Real-time visualization of dextran extravasation in intermittent hypoxia mice using noninvasive SWIR imaging.

Imaging tools are crucial for studying the vascular network and its barrier function in various physiopathological conditions. Shortwave infrared (SWIR) window optical imaging allows noninvasive, in-depth exploration. We applied SWIR imaging, combined with vessel segmentation and deep learning analyses, to study real-time dextran probe extravasation in mice experiencing intermittent hypoxia (IH)-a characteristic of obstructive sleep apnea associated with potential cardiovascular alterations due to early vascular permeability. Evidence for permeability in this context is limited, making our investigation significant. C57Bl/6 mice were exposed to normoxia or intermittent hypoxia for 14 days. Then SWIR imaging between 1,250 and 1,700 nm was performed on the saphenous artery and vein and on the surrounding tissue after intravenous injection of labeled dextrans of two different sizes (10 or 70 kDa). Postprocessing and segmentation of the SWIR images were conducted using deep learning treatment. We monitored high-resolution signals, distinguishing arteries, veins, and surrounding tissues. In the saphenous artery and vein, after 70-kD dextran injection, tissue/vessel ratio was higher after intermittent hypoxia (IH) than normoxia (N) over 500 seconds (P < 0.05). However, the ratio was similar in N and IH after 10-kD dextran injection. The SWIR imaging technique allows noninvasive, real-time monitoring of dextran extravasation in vivo. Dextran 70 extravasation is increased after exposure to IH, suggesting an increased vessel permeability in this mice model of obstructive sleep apnea.NEW & NOTEWORTHY We demonstrate that SWIR imaging technique is a useful tool to monitor real-time dextran extravasation from vessels in vivo, with a high resolution. We report for the first time an increased real-time dextran (70 kD) extravasation in mice exposed to intermittent hypoxia for 14 days compared with normoxic controls.

Targeting Tn-Antigen-Positive Human Tumors with a Recombinant Human Macrophage Galactose C-Type Lectin.

Alterations in glycosylation cause the emergence of tumor-associated carbohydrate antigens (TACAs) during tumorigenesis. Truncation of O-glycans reveals the Thomsen nouveau (Tn) antigen, an N-acetylgalactosamine (GalNAc) frequently attached to serine or threonine amino acids, that is accessible on the surface of cancer cells but not on healthy cells. Interestingly, GalNac can be recognized by macrophage galactose lectin (MGL), a type C lectin receptor expressed in immune cells. In this study, recombinant MGL fragments were tested in vitro for their cancer cell-targeting efficiency by flow cytometry and confocal microscopy and in vivo after administration of fluorescent MGL to tumor-bearing mice. Our results demonstrate the ability of MGL to target Tn-positive human tumors without inducing toxicity. This outcome makes MGL, a fragment of a normal human protein, the first vector candidate for in vivo diagnosis and imaging of human tumors and, possibly, for therapeutic applications.

Distinction between arterial and venous occlusion with tissue oxygen pressure in a porcine fascio-cutaneous flap model.

BACKGROUND: In recent years, many devices have been developed to monitor free flaps. The Licox probe, which measures tissue oxygen pressure (PtO2 ), is one of the available devices. Our aim was to demonstrate that PtO2 could distinguish arterial from venous occlusion in a porcine fascio-cutaneous flap model. MATERIALS AND METHODS: Twenty pigs (Sus scrofa domestica, Youna strain, males) were included in this study. The median weight was 87.6 kg (84.6-90.8). Bilateral fascio-cutaneous flaps based on the superficial inferior epigastric pedicle were harvested from each pig. Thirty-eight flaps were analyzed in this study and were monitored by a Licox system during vascular occlusion. The flaps were randomized into two groups according to the clamped vessel: the arterial group (n = 19) and the venous group (n = 19). After a stabilization period of almost 40 min, vascular clamping (arterial or venous) was performed using a microvascular clamp for almost 20 min. The curve profiles were compared between arterial and venous occlusion. RESULTS: The inflection point was reached significantly faster in the arterial group: 11 min (9-16) for arterial clamping and 17 min (13-23) for venous clamping (p = .001). A total of 18/19 (95%) pigs in the arterial group and 13/19 (68%) in the venous group (p = .09) reached a level lower than 10 mmHg. The median duration for pressure to drop below 10 mmHg was 9 min (6-12) for arterial clamping and 10 min (9-16) for venous clamping (p = .06). CONCLUSION: We showed that PtO2 decreased faster in cases of arterial occlusion than in cases of venous occlusion in a pig model. Based on this observation, it may be possible to distinguish arterial from venous occlusion.

