Fluorescence Imaging In Mice Research Articles

A novel low-background nitroreductase fluorescent probe for real-time fluorescence imaging and surgical guidance of thyroid cancer resection

Thyroid cancer always appears insidiously with few noticeable clinical symptoms. Due to its limitations, conventional ultrasound imaging can lead to missed or misdiagnosed cases. Surgery is still the primary treatment method of thyroid cancer, but removal of surrounding healthy tissues to minimize recurrence leads to overtreatment and added patient suffering. To address this challenge, herein, a nitroreductase (NTR) fluorescent probe, Ox-NTR, has been developed for detecting thyroid cancer and tracking the surgical removal of thyroid tumors by fluorescence imaging. The conjugated structure of oxazine 1 was disrupted, significantly reducing the issue of high background signals, thus effectively achieving low background fluorescence. Under hypoxic conditions, the nitro group of Ox-NTR can be reduced to an amine and subsequently decomposed into oxazine 1, emitting intense red fluorescence. Ox-NTR has a low detection limit of 0.09 μg/mL for NTR with excellent photostability and selectivity. Cellular studies show that Ox-NTR can effectively detect NTR levels in hypoxic thyroid cancer cells. Moreover, the ability of Ox-NTR of rapid response to thyroid cancer in vivo is confirmed by fluorescence imaging in mice, distinguishing tumors from normal tissues due to its superior low background fluorescence. Utilizing this fluorescence imaging method during surgical resection can guide the removal of tumors, preventing both missed tumor tissues and accidental removal of healthy tissue. In summary, the novel Ox-NTR offers precise detection capabilities that provide significant advantages over traditional imaging methods for thyroid cancer diagnosis and treatment, making it a valuable tool to guide tumor removal in surgical procedures.

Rapid method to screen biomaterial angiogenesis in vivo using fluorescence imaging in mice.

Effective vascularization is crucial for repairing and enhancing the longevity of engineered tissues and organs. As the field advances, there is a vital need for efficient and reliable methods for assessing vascularization in real-time. The integration and performance of constructed biomaterials in living organisms rely on angiogenesis and vascularization, making it essential to evaluate vascular development and networks within biomaterials. Current histology-based methods are limited and labor-intensive. On the other hand, fluorescence imaging offers promise for efficient, real-time evaluation of angiogenesis, reducing the time needed for screening many compounds and offering a high-throughput alternative to histology-based methods. Here, we investigated a novel, non-invasive method for quick and repeated analysis of the angiogenic and vascularization process in biomaterials via fluorescence IVIS imaging. Multi-domain peptides (MDPs), self-assembling peptide hydrogels that can possess pro-angiogenic properties depending on their primary sequence, were synthesized and utilized as angiogenic biomaterials and screened with a fluorescence IVIS probe to demonstrate real-time rapid angiogenesis in vivo. The fluorescence-based imaging showed the influence of the peptide chemistry, volume, and concentration on angiogenesis, with one particular MDP, SLanc, promoting robust angiogenesis after one week at 2 w/v%. Through this method, we were able to identify the optimal peptide for rapid and sustained angiogenesis. This approach enables real-time monitoring of angiogenic responses and vascularization processes in the same living subject. It promotes the development of new biomaterials that facilitate vascularization and validates an advanced in vivo screening technique for angiogenesis.

Neutrophil membrane camouflaged nanoprobes for NIR-II fluorescence imaging of inflamed, high-risk atherosclerotic plaques in mouse and rabbit models

