Smart-Dust-Nanorice for Enhancement of Endogenous Raman Signal, Contrast in Photoacoustic Imaging, and T2-Shortening in Magnetic Resonance Imaging.

Raman microspectroscopy provides chemo-selective image contrast, sub-micrometer resolution, and multiplexing capabilities. However, it suffers from weak signals resulting in image-acquisition times of up to several hours. Surface-enhanced Raman scattering (SERS) can dramatically enhance signals of molecules in close vicinity of metallic surfaces and overcome this limitation. Multimodal, SERS-active nanoparticles are usually labeled with Raman marker molecules, limiting SERS to the coating material. In order to realize multimodal imaging while acquiring the rich endogenous vibronic information of the specimen, a core-shell particle based on "Nanorice", where a spindle-shaped iron oxide core is encapsulated by a closed gold shell, is developed. An ultrathin layer of silica prevents agglomeration and unwanted chemical interaction with the specimen. This approach provides Raman signal enhancement due to plasmon resonance effects of the shell while the optical absorption in the near-infrared spectral region provides contrast in photoacoustic tomography. Finally, T2-relaxation of a magnetic resonance imaging (MRI) experiment is altered by taking advantage of the iron oxide core. The feasibility for Raman imaging is evaluated by nearfield simulations and experimental studies on the primate cell line COS1. MRI and photoacoustics are demonstrated in agarose phantoms illustrating the promising translational nature of this strategy for clinical applications in radiology.

Functional DNA Delivery Enabled by Lipid-Modified Charge-Altering Releasable Transporters (CARTs).

Safe and effective DNA delivery systems are required to enable or enhance clinical strategies and research involving gene therapy and DNA vaccinations. To address this delivery problem, a series of charge-altering releasable transporters (CARTs) with varied lipid content were prepared and evaluated for plasmid DNA (pDNA) delivery into cultured cells. These lipid-modified CART co-oligomers were synthesized in only two steps via sequential organocatalytic ring-opening polymerization of lipid-containing cyclic carbonate monomers and morpholinone monomers. Lipid variations of the CARTs substantially impacted the delivery efficiency of pDNA, with oleyl- and linoleyl-based CARTs showing enhanced performance relative to the commercial transfection agent Lipofectamine 2000 (L2000). The best-performing oleyl CART was carried forward to study stable luciferase transfection with a Sleeping Beauty ( SB) transposon system. The oleyl CART outperformed the L2000 positive control with respect to stable transfection efficiency. CART-pDNA complexes represent a new DNA delivery system for research and clinical applications.

New imaging probes to track cell fate: reporter genes in stem cell research.

Cell fate is a concept used to describe the differentiation and development of a cell in its organismal context over time. It is important in the field of regenerative medicine, where stem cell therapy holds much promise but is limited by our ability to assess its efficacy, which is mainly due to the inability to monitor what happens to the cells upon engraftment to the damaged tissue. Currently, several imaging modalities can be used to track cells in the clinical setting; however, they do not satisfy many of the criteria necessary to accurately assess several aspects of cell fate. In recent years, reporter genes have become a popular option for tracking transplanted cells, via various imaging modalities in small mammalian animal models. This review article examines the reporter gene strategies used in imaging modalities such as MRI, SPECT/PET, Optoacoustic and Bioluminescence Imaging. Strengths and limitations of the use of reporter genes in each modality are discussed.

Differential fates of biomolecules delivered to target cells via extracellular vesicles.

