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Detection of enzyme activity or transgene expression offers potential insight into developmental biology, disease progression, and potentially personalized medicine. Historically, the lacZ gene encoding the enzyme beta-galactosidase has been the most common reporter gene and many chromogenic and fluorogenic substrates are well established, but limited to histology or in vitro assays. We now present a novel approach for in vivo detection of beta-galactosidase using optical imaging to detect light emission following administration of the chemiluminescent 1,2-dioxetane substrate Galacto-Light PlusTM. B-gal activity was visualized in stably transfected human MCF7-lacZ tumors growing in mice. LacZ tumors were identified versus contralateral wild type tumors as controls, based on two- to tenfold greater light emission following direct intra tumoral or intravenous administration of reporter substrate. The 1,2-dioxetane substrate is commercially available as a kit for microplate-based assays for beta-gal detection, and we have adapted it for in vivo application. Typically, 100 microl substrate mixture was administered intravenously and light emission was detected from the lacZ tumor immediately with gradual decrease over the next 20 mins. Imaging was also undertaken in transgenic ROSA26 mice following subcutaneous or intravenous injection of substrate mixture. Light emission was detectable using standard instrumentation designed for more traditional bioluminescent imaging. Use of 1,2-dioxetane substrates to detect enzyme activity offers a new paradigm for non-invasive biochemistry in vivo.

Tumor vasculature proliferates rapidly, generally lacks pericyte coverage, and is uniquely fragile making it an attractive therapeutic target. A subset of small-molecule tubulin binding agents cause disaggregation of the endothelial cytoskeleton leading to enhanced vascular permeability generating increased interstitial pressure. The resulting vascular collapse and ischemia cause downstream hypoxia, ultimately leading to cell death and necrosis. Thus, local damage generates massive amplification and tumor destruction. The tumor vasculature is readily accessed and potentially a common target irrespective of disease site in the body. Development of a therapeutic approach and particularly next generation agents benefits from effective non-invasive assays. Imaging technologies offer varying degrees of sophistication and ease of implementation. This review considers technological strengths and weaknesses with examples from our own laboratory. Methods reveal vascular extent and patency, as well as insights into tissue viability, proliferation and necrosis. Spatiotemporal resolution ranges from cellular microscopy to single slice tomography and full three-dimensional views of whole tumors and measurements can be sufficiently rapid to reveal acute changes or long-term outcomes. Since imaging is non-invasive, each tumor may serve as its own control making investigations particularly efficient and rigorous. The concept of tumor vascular disruption was proposed over 30 years ago and it remains an active area of research.

None of the current biodegradable polymers can function as both implant materials and fluorescent imaging probes. The objective of this study was to develop aliphatic biodegradable photoluminescent polymers (BPLPs) and their associated cross-linked variants (CBPLPs) for biomedical applications. BPLPs are degradable oligomers synthesized from biocompatible monomers including citric acid, aliphatic diols, and various amino acids via a convenient and cost-effective polycondensation reaction. BPLPs can be further cross-linked into elastomeric cross-linked polymers, CBPLPs. We have shown representatively that BPLP-cysteine (BPLP-Cys) and BPLP-serine (BPLP-Ser) offer advantages over the traditional fluorescent organic dyes and quantum dots because of their preliminarily demonstrated cytocompatibility in vitro, minimal chronic inflammatory responses in vivo, controlled degradability and high quantum yields (up to 62.33%), tunable fluorescence emission (up to 725 nm), and photostability. The tensile strength of CBPLP-Cys film ranged from 3.25 ± 0.13 MPa to 6.5 ± 0.8 MPa and the initial Modulus was in a range of 3.34 ± 0.15 MPa to 7.02 ± 1.40 MPa. Elastic CBPLP-Cys could be elongated up to 240 ± 36%. The compressive modulus of BPLP-Cys (0.6) (1:1:0.6 OD:CA:Cys) porous scaffold was 39.60 ± 5.90 KPa confirming the soft nature of the scaffolds. BPLPs also possess great processability for micro/nano-fabrication. We demonstrate the feasibility of using BPLP-Ser nanoparticles (\"biodegradable quantum dots\") for in vitro cellular labeling and noninvasive in vivo imaging of tissue engineering scaffolds. The development of BPLPs and CBPLPs represents a new direction in developing fluorescent biomaterials and could impact tissue engineering, drug delivery, bioimaging.

Multispectral photoacoustic tomography enables the resolution of spectral components of a tissue or sample at high spatiotemporal resolution. With the availability of commercial instruments, the acquisition of data using this modality has become consistent and standardized. However, the analysis of such data is often hampered by opaque processing algorithms, which are challenging to verify and validate from a user perspective. Furthermore, such tools are inflexible, often locking users into a restricted set of processing motifs, which may not be able to accommodate the demands of diverse experiments. To address these needs, we have developed a Reconstruction, Analysis, and Filtering Toolbox to support the analysis of photoacoustic imaging data. The toolbox includes several algorithms to improve the overall quantification of photoacoustic imaging, including non-negative constraints and multispectral filters. We demonstrate various use cases, including dynamic imaging challenges and quantification of drug effect, and describe the ability of the toolbox to be parallelized on a high performance computing cluster.