Atherosclerosis is the leading cause of vascular death worldwide, and inflammation plays a crucial role in atherosclerotic plaque progression and rupture. Fluorescent nanoprobes that can detect inflamed, high-risk atherosclerotic plaques and evaluate the therapeutic efficacy at the animal level in vivo are particularly meaningful. Herein, we developed a kind of neutrophil membrane-camouflaged nanoprobe in the second near-infrared spectral region (NIR-II) for the imaging of inflamed, high-risk atherosclerotic plaques in both mouse and rabbit models. Compared with free indocyanine green (ICG), the NIR-II fluorescence intensity of neutrophil biomimetic nanoparticles (Neu-NPs) was increased by 276% and had good stability in serum. Neu-NPs could specifically target inflamed, high-risk atherosclerotic plaques through the interaction of the chemokine receptor LFA-1with ICAM-1, which are highly expressed in inflamed sites. In the ApoE−/− mouse model, the signal-to background (S/B) ratio of the Neu-NP-treated mice was 1.67-fold higher than that of NP-treated mice. Even in large animals, such as the New Zealand white rabbit atherosclerosis model, Neu-NPs exhibited excellent targeting to high-risk atherosclerotic plaques. Moreover, the Neu-NPs could be used to reflect the reduction in plaque inflammation after the treatment and could thus be used for therapeutic evaluation and drug screening. Therefore, this highly biocompatible neutrophil membrane-camouflaged nanoprobe may be a new nanocarrier for the treatment of chronic inflammatory diseases such as atherosclerosis. Moreover, the use of NIR-II fluorescence imaging in mice and rabbits with atherosclerosis extends the scope of this emerging imaging technology.

Visualization of degradation of injectable thermosensitive hydroxypropyl chitin modified by aggregation-induced emission

The reversible thermosensitive hydroxypropyl chitin (HPCH) has been widely investigated for drug delivery, tissue engineering, wound repair and antibacterial hemostasis. To achieve non-invasive and real-time visualization of in vivo degradation of HPCH, aggregation-induced emission fluorogen (AIEgen) was introduced covalently in HPCH in water to obtain thermosensitive fluorescent hydroxypropyl chitin (FHPCH) avoiding using any organic solvent. The obtained fluorescent FHPCH hydrogel showed strong yellow-green fluorescence feature under UV irradiation, enabling visualization of the hydrogel with reversible thermosensitivity and reliable injectability. The in vitro enzymatic degradation study of FHPCH hydrogel showed that the attenuation of fluorescence intensity well matched with hydrogel weight loss. The injection through a 26-gauge needle for real-time fluorescence imaging in mice indeed indicated that AIE modified injectable thermosensitive FHPCH can be used for non-invasive, continuous and real-time visualization and quantitative analysis of in vivo degradation. This provides a new method for non-invasive real-time monitoring of similar implantable materials.

Erythrocyte membrane-camouflaged nanoworms with on-demand antibiotic release for eradicating biofilms using near-infrared irradiation

The increase in the number of resistant bacteria caused by the abuse of antibiotics and the emergence of biofilms significantly reduce the effectiveness of antibiotics. Bacterial infections are detrimental to our life and health. To reduce the abuse of antibiotics and treat biofilm-related bacterial infections, a biomimetic nano-antibacterial system, RBCM-NW-G namely, that controls the release of antibiotics through near infrared was prepared. The hollow porous structure and the high surface activity of nanoworms are used to realize antibiotic loading, and then, biomimetics are applied with red blood cell membranes (RBCM). RBCM-NW-G, which retains the performance of RBCM, shows enhanced permeability and retention effects. Fluorescence imaging in mice showed the effective accumulation of RBCM-NW-G at the site of infection. In addition, the biomimetic nanoparticles showed a longer blood circulation time and good biocompatibility. Anti-biofilm test results showed damage to biofilms due to a photothermal effect and a highly efficient antibacterial performance under the synergy of the photothermal effect, silver iron, and antibiotics. Finally, by constructing a mouse infection model, the great potential of RBCM-NW-G in the treatment of in vivo infections was confirmed.

Multimodal Tracking of Controlled Staphylococcus aureus Infections in Mice.

There is a need to develop diagnostic and analytical tools that allow noninvasive monitoring of bacterial growth and dissemination in vivo. For such cell-tracking studies to hold translational value to controlled human infections, in which volunteers are experimentally colonized, they should not require genetic modification, and they should allow tracking over a number of replication cycles. To gauge if an antimicrobial peptide tracer, 99mTc-UBI29–41-Cy5, which contains both a fluorescent and a radioactive moiety, could be used for such in vivo bacterial tracking, we performed longitudinal imaging of a thigh-muscle infection with 99mTc-UBI29–41-Cy5-labeled Staphylococcus aureus. Mice were imaged using SPECT and fluorescence-imaging modalities at various intervals during a 28 h period. Biodistribution analyses were performed to quantitate radioactivity in the abscess and other tissues. SPECT and fluorescence imaging in mice showed clear retention of the 99mTc-UBI29–41-Cy5-labeled bacteria following inoculation in the thigh muscle. Despite bacterial replication, the signal intensity in the abscess only modestly decreased within a 28 h period: 52% of the total injected radioactivity per gram of tissue (%ID/g) at 4 h postinfection (pi) versus 44%ID/g at 28 h pi (15% decrease). After inoculation, a portion of the bacteria disseminated from the abscess, and S. aureus cultures were obtained from radioactive urine samples. Bacterial staining with 99mTc-UBI29–41-Cy5 allowed noninvasive bacterial-cell tracking during a 28 h period. Given the versatility of the presented bacterial-tracking method, we believe that this concept could pave the way for precise imaging capabilities during controlled-human-infection studies.