Extracellular vesicles (EVs), specifically exosomes and microvesicles (MVs), are presumed to play key roles in cell-cell communication via transfer of biomolecules between cells. The biogenesis of these two types of EVs differs as they originate from either the endosomal (exosomes) or plasma (MVs) membranes. To elucidate the primary means through which EVs mediate intercellular communication, we characterized their ability to encapsulate and deliver different types of macromolecules from transiently transfected cells. Both EV types encapsulated reporter proteins and mRNA but only MVs transferred the reporter function to recipient cells. De novo reporter protein expression in recipient cells resulted only from plasmid DNA (pDNA) after delivery via MVs. Reporter mRNA was delivered to recipient cells by both EV types, but was rapidly degraded without being translated. MVs also mediated delivery of functional pDNA encoding Cre recombinase in vivo to tissues in transgenic Cre-lox reporter mice. Within the parameters of this study, MVs delivered functional pDNA, but not RNA, whereas exosomes from the same source did not deliver functional nucleic acids. These results have significant implications for understanding the role of EVs in cellular communication and for development of EVs as delivery tools. Moreover, studies using EVs from transiently transfected cells may be confounded by a predominance of pDNA transfer.

Local estrogen axis in the human bone microenvironment regulates estrogen receptor-positive breast cancer cells.

BACKGROUND: Approximately 70% of all breast cancers express the estrogen receptor, and are regulated by estrogen. While the ovaries are the primary source of estrogen in premenopausal women, most breast cancer is diagnosed following menopause, when systemic levels of this hormone decline. Estrogen production from androgen precursors is catalyzed by the aromatase enzyme. Although aromatase expression and local estrogen production in breast adipose tissue have been implicated in the development of primary breast cancer, the source of estrogen involved in the regulation of estrogen receptor-positive (ER+) metastatic breast cancer progression is less clear. METHODS: Bone is the most common distant site of breast cancer metastasis, particularly for ER+ breast cancers. We employed a co-culture model using trabecular  bone tissues obtained from total hip replacement (THR) surgery specimens to study ER+ and estrogen receptor-negative (ER-) breast cancer cells within the human bone microenvironment. Luciferase-expressing ER+ (MCF-7, T-47D, ZR-75) and ER- (SK-BR-3, MDA-MB-231, MCF-10A) breast cancer cells were cultured directly on bone tissue fragments or in bone tissue-conditioned media, and monitored over time with bioluminescence imaging (BLI). Bone tissue-conditioned media were generated in the presence vs. absence of aromatase inhibitors, and testosterone. Bone tissue fragments were analyzed for aromatase expression by immunohistochemistry. RESULTS: ER+ breast cancer cells were preferentially sustained in co-cultures with bone tissues and bone tissue-conditioned media relative to ER- cells. Bone fragments analyzed by immunohistochemistry revealed expression of the aromatase enzyme. Bone tissue-conditioned media generated in the presence of testosterone had increased estrogen levels and heightened capacity to stimulate ER+ breast cancer cell proliferation. Pretreatment of cultured bone tissues with aromatase inhibitors, which inhibited estrogen production, reduced the capacity of conditioned media to stimulate ER+ cell proliferation. CONCLUSIONS: These results suggest that a local estrogen signaling axis regulates ER+ breast cancer cell viability and proliferation within the bone metastatic niche, and that aromatase inhibitors modulate this axis. Although endocrine therapies are highly effective in the treatment of ER+ breast cancer, resistance to these treatments reduces their efficacy. Characterization of estrogen signaling networks within the bone microenvironment will identify new strategies for combating metastatic progression and endocrine resistance.

A truncated human NKG2D splice isoform negatively regulates NKG2D-mediated function.

Natural killer group 2, member D (NKG2D) is a stimulatory receptor expressed by NK cells and a subset of T cells. NKG2D is crucial in diverse aspects of innate and adaptive immune functions. In this study, we characterize a novel splice variant of human NKG2D that encodes a truncated receptor lacking the ligand-binding ectodomain. This truncated NKG2D (NKG2D(TR)) isoform was detected in primary human NK and CD8(+) T cells. Overexpression of NKG2D(TR) severely attenuated cell killing and IFN-γ release mediated by full-length NKG2D (NKG2D(FL)). In contrast, specific knockdown of endogenously expressed NKG2D(TR) enhanced NKG2D-mediated cytotoxicity, suggesting that NKG2D(TR) is a negative regulator of NKG2D(FL). Biochemical studies demonstrated that NKG2D(TR) was bound to DNAX-activated protein of 10 kDa (DAP10) and interfered with the interaction of DAP10 with NKG2D(FL). In addition, NKG2D(TR) associated with NKG2D(FL), which led to forced intracellular retention, resulting in decreased surface NKG2D expression. Taken together, these data suggest that competitive interference of NKG2D/DAP10 complexes by NKG2D(TR) constitutes a novel mechanism for regulation of NKG2D-mediated function in human CD8(+) T cells and NK cells.