It is recognized that cancer cells exhibit highly elevated glucose metabolism compared to non-tumor cells. We have applied in vivo optical imaging to study dynamic uptake of a near-infrared dye-labeled glucose analogue, 2-deoxyglucose (2-DG) by orthotopic glioma in a mouse model. The orthotopic glioma model was established by surgically implanting U87-luc glioma cells into the right caudal nuclear area of nude mice. Intracranial tumor growth was monitored longitudinally by bioluminescence imaging and MRI. When tumor size reached >4 mm diameter, dynamic fluorescence imaging was performed after an injection of the NIR labeled 2-DG, IRDye800CW 2-DG. Real-time whole body images acquired immediately after i.v. infusion clearly visualized the near-infrared dye circulating into various internal organs sequentially. Dynamic fluorescence imaging revealed significantly higher signal intensity in the tumor side of the brain than the contralateral normal brain 24 h after injection (tumor/normal ratio, TNR = 2.8+/-0.7). Even stronger contrast was achieved by removing the scalp (TNR = 3.7+/-1.1) and skull (TNR = 4.2+/-1.1) of the mice. In contrast, a control dye, IRDye800CW carboxylate, showed little difference (1.1+/-0.2). Ex vivo fluorescence imaging performed on ultrathin cryosections (20 microm) of tumor bearing whole brain revealed distinct tumor margins. Microscopic imaging identified cytoplasmic locations of the 2-DG dye in tumor cells. Our results suggest that the near-infrared dye labeled 2-DG may serve as a useful fluorescence imaging probe to noninvasively assess intracranial tumor burden in preclinical animal models.

Small animal imaging provides diverse methods for evaluating tumor growth and acute response to therapy. This study compared the utility of non-invasive optical and ultrasound imaging to monitor growth of three diverse human tumor xenografts (brain U87-luc-mCherry, mammary MCF7-luc-mCherry, and prostate PC3-luc) growing in nude mice. Bioluminescence imaging (BLI), fluorescence imaging (FLI), and Power Doppler ultrasound (PD US) were then applied to examine acute vascular disruption following administration of arsenic trioxide (ATO).During initial tumor growth, strong correlations were found between manual caliper measured tumor volume and FLI intensity, BLI intensity following luciferin injection, and traditional B-mode US. Administration of ATO to established U87 tumors caused significant vascular shutdown within 2 hrs at all doses in the range 5 to 10 mg/kg in a dose dependant manner, as revealed by depressed bioluminescent light emission. At lower doses substantial recovery was seen within 4 hrs. At 8 mg/kg there was >85% reduction in tumor vascular perfusion, which remained depressed after 6 hrs, but showed some recovery after 24 hrs. Similar response was observed in MCF7 and PC3 tumors. Dynamic BLI and PD US each showed similar duration and percent reductions in tumor blood flow, but FLI showed no significant changes during the first 24 hrs.The results provide further evidence for comparable utility of optical and ultrasound imaging for monitoring tumor growth, More specifically, they confirm the utility of BLI and ultrasound imaging as facile assays of the vascular disruption in solid tumors based on ATO as a model agent.

Multi-modality image-guided radiotherapy is the standard of care in contemporary cancer management; however, it is not common in preclinical settings due to both hardware and software limitations. Soft tissue lesions, such as orthotopic prostate tumors, are difficult to identify using cone beam computed tomography (CBCT) imaging alone. In this study, we characterized a research magnetic resonance (MR) scanner for preclinical studies and created a protocol for combined MR-CBCT image-guided small animal radiotherapy. Two in-house dual-modality, MR and CBCT compatible, phantoms were designed and manufactured using 3D printing technology. The phantoms were used for quality assurance tests and to facilitate end-to-end testing for combined preclinical MR and CBCT based treatment planning. MR and CBCT images of the phantoms were acquired utilizing a Varian 4.7 T scanner and XRad-225Cx irradiator, respectively. The geometry distortion was assessed by comparing MR images to phantom blueprints and CBCT. The corrected MR scans were co-registered with CBCT and subsequently used for treatment planning. The fidelity of 3D printed phantoms compared to the blueprint design yielded favorable agreement as verified with the CBCT measurements. The geometric distortion, which varied between -5% and 11% throughout the scanning volume, was substantially reduced to within 0.4% after correction. The distortion free MR images were co-registered with the corresponding CBCT images and imported into a commercial treatment planning software SmART Plan. The planning target volume (PTV) was on average 19% smaller when contoured on the corrected MR-CBCT images relative to raw images without distortion correction. An MR-CBCT based preclinical workflow was successfully designed and implemented for small animal radiotherapy. Combined MR-CBCT image-guided radiotherapy for preclinical research potentially delivers enhanced relevance to human radiotherapy for various disease sites. This novel protocol is wide-ranging and not limited to the orthotopic prostate tumor study presented in the study.