Near-Infrared Optical Imaging of Nucleic Acid Nanocarriers In Vivo.

Noninvasive, real-time optical imaging methods are well suited to follow the in vivo distribution of nucleic acid nanocarriers, their dissociation and the resulting gene expression or inhibition. Indeed, most small animal imaging devices are performing bioluminescence and fluorescence measurements without moving the animal, allowing a simple, rapid, and cost-effective method of investigation of several parameters at a time, in longitudinal experiments that can last for days or weeks.Here we help the reader in choosing adapted near-infrared (NIR) fluorophores or pairs of fluorophores for FRET assays, imaging of reporter genes as well as nanocarriers for in vivo gene and siRNA delivery. In addition, we present the labeling methods of these macromolecules, and of their payload and the protocols to detect them using bioluminescence and NIR fluorescence imaging in mice.

Perfluorocarbon nanodroplets can reoxygenate hypoxic tumors in vivo without carbogen breathing.

Nanoscale perfluorocarbon (PFC) droplets have enormous potential as clinical theranostic agents. They are biocompatible and are currently used in vivo as contrast agents for a variety of medical imaging modalities, including ultrasound, computed tomography, photoacoustic and 19F-magnetic resonance imaging. PFC nanodroplets can also carry molecular and nanoparticulate drugs and be activated in situ by ultrasound or light for targeted therapy. Recently, there has been renewed interest in using PFC nanodroplets for hypoxic tumor reoxygenation towards radiosensitization based on the high oxygen solubility of PFCs. Previous studies showed that tumor oxygenation using PFC agents only occurs in combination with enhanced oxygen breathing. However, recent studies suggest that PFC agents that accumulate in solid tumors can contribute to radiosensitization, presumably due to tumor reoxygenation without enhanced oxygen breathing. In this study, we quantify the impact of oxygenation due to PFC nanodroplet accumulation in tumors alone in comparison with other reoxygenation methodologies, in particular, carbogen breathing.Methods: Lipid-stabilized, PFC (i.e., perfluorooctyl bromide, CF3(CF2)7Br, PFOB) nanoscale droplets were synthesized and evaluated in xenograft prostate (DU145) tumors in male mice. Biodistribution assessment of the nanodroplets was achieved using a fluorescent lipophilic indocarbocyanine dye label (i.e., DiI dye) on the lipid shell in combination with fluorescence imaging in mice (n≥3 per group). Hypoxia reduction in tumors was measured using PET imaging and a known hypoxia radiotracer, [18F]FAZA (n≥ 3 per group).Results: Lipid-stabilized nanoscale PFOB emulsions (mean diameter of ~250 nm), accumulated in the xenograft prostate tumors in mice 24 hours post-injection. In vivo PET imaging with [18F]FAZA showed that the accumulation of the PFOB nanodroplets in the tumor tissues alone significantly reduced tumor hypoxia, without enhanced oxygen (i.e., carbogen) breathing. This reoxygenation effect was found to be comparable with carbogen breathing alone.Conclusion: Accumulation of nanoscale PFOB agents in solid tumors alone successfully reoxygenated hypoxic tumors to levels comparable with carbogen breathing alone, an established tumor oxygenation method. This study confirms that PFC agents can be used to reoxygenate hypoxic tumors in addition to their current applications as multifunctional theranostic agents.