Functional imaging of legumain in cancer using a new quenched activity-based probe.

Legumain is a lysosomal cysteine protease whose biological function remains poorly defined. Legumain activity is up-regulated in most human cancers and inflammatory diseases most likely as the result of high expression in populations of activated macrophages. Within the tumor microenvironment, legumain activity is thought to promote tumorigenesis. To obtain a greater understanding of the role of legumain activity during cancer progression and inflammation, we developed an activity-based probe that becomes fluorescent only upon binding active legumain. This probe is highly selective for legumain, even in the context of whole cells and tissues, and is also a more effective label of legumain than previously reported probes. Here we present the synthesis and application of our probe to the analysis of legumain activity in primary macrophages and in two mouse models of cancer. We find that legumain activity is highly correlated with macrophage activation and furthermore that it is an ideal marker for primary tumor inflammation and early stage metastatic lesions.

Cancer stem cells from human breast tumors are involved in spontaneous metastases in orthotopic mouse models.

To examine the role of breast cancer stem cells (BCSCs) in metastasis, we generated human-in-mouse breast cancer orthotopic models using patient tumor specimens, labeled with optical reporter fusion genes. These models recapitulate human cancer features not captured with previous models, including spontaneous metastasis in particular, and provide a useful platform for studies of breast tumor initiation and progression. With noninvasive imaging approaches, as few as 10 cells of stably labeled BCSCs could be tracked in vivo, enabling studies of early tumor growth and spontaneous metastasis. These advances in BCSC imaging revealed that CD44(+) cells from both primary tumors and lung metastases are highly enriched for tumor-initiating cells. Our metastatic cancer models, combined with noninvasive imaging techniques, constitute an integrated approach that could be applied to dissect the molecular mechanisms underlying the dissemination of metastatic CSCs (MCSCs) and to explore therapeutic strategies targeting MCSCs in general or to evaluate individual patient tumor cells and predict response to therapy.

In Vivo Analysis of Heterogeneous Extracellular Vesicles Using a Red-Shifted Bioluminescence Resonance Energy Transfer Reporter Protein.

Current methods for characterizing the biodistribution of extracellular vesicles (EVs) are not sensitive enough to track EVs in vivo, despite significant advances over the past decade. Commonly used lipophilic fluorescent dyes are convenient, but lack specificity and yield inaccurate spatiotemporal images in the long-term tracking of EVs. In contrast, protein-based fluorescent or bioluminescent EV reporters have more accurately revealed their distribution in cells and mouse models. Here, we describe a red-shifted bioluminescence resonance energy transfer (BRET) EV reporter, PalmReNL, to analyze the trafficking of small EVs (<200 nm; sEVs) and medium/large EVs (>200 nm; m/lEVs) in mice. Its advantages are that (i) background signals in bioluminescence imaging (BLI) are negligible and (ii) the photons PalmReNL emits have spectral wavelengths longer than 600 nm and can more efficiently penetrate tissues than reporters emitting shorter wavelength light.

Phytochemical drug discovery for COVID-19 using high-resolution computational docking and machine learning assisted binder prediction.

