that the glutamate group in GSH is directly derived from glutamine, linking glutamine utilization intimately to GSH syntheses. MATERIALS AND METHODS: To understand the possible mechanistic link between glutamine consumption and GSH excretion, we studied GSH metabolism in more detail. Inhibition of glutaminase (GLS) with BPTES, a GLS-specific inhibitor, effectively abolished GSH synthesis and excretion. Since our previous work, several novel GLS inhibitors became available and we report herein effects of CB-839 in A427, H460 and A549 lung tumor cells and human lungtumor xenografts in mice. RESULTS: Inhibition of GLS markedly reduced cell viability, producing ED50 values for inhibition of colony formation of 9, 27 and 217 nM in A427, A549 and H460, respectively. Inhibition of GLS is accompanied by ∼30% increased response to radiation, suggesting an important role of glutamine-derived GSH in protecting tumor cells against radiation-induced injury. In subsequent mouse xenografts, short-term CB-839 treatments reduced serum GSH by >50% and increased response to radiotherapy of H460-derived tumor xenografts by 30%. CONCLUSION: The results support the proposed mechanistic link between GLS activity and GSH synthesis and suggest that GLS inhibitors are effective radiosensitizers.