Abstract 3997: Interleukin-4 receptor-targeted cytotoxic T cells enhances the therapeutic efficacy of adoptive T cell therapy against melanoma

Abstract Melanoma, the deadliest form of skin cancer, has long been a major focus of immunotherapy. Adoptive T cell therapy-based cancer immunotherapy has been used to enhance the specificity and potential of host immune system to treat melanoma. However, the infiltration and antitumor function of cytotoxic T cells (CTLs) are blocked or down-regulated by various signals derived from tumor cells and cells in tumor microenvironment. A novel approach to specify and enhance the homing ability of CTLs to tumor is of high demand. Interleukin-4 receptor (IL-4R) is over-expressed on many types of cancer cells, including melanoma, and has been widely employed for targeted drug delivery. In this work, we examined whether the tumor homing efficiency and anti-tumor therapeutic efficacy of CTLs could be heighten by labeling CTLs with IL4RPep-1, a targeting peptide that binds to IL-4R. The labeling was empowered by conjugating IL4RPep-1 with Dioleylphosphatidylethanolamine-biological anchor for membrane (DOPE-BAM), an oleyl acid-derived membrane anchor that incorporates into lipid membrane of cells. Tumor-specific CTLs isolated from the mice immunized by irradiated B16F10 melanoma cells were highly populated with CD62L+CD44+ activated T cells. After labeling with IL4RPep-1 via DOPE-BAM, CTLs were still functionally active, as determined by intracellular interferon-γ and granzyme release assays. Moreover, such labeling did not affect the proliferation of T cells. Adoptive transfer of IL4RPep-1-labeled, IL-4R-targeted CD45.1+CTLs into CD45.2 mice bearing B16F10 melanoma enhanced their accumulation at tumor compared to that of unlabeled T cells. Tumor homing of IL4RPep-1-labeled T cells was further validated by in vivo fluorescence imaging of mice bearing 4T1 tumor at mammary gland. Adoptive transfer of IL-4R-targeted CTLs showed remarkable anti-tumor growth activity in mice bearing B16F10 tumor compared to untargeted CTLs. After the transfer, flow cytometric study of immune cells showed significant increase of CD8+ T cells and F4/80+CD86+M1-polarized macrophages with noticeable decrease in tumor-promoting immune cells, such as F4/80+CD206+M2-polarized macrophages, Gr1+CD11b+ myeloid derived suppressor cells, and CD3+CD4+ T cells. The CTLs accumulated at tumor were mostly exogenous CD45.1+ T cells. These results implicate that IL-4R-targeted CTLs, which is enabled by labeling of with IL4RPep-1, can be a promising strategy in the field of adoptive T cell therapy against IL-4R-overexpressing tumor. Citation Format: Gunassekaran Gowri Rangaswamy, Poongkavithai Vadevoo Sri Murugan, Guruprasath Padmanaban, Lianhua Chi, Ha-Jeong Kim, Byung Heon Lee. Interleukin-4 receptor-targeted cytotoxic T cells enhances the therapeutic efficacy of adoptive T cell therapy against melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3997. doi:10.1158/1538-7445.AM2017-3997

P1.07-033 Trastuzumab Emtansine (T-DM1) Suppresses the Growth of HER2-Positive Small-Cell Lung Cancer in Preclinical Models

To overcome chemoresistance is indispensable to bring about better prognosis in small-cell lung cancer (SCLC). We have reported that HER2 is upregulated when HER2-positive SCLC cells acquire chemoresistance. Moreover, HER2-upregulated cisplatin- or etoposide-resistant SCLC cells were sensitive to trastuzumab-mediated antibody-dependent cell-mediated cytotoxicity (ADCC). However, irinotecan-resistant SCLC cells, e.g. SBC-3/SN-38, were refractory to trastuzumab despite high HER2 expression. To address this issue, we studied the antitumor efficacy of trastuzumab emtansine (T-DM1) on trastuzumab-resistant HER2-positive SCLC. SBC-3/SN-38 (HER2-positive) or OS2RA (HER2-negative) SCLC cells were inoculated subcutaneously in the flank of six-week-old male nude mice. Tumor size was measured with a caliper two or three times per week. When tumor volume reached about 150-200 mm3, mice were randomly assigned to three treatment groups. Each group was treated with intravenous injection of T-DM1 15 mg/kg, intraperitoneal injection of trastuzumab 30 mg/kg, or saline as control. To confirm the HER2-specific delivery of T-DM1, in vivo imaging was performed. Namely, several tumor-bearing mice were intravenously administered with fluorescence-labeled T-DM1. Fluorescence images of mice were captured with IVIS® Lumina II system (PerkinElmer). Treatment with T-DM1 significantly suppressed the growth of SBC-3/SN-38 xenografts compared with trastuzumab and saline groups. Histological analysis revealed that T-DM1 remarkably induced apoptosis and inhibited proliferation. Fluorescence-labeled T-DM1 definitely accumulated to the xenografts in a HER2-selective fashion. T-DM1 treatment could be an attractive therapeutic option in trastuzumab-resistant HER2-positive SCLC where trastuzumab cannot induce enough ADCC activity. Delivery of a cytotoxic agent DM1 to the inside of cells via HER2-mediated internalization is expected and crucial to exert antitumor effect in such ADCC-lacking SCLC cells.