BPR0L075, 6-methoxy-3-(3',4',5'-trimethoxy-benzoyl)-1H-indole, is a tubulin-binding agent that inhibits tubulin polymerization by binding to the colchicine-binding site. BPR0L075 has shown antimitotic and antiangiogenic activity in vitro. The current study evaluated the vascular-disrupting activity of BPR0L075 in human breast cancer mammary fat pad xenografts using dynamic bioluminescence imaging. A single dose of BPR0L075 (50 mg/kg, intraperitoneally (i.p.)) induced rapid, temporary tumor vascular shutdown (at 2, 4, and 6 hours); evidenced by rapid and reproducible decrease of light emission from luciferase-expressing orthotopic MCF7 and MDA-MB-231 breast tumors after administration of luciferin substrate. A time-dependent reduction of tumor perfusion after BPR0L075 treatment was confirmed by immunohistological staining of the perfusion marker Hoechst 33342 and tumor vasculature marker CD31. The vasculature showed distinct recovery within 24 hours post therapy. A single i.p. injection of 50 mg/kg of BPR0L075 initially produced plasma concentrations in the micromolar range within 6 hours, but subsequent drug distribution and elimination caused BPR0L075 plasma levels to drop rapidly into the nanomolar range within 24 h. Tests with human umbilical vein endothelial (HUVEC) cells and tumor cells in culture showed that BPR0L075 was cytotoxic to both tumor cells and proliferating endothelial cells, and disrupted pre-established vessels in vitro and ex vivo. In conclusion, BPR0L075 caused rapid, albeit, temporary tumor vascular shutdown and led to reduction of tumor perfusion in orthotopic human breast cancer xenografts, suggesting that this antimitotic agent may be useful as a vascular-disrupting cancer therapy.

Hypoxia, a condition of insufficient O2 to support metabolism, occurs when the vascular supply is interrupted, as in stroke or myocardial infarction, or when a tumor outgrows its vascular supply. When otherwise healthy tissues lose their O2 supply acutely, the cells usually die, whereas when cells gradually become hypoxic, they adapt by up-regulating the production of numerous proteins that promote their survival. These proteins slow the rate of growth, switch the mitochondria to glycolysis, stimulate growth of new vasculature, inhibit apoptosis, and promote metastatic spread. The consequence of these changes is that patients with hypoxic tumors invariably experience poor outcome to treatment. This has led the molecular imaging community to develop assays for hypoxia in patients, including regional measurements from O2 electrodes placed under CT guidance, several nuclear medicine approaches with imaging agents that accumulate with an inverse relationship to O2, MRI methods that measure either oxygenation directly or lactate production as a consequence of hypoxia, and optical methods with NIR and bioluminescence. The advantages and disadvantages of these approaches are reviewed, along with the individual strategies for validating different imaging methods. Ultimately the proof of value is in the clinical performance to predict outcome, select an appropriate cohort of patients to benefit from a hypoxia-directed treatment, or plan radiation fields that result in better local control. Hypoxia imaging in support of molecular medicine has become an important success story over the last decade and provides a model and some important lessons for development of new molecular imaging probes or techniques.

Non-invasive monitoring of disease progression in kidney disease is still a major challenge in clinical practice. In vivo near-infrared (NIR) imaging provides a new tool for studying disease mechanisms and non-invasive monitoring of disease development, even in deep organs. The LI-COR IRDye® 800CW RGD optical probe (RGD probe) is a NIR fluorophore, that can target integrin alpha v beta 3 (α(v)β(3)) in tissues. This study aims to monitor renal disease progression in an anti-glomerular basement membrane (GBM) nephritis mouse model. Anti-GBM nephritis was induced in 129x1/svJ mice by anti-GBM serum challenge. The expression of integrin α(v)β(3) in the diseased kidney was examined by immunohistochemistry and quantitative polymerase chain reaction. The RGD probe and control fluorophores, the 800CW dye, and the BSA-conjugated 800CW dye, were administered into anti-GBM nephritic mice. LI-COR Pearl® Impulse imaging system was used for in vivo imaging; while ex vivo organ imaging was acquired using the Maestro(TM) imaging system. Kidney tissue from anti-GBM nephritic mice showed higher levels of integrin α(v)β(3) expression at both the protein and the mRNA level compared to normal mice. The RGD probe allowed in vivo renal imaging and the fluorescent signal could be specifically captured in the diseased kidneys up to 14 days, reflecting longitudinal changes in renal function. The infrared RGD molecular probe that tracks integrin expression can be successfully used to monitor renal disease progression following immune-mediated nephritis.