The COVID-19 pandemic has resulted in millions of deaths around the world. Multiple vaccines are in use, but there are many underserved locations that do not have adequate access to them. Variants may emerge that are highly resistant to existing vaccines, and therefore cheap and readily obtainable therapeutics are needed. Phytochemicals, or plant chemicals, can possibly be such therapeutics. Phytochemicals can be used in a polypharmacological approach, where multiple viral proteins are inhibited and escape mutations are made less likely. Finding the right phytochemicals for viral protein inhibition is challenging, but in-silico screening methods can make this a more tractable problem. In this study, we screen a wide range of natural drug products against a comprehensive set of SARS-CoV-2 proteins using a high-resolution computational workflow. This workflow consists of a structure-based virtual screening (SBVS), where an initial phytochemical library was docked against all selected protein structures. Subsequently, ligand-based virtual screening (LBVS) was employed, where chemical features of 34 lead compounds obtained from the SBVS were used to predict 53 lead compounds from a larger phytochemical library via supervised learning. A computational docking validation of the 53 predicted leads obtained from LBVS revealed that 28 of them elicit strong binding interactions with SARS-CoV-2 proteins. Thus, the inclusion of LBVS resulted in a 4-fold increase in the lead discovery rate. Of the total 62 leads, 18 showed promising pharmacokinetic properties in a computational ADME screening. Collectively, this study demonstrates the advantage of incorporating machine learning elements into a virtual screening workflow.Communicated by Ramaswamy H. Sarma.

In Vitro and In Vivo Analysis of Extracellular Vesicle-Mediated Metastasis Using a Bright, Red-Shifted Bioluminescent Reporter Protein.

Cancer cells produce heterogeneous extracellular vesicles (EVs) as mediators of intercellular communication. This study focuses on a novel method to image EV subtypes and their biodistribution in vivo. A red-shifted bioluminescence resonance energy transfer (BRET) EV reporter is developed, called PalmReNL, which allows for highly sensitive EV tracking in vitro and in vivo. PalmReNL enables the authors to study the common surface molecules across EV subtypes that determine EV organotropism and their functional differences in cancer progression. Regardless of injection routes, whether retro-orbital or intraperitoneal, PalmReNL positive EVs, isolated from murine mammary carcinoma cells, localized to the lungs. The early appearance of metastatic foci in the lungs of mammary tumor-bearing mice following multiple intraperitoneal injections of the medium and large EV (m/lEV)-enriched fraction derived from mammary carcinoma cells is demonstrated. In addition, the results presented here show that tumor cell-derived m/lEVs act on distant tissues through upregulating LC3 expression within the lung.

Panel of prototypical raltegravir-resistant infectious molecular clones in a novel integrase-deleted cloning vector.

We created an HIV-1 cloning vector, pNL4.3DeltaIN, to generate recombinant infectious molecular clones for analysis of patient-derived HIV-1 integrase coding regions. Using this vector, we constructed a panel of clinically derived viruses with the canonical patterns of raltegravir resistance mutations and submitted the panel to the NIH AIDS Research and Reference Reagent Program. Investigational integrase inhibitors with activity against these clones are likely to retain activity against the most clinically relevant raltegravir-resistant variants.

MYC inactivation uncovers pluripotent differentiation and tumour dormancy in hepatocellular cancer.

Hepatocellular carcinoma is generally refractory to clinical treatment. Here, we report that inactivation of the MYC oncogene is sufficient to induce sustained regression of invasive liver cancers. MYC inactivation resulted en masse in tumour cells differentiating into hepatocytes and biliary cells forming bile duct structures, and this was associated with rapid loss of expression of the tumour marker alpha-fetoprotein, the increase in expression of liver cell markers cytokeratin 8 and carcinoembryonic antigen, and in some cells the liver stem cell marker cytokeratin 19. Using in vivo bioluminescence imaging we found that many of these tumour cells remained dormant as long as MYC remain inactivated; however, MYC reactivation immediately restored their neoplastic features. Using array comparative genomic hybridization we confirmed that these dormant liver cells and the restored tumour retained the identical molecular signature and hence were clonally derived from the tumour cells. Our results show how oncogene inactivation may reverse tumorigenesis in the most clinically difficult cancers. Oncogene inactivation uncovers the pluripotent capacity of tumours to differentiate into normal cellular lineages and tissue structures, while retaining their latent potential to become cancerous, and hence existing in a state of tumour dormancy.