Optical tracer size differences allow quantitation of active pumping rate versus Stokes-Einstein diffusion in lymphatic transport.

Lymphatic uptake of interstitially administered agents occurs by passive convective–diffusive inflow driven by interstitial concentration and pressure, while the downstream lymphatic transport is facilitated by active propulsive contractions of lymphatic vessel walls. Near-infrared fluorescence imaging in mice was used to measure these central components of lymphatic transport for the first time, using two different-sized molecules––methylene blue (MB) and fluorescence-labeled antibody immunoglobulin G (IgG)-IRDye 680RD. This work confirms the hypothesis that lymphatic passive inflow and active propulsion rates can be separated based upon the relative differences in Stokes–Einstein diffusion coefficient. This coefficient specifically affects the passive-diffusive uptake when the interstitial volume and pressure are constant. Parameters such as mean time-to-peak signal, overall fluorescence signal intensities, and number of active peristaltic pulses, were estimated from temporal imaging data. While the mean time to attain peak signal representative of diffusion-dominated flow in the lymph vessels was 0.6±0.2??min for MB and 8±6??min for IgG, showing a size dependence, the active propulsion rates were 3.4±0.8??pulses/min and 3.3±0.5??pulses/min, respectively, appearing size independent. The propulsion rates for both dyes decreased with clearance from the interstitial injection-site, indicating intrinsic control of the smooth muscles in response to interstitial pressure. This approach to size-comparative agent flow imaging of lymphatic function can enable noninvasive characterization of diseases related to uptake and flow in lymph networks.

Auditory cortical field coding long-lasting tonal offsets in mice.

Although temporal information processing is important in auditory perception, the mechanisms for coding tonal offsets are unknown. We investigated cortical responses elicited at the offset of tonal stimuli using flavoprotein fluorescence imaging in mice. Off-responses were clearly observed at the offset of tonal stimuli lasting for 7 s, but not after stimuli lasting for 1 s. Off-responses to the short stimuli appeared in a similar cortical region, when conditioning tonal stimuli lasting for 5–20 s preceded the stimuli. MK-801, an inhibitor of NMDA receptors, suppressed the two types of off-responses, suggesting that disinhibition produced by NMDA receptor-dependent synaptic depression might be involved in the off-responses. The peak off-responses were localized in a small region adjacent to the primary auditory cortex, and no frequency-dependent shift of the response peaks was found. Frequency matching of preceding tonal stimuli with short test stimuli was not required for inducing off-responses to short stimuli. Two-photon calcium imaging demonstrated significantly larger neuronal off-responses to stimuli lasting for 7 s in this field, compared with off-responses to stimuli lasting for 1 s. The present results indicate the presence of an auditory cortical field responding to long-lasting tonal offsets, possibly for temporal information processing.

Preclinical evaluation of near-infrared (NIR) fluorescently labeled cetuximab as a potential tool for fluorescence-guided surgery.

The high rate of recurrence in patients with pancreatic ductal adenocarcinoma (PDAC) could be reduced by supporting the surgeons in discriminating healthy from diseased tissues with intraoperative fluorescence-guidance. Here, we studied the suitability of Cetuximab, a therapeutic monoclonal antibody targeting the human epidermal growth factor receptor (EGFR), near-infrared (NIR) fluorescently labeled as a new tool for fluorescence-guided surgery. Distribution and binding of systemically injected Cetuximab Alexa Fluor 647 conjugate (Cetux-Alexa-647) and the co-injected control human IgG Alexa Fluor 750 conjugate (hIgG-Alexa-750) was studied over 48 h by NIR fluorescence imaging in mice bearing human orthotopic AsPC-1 and MIA PaCa-2 PDAC tumors. Cetux-Alexa-647, but not the control hIgG-Alexa-750 fluorescence, was specifically detected in vivo in both primary pancreatic tumors with maximum fluorescence intensities at 24 h, and in metastases of AsPC-1 tumors as small as 1 mm. Lifetime analysis and NIR fluorescence microscopy of tumor sections confirmed the binding specificity of Cetux-Alexa-647 to PDAC cells. Comparable results were obtained with Cetuximab conjugated to Alexa Fluor 750 dye (Cetux-Alexa-750). Fluorescence-guided dissection, performed 24 h after injection of Cetuximab conjugated to IRDye 800CW (Cetux-800CW), enabled a real-time delineation of AsPC-1 tumor margins, and small metastases. Odyssey scans revealed that only the vital part of the tumor, but not the necrotic part was stained with Cetux-800CW. NIR fluorescently labeled Cetuximab may be a promising tool that can be applied for fluorescence-guided surgery to visualize tumor margins and metastatic sites in order to allow a precise surgical resection.

Establishment of an mKate2-Expressing Cell Line for Non-Invasive Real-Time Breast Cancer In Vivo Imaging.

Non-invasive real-time in vivo imaging experiments using mice as animal models have become crucial for understanding cancer development and treatment. In this study, we have developed and validated a new breast cancer cell line MDA-MB-435s that stably express a far-red fluorescence protein (mKate2) and that could serve as a highly valuable cell model for studying breast cancer detection and therapy using in vivo fluorescence imaging in nude mice. The new cell line (MDA-MB-435s-mKate2) was constructed by plasmid transfection. The stability and sensitivity of mKate2, and the cell biological activities, were tested in vitro using different experimental approaches. For its potential use in tumor growth research and drug therapy in vivo, MDA-MB-435s-mKate2 was validated using the immunocompromised Balb/c nude mice tumor model. In addition, the new cell line has been characterized as a luteinizing hormone-releasing hormone receptor (LHRHR) positive cell line. Firstly, MDA-MB-435s-mKate2 has shown a stable chromosomal integration of the amplified mKate2 gene and good fluorescence sensitivity for detection using a fluorescence reflectance imaging (FRI) device. Compared to its parental cell line, no significant difference in cell migration, proliferation, and clone formation was observed in vitro. Secondly, using the quantification of tumor-fluorescence surface area in live animals, we were able to monitor and detect the tumor progress or tumor inhibition rate (by Paclitaxel treatment) non-invasively and in real-time. Furthermore, MDA-MB-435s-mKate2 has been positively tested for LHRHR; these findings open the possibility to use this cell line for future studies of breast cancer therapy based on LHRH analogs in vivo. In the present research, we have successfully built the MDA-MB-435s-mKate2 cell line that can be used as a suitable cell model for breast cancer therapy and anti-cancer drug evaluation by non-invasive fluorescence imaging in mice.

Tumor imaging technologies in mouse models.

In this chapter, we describe protocols for tumor imaging technologies in mouse models. These models utilize human cancer cell lines which have been genetically engineered to selectively express high levels of green fluorescent protein (GFP) or red fluorescent protein (RFP). Tumors with fluorescent genetic reporters are established subcutaneously in nude mice, and fragments of the subcutaneous tumors are then surgically transplanted onto the orthotopic organ. Locoregional tumor growth and distant metastasis of these orthotopic implants occur spontaneously and rapidly throughout the abdomen in a manner consistent with clinical human disease. Highly specific, high-resolution, real-time quantitative fluorescence imaging of tumor growth and metastasis may be achieved in vivo without the need for contrast agents, invasive techniques, or expensive imaging equipment. Transplantation of RFP-expressing tumor fragments onto the pancreas of GFP- or cyan fluorescent protein (CFP)-expressing transgenic nude mice was used to facilitate visualization of tumor-host interaction between the pancreatic cancer cells and host-derived stroma and vasculature. Such in vivo models have enabled us to visualize in real time and acquire images of the progression of pancreatic cancer in the live animal, and to demonstrate the real-time antitumor and antimetastatic effects of several novel therapeutic strategies on a variety of malignancies. We discuss studies from our laboratory that demonstrate that fluorescence imaging in mice is complementary to other modalities such as magnetic resonance imaging (MRI) or ultrasound. These fluorescent models are powerful and reliable tools with which to investigate metastatic human cancer and novel therapeutic strategies directed against it.