CNOB/ChrR6, a new prodrug enzyme cancer chemotherapy.

We report the discovery of a new prodrug, 6-chloro-9-nitro-5-oxo-5H-benzo(a)phenoxazine (CNOB). This prodrug is efficiently activated by ChrR6, the highly active prodrug activating bacterial enzyme we have previously developed. The CNOB/ChrR6 therapy was effective in killing several cancer cell lines in vitro. It also efficiently treated tumors in mice with up to 40% complete remission. 9-Amino-6-chloro-5H-benzo(a)phenoxazine-5-one (MCHB) was the only product of CNOB reduction by ChrR6. MCHB binds DNA; at nonlethal concentration, it causes cell accumulation in the S phase, and at lethal dose, it induces cell surface Annexin V and caspase-3 and caspase-9 activities. Further, MCHB colocalizes with mitochondria and disrupts their electrochemical potential. Thus, killing by CNOB involves MCHB, which likely induces apoptosis through the mitochondrial pathway. An attractive feature of the CNOB/ChrR6 regimen is that its toxic product, MCHB, is fluorescent. This feature proved helpful in in vitro studies because simple fluorescence measurements provided information on the kinetics of CNOB activation within the cells, MCHB killing mechanism, its generally efficient bystander effect in cells and cell spheroids, and its biodistribution. The emission wavelength of MCHB also permitted its visualization in live animals, allowing noninvasive qualitative imaging of MCHB in mice and the tumor microenvironment. This feature may simplify exploration of barriers to the penetration of MCHB in tumors and their amelioration.

Human immunodeficiency virus type 1 isolates with the reverse transcriptase (RT) mutation Q145M retain nucleoside and nonnucleoside RT inhibitor susceptibility.

Q145M, a mutation in a conserved human immunodeficiency virus type 1 reverse transcriptase (RT) region, was reported to decrease susceptibility to multiple RT inhibitors. We report that Q145M and other Q145 mutations do not emerge with RT inhibitors nor decrease RT inhibitor susceptibility. Q145M should not, therefore, be considered an RT inhibitor resistance mutation.

Microvesicle-Mediated Delivery of Minicircle DNA Results in Effective Gene-Directed Enzyme Prodrug Cancer Therapy.

An emerging approach for cancer treatment employs the use of extracellular vesicles, specifically exosomes and microvesicles, as delivery vehicles. We previously demonstrated that microvesicles can functionally deliver plasmid DNA to cells and showed that plasmid size and sequence, in part, determine the delivery efficiency. In this study, delivery vehicles comprised of microvesicles loaded with engineered minicircle (MC) DNA that encodes prodrug converting enzymes developed as a cancer therapy in mammary carcinoma models. We demonstrated that MCs can be loaded into shed microvesicles with greater efficiency than their parental plasmid counterparts and that microvesicle-mediated MC delivery led to significantly higher and more prolonged transgene expression in recipient cells than microvesicles loaded with the parental plasmid. Microvesicles loaded with MCs encoding a thymidine kinase (TK)/nitroreductase (NTR) fusion protein produced prolonged TK-NTR expression in mammary carcinoma cells. In vivo delivery of TK-NTR and administration of prodrugs led to the effective killing of both targeted cells and surrounding tumor cells via TK-NTR-mediated conversion of codelivered prodrugs into active cytotoxic agents. In vivo evaluation of the bystander effect in mouse models demonstrated that for effective therapy, at least 1% of tumor cells need to be delivered with TK-NTR-encoding MCs. These results suggest that MC delivery via microvesicles can mediate gene transfer to an extent that enables effective prodrug conversion and tumor cell death such that it comprises a promising approach to cancer therapy.