Matrix-metalloproteinases in head and neck carcinoma-cancer genome atlas analysis and fluorescence imaging in mice.

(1) Obtain matrix-metalloproteinase (MMP) expression profiles for head and neck squamous cell carcinoma (HNSCC) specimens from the Cancer Genomic Atlas (TCGA). (2) Demonstrate HNSCC imaging using MMP-cleavable, fluorescently labeled ratiometric activatable cell-penetrating peptide (RACPP). Retrospective human cohort study; prospective animal study. Translational research laboratory. Patient clinical data and mRNA expression levels of MMP genes were downloaded from TCGA data portal. RACPP provides complementary ratiometric fluorescent contrast (increased Cy5 and decreased Cy7 intensities) when cleaved by MMP2/9. HNSCC-tumor bearing mice were imaged in vivo after RACPP injection. Histology was evaluated by a pathologist blinded to experimental conditions. Zymography confirmed MMP-2/9 activity in xenografts. RACPP was applied to homogenized human HNSCC specimens, and ratiometric fluorescent signal was measured on a microplate reader for ex vivo analysis. Expression of multiple MMPs including MMP2/9 is greater in patient HNSCC tumors than matched control tissue. In patients with human papilloma virus positive (HPV+) tumors, higher MMP2 and MMP14 expression correlates with worse 5-year survival. Orthotopic tongue HNSCC xenografts showed excellent ratiometric fluorescent labeling with MMP2/9-cleavable RACPP (sensitivity = 95.4%, specificity = 95.0%). Fluorescence ratios were greater in areas of higher tumor burden (P < .03), which is useful for intraoperative margin assessment. Ex vivo, human HNSCC specimens showed greater cleavage of RACPP when compared to control tissue (P = .009). Human HNSCC tumors show increased mRNA expression of multiple MMPs including MMP2/9. We used RACPP, a ratiometric fluorescence assay of MMP2/9 activity, to show improved occult tumor identification and margin clearance. Ex vivo assays using RACPP in biopsy specimens may identify patients who will benefit from intraoperative RACPP use.

The application of anti‐ESAT‐6 monoclonal antibody fluorescent probe in ex vivo near‐infrared fluorescence imaging in mice with pulmonary tuberculosis

Here, we aimed to assess the feasibility of anti-ESAT-6 monoclonal antibody (mAb) coupling with IR783 and rhodamine fluorescent probe in the detection of ESAT-6 expression in tuberculosis tissue of mice using near-infrared fluorescence imaging. IR783 and rhodamine were conjugated to the anti-ESAT-6 mAb or IgG. Mice in the experimental group were injected with fluorescence-labeled mAb probe, and mice in the control group were injected with fluorescence-labeled non-specific IgG antibody. Twenty-four hours later, the lung tissue of mice was examined using ex vivo near-infrared fluorescence imaging. In addition, the contrast-to-noise ratio (CNR) was calculated by measuring the signal intensities of the pulmonary lesions, normal lung tissue and background noise. The frozen lung tissue section was examined under fluorescence microscopy and compared with hemoxylin and eosin (HE) staining. The ex vivo near-infrared fluorescence imaging showed that the fluorescence signal in the lung tuberculosis lesions in the experimental group was significantly enhanced, whereas there was only a weak fluorescence signal or even no fluorescence signal in the control group. CNR values were 64.40 ± 7.02 (n = 6) and 8.75 ± 3.87 (n = 6), respectively (t = 17.01, p < 0.001). The fluorescence accumulation distribution detected under fluorescence microscopy was consistent with HE staining of the tuberculosis region. In conclusion, anti-ESAT-6 mAb fluorescent probe could target and be applied in specific ex vivo imaging of mice tuberculosis, and may be of further use in tuberculosis in living mice.