In vivo analysis of heat-shock-protein-70 induction following pulsed laser irradiation in a transgenic reporter mouse.

Induction of heat shock protein (Hsp) expression appears to correlate with a cytoprotective effect in cultured cells and with improved healing of damaged tissues in animal models and in humans. This family of proteins can also serve as indicators of thermal stress in cases of burn injury or surgical procedures that produce heat. Thus, a rapid in vivo readout for induction of Hsp transcription would facilitate studies of Hsp genes and their encoded proteins as mediators of therapeutic effects and as reporters of thermal damage to tissues. We created a transgenic reporter mouse where expression of luciferase is controlled by the regulatory region of the inducible 70 kDa Hsp, and assessed activation of Hsp70 transcription in live animals in response to rapid, high temperature stresses using in vivo bioluminescence imaging (BLI). This model can be used to noninvasively reveal levels of Hsp70 transcription in living tissues, and has utility in studies of the predictive and protective effects of Hsp70 expression, and of various stress responses in tissues.

An intravascular magnetic wire for the high-throughput retrieval of circulating tumour cells in vivo.

The detection and analysis of rare blood biomarkers is necessary for early diagnosis of cancer and to facilitate the development of tailored therapies. However, current methods for the isolation of circulating tumour cells (CTCs) or nucleic acids present in a standard clinical sample of only 5-10 ml of blood provide inadequate yields for early cancer detection and comprehensive molecular profiling. Here, we report the development of a flexible magnetic wire that can retrieve rare biomarkers from the subject's blood in vivo at a much higher yield. The wire is inserted and removed through a standard intravenous catheter and captures biomarkers that have been previously labelled with injected magnetic particles. In a proof-of-concept experiment in a live porcine model, we demonstrate the in vivo labelling and single-pass capture of viable model CTCs in less than 10 s. The wire achieves capture efficiencies that correspond to enrichments of 10-80 times the amount of CTCs in a 5-ml blood draw, and 500-5,000 times the enrichments achieved using the commercially available Gilupi CellCollector.

Signaling by Extracellular Vesicles Advances Cancer Hallmarks.

Mammalian cells secrete various extracellular vesicles (EVs; exosomes, microvesicles, and apoptotic bodies) that differ in biogenesis, composition, and function. Each vesicle type can originate from normal or cancerous cells, transfer molecular cargo to both neighboring and distant cells, and modulate cellular behaviors involved in eubiology and pathology, such as tumor development. Here, we review evidence for the role of EVs in the establishment and maintenance of cancer hallmarks, including sustaining proliferative signaling, evading growth suppression, resisting cell death, reprogramming energy metabolism, acquiring genomic instability, and remodeling the tumor microenvironment. We also discuss how EVs are implicated in the induction of angiogenesis, control of cellular invasion, initiation of premetastatic niches, maintenance of inflammation, and evasion of immune surveillance. The deeper understanding of the biology of EVs and their contribution to the development and progression of tumors is leading to new opportunities in the diagnosis and treatment of cancer.

Molecular imaging using labeled donor tissues reveals patterns of engraftment, rejection, and survival in transplantation.

Tissue regeneration and transplantation of solid organs involve complex processes that can only be studied in the context of the living organism, and methods of analyzing these processes in vivo are essential for development of effective transplantation and regeneration procedures. We utilized in vivo bioluminescence imaging (BLI) to noninvasively visualize engraftment, survival, and rejection of transplanted tissues from a transgenic donor mouse that constitutively expresses luciferase. Dynamic early events of hematopoietic reconstitution were accessible and engraftment from as few as 200 transplanted whole bone marrow (BM) cells resulted in bioluminescent foci in lethally irradiated, syngeneic recipients. The transplantation of autologous pancreatic Langerhans islets and of allogeneic heart revealed the tempo of transplant degeneration or immune rejection over time. This imaging approach is sensitive and reproducible, permits study of the dynamic range of the entire process of transplantation, and will greatly enhance studies across various disciplines involving transplantation.