Monitoring transplanted adipose tissue-derived stem cells combined with heparin in the liver by fluorescence imaging using quantum dots

Adipose tissue-derived stem cell (ASC) transplantation, when used in combination with heparin, has proven to be an effective treatment for acute liver failure in mice. However, the behavior and organ-specific accumulation of transplanted ASCs alone or in combination with heparin is poorly understood. In this paper, we investigated whether quantum dots (QDs) labeling using octa-arginine peptide (R8) for ASCs could be applied for in vivo fluorescence imaging in mice with acute liver failure, and analyzed the behavior and organ-specific accumulation of ASCs that were transplanted alone or in combination with heparin using an IVIS ® Spectrum analysis. Almost all of the transplanted ASCs were observed to accumulate in the lungs within 10 min without heparin. However, when heparin was used in combination with the ASCs, the accumulation of the transplanted ASCs was found not only in the lungs but also in the liver. The region of interest (ROI) analysis of ex vivo fluorescence imaging showed that the accumulation rate of transplanted ASCs in the liver increased to about 30%. In the time course analysis, the accumulation rate of ASCs in the liver was about 10% in 1 day and was maintained at that level for at least 2 day. We observed that heparin was effective for increasing the accumulation of transplanted ASCs in the liver using fluorescence imaging technology. We suggest that fluorescence imaging by means of QDs labeling using R8 can be useful for tracing the transplanted cells.

Influence of glioma tumour microenvironment on the transport of ANG1005 via low-density lipoprotein receptor-related protein 1

Background:ANG1005 consists of three molecules of paclitaxel conjugated via ester bonds to the 19-amino-acid peptide Angiopep-2. The new chemical agent has been shown to cross the blood–brain barrier (BBB) by receptor-mediated transcytosis via low-density lipoprotein receptor-related protein 1 (LRP1). The experiments here examined the role of LRP1 in the subsequent endocytosis of drug into cancer cells.Methods:Localisation of ANG1005 and Angiopep-2 was examined by immunohistochemistry and in-vivo near-infrared fluorescence imaging in mice carrying orthotopic glioma tumours. Transport of ANG1005 and Angiopep-2 was examined in U87 glioblastoma cell lines.Results:Systemically administered ANG1005 and Cy5.5Angiopep-2 localised to orthotopic glioma tumours in mice. The glioma transplants correlated with high expression levels of LRP1. Decreasing LRP1 activity, by RNA silencing or LRP1 competitors, decreased uptake of ANG1005 and Angiopep-2 into U87 glioblastoma cells. Conversely, LRP1 expression and endocytosis rates for ANG1005 and Angiopep-2 increased in U87 cells under conditions that mimicked the microenvironment near aggressive tumours, that is, hypoxic and acidic conditions.Conclusion:ANG1005 might be a particularly effective chemotherapeutic agent for the wide array of known LRP1-expressing brain and non-brain cancers, in particular those with an aggressive phenotype.

Initial Phase of Neuropathic Pain within a Few Hours after Nerve Injury in Mice

We tested a hypothesis that the spinal plasticity induced within a few hours after nerve injury may produce changes in cortical activities and an initial phase of neuropathic pain. Somatosensory cortical responses elicited by vibratory stimulation were visualized by transcranial flavoprotein fluorescence imaging in mice. These responses were reduced immediately after cutting the sensory nerves. However, the remaining cortical responses mediated by nearby nerves were potentiated within a few hours after nerve cutting. Nerve injury induces neuropathic pain. In the present study, mice exhibited tactile allodynia 1-2 weeks after nerve injury. Lesioning of the ipsilateral dorsal column, mediating tactile cortical responses, abolished somatic cortical responses to tactile stimuli. However, nontactile cortical responses appeared in response to the same tactile stimuli within a few hours after nerve injury, indicating that tactile allodynia was acutely initiated. We investigated the trigger mechanisms underlying the cortical changes. Endogenous glial cell line-derived neurotrophic factor (GDNF), found in the Meissner corpuscles, induced basal firing ∼0.1 Hz or less in its Aβ tactile afferents, and disruption of the basal firing triggered the potentiation of nontactile cortical responses. Application of 10 nm LY341495 [(2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid], a specific antagonist of group II metabotropic glutamate receptors (mGluRs), on to the surface of the spinal cord also induced the potentiation of nontactile cortical responses. Together, it is suggested that low-frequency afferent firing produced by GDNF in touch-sensitive nerve fibers continuously activated spinal group II mGluRs and that failure of this activation triggered tactile allodynia.