Gold nanoclusters as a contrast agent for image-guided surgery of head and neck tumors.

With the objective to evaluate the potential of ultra-small gold (Au) nanoclusters (NCs) for optical image-guided surgery, we synthesized and characterized AuNCs shelled by zwitterionic or pegylated ligands. The toxicity of the different AuNCs was evaluated on the Head and Neck Squamous Cell Carcinoma (HNSCC) CAL-33 and SQ20B cell lines in vitro. The safer AuNCs were administrated intravenously to mice for the determination of the pharmacokinetic properties. Biodistributions were performed on orthotopic CAL-33 HNSCC-bearing mice. Finally, the AuNCs were used for image-guided surgery, allowing the increase of the survival time vs. control animals, and the number of animals without any local recurrence.

Design of RGD-ATWLPPR peptide conjugates for the dual targeting of αVß3 integrin and neuropilin-1.

Targeting the tumour microenvironment is a promising strategy to detect and/or treat cancer. The design of selective compounds that co-target several receptors frequently overexpressed in solid tumours may allow a reliable and selective detection of tumours. Here we report the modular synthesis of compounds encompassing ligands of αVß3 integrin and neuropilin-1 that are overexpressed in the tumour microenvironment. These compounds were then evaluated through cellular experiments and imaging of tumours in mice. We observed that the peptide that displays both ligands is more specifically accumulating in the tumours than in controls. Simultaneous interaction with αVß3 integrin and NRP1 induces NRP1 stabilization at the cell membrane surface which is not observed with the co-injection of the controls.

Systemic Delivery of Tumor-Targeted Bax-Derived Membrane-Active Peptides for the Treatment of Melanoma Tumors in a Humanized SCID Mouse Model.

Melanoma is a highly metastatic and deadly form of cancer. Invasive melanoma cells overexpress integrin αvß3, which is a well-known target for Arg-Gly-Asp-based (RGD) peptides. We developed a sophisticated method to synthetize milligram amounts of a targeted vector that allows the RGD-mediated targeting, internalization, and release of a mitochondria-disruptive peptide derived from the pro-apoptotic Bax protein. We found that 2.5 µM Bax[109-127] was sufficient to destabilize the mitochondria in ten different tumor cell lines, even in the presence of the anti-apoptotic Bcl2 protein, which is often involved in tumor resistance. This pore-forming peptide displayed antitumor activity when it was covalently linked by a disulfide bridge to the tetrameric RAFT-c[RGD]4-platform and after intravenous injection in a human melanoma tumor model established in humanized immuno-competent mice. In addition to its direct toxic effect, treatment with this combination induced the release of the immuno-stimulating factor monocyte chimoattractant protein 1 (MCP1) in the blood and a decrease in the level of the pro-angiogenic factor FGF2. Our novel multifunctional, apoptosis-inducing agent could be further customized and assayed for potential use in tumor-targeted therapy.

"Polymultivalent" Polymer-Peptide Cluster Conjugates for an Enhanced Targeting of Cells Expressing αvß3 Integrins.

A new class of "polymultivalent" ligands combining several ligand clusters and a water-soluble biocompatible polymer is introduced. These original conjugates bear two levels of multivalency. They are prepared by covalent coupling of a controlled number of tetrameric cRGD peptide clusters along a well-defined copolymer synthesized by RAFT polymerization. The presence of multiple copies of peptide clusters on the same polymer backbone resulted in a much-higher relative potency than the free cluster reference. Thanks to the "polymultivalency", up to ∼2 orders of magnitude potency enhancement was reached in a competitive cell adhesion assay (nanomolar-range IC50 values). In addition, confocal microscopy and flow cytometry demonstrated that fluorescent "polymultivalent" conjugates (emitting in the far-red/near-infrared region) were able to specifically and selectively label cells expressing αvß3-integrin, the natural receptor of cRGD.

Targeting CD44 receptor-positive lung tumors using polysaccharide-based nanocarriers: Influence of nanoparticle size and administration route.

New approaches that are more efficient and able to specifically reach lung tumors are needed. We developed new hyaluronan-based nanoparticles targeting CD44 receptors of two different sizes and compared their lung cancer cells targeting efficacy in vitro and in vivo. The nanoparticles' cellular uptake was dose-dependent, and specific to hyaluronan receptors, particularly CD44. The binding and internalization differed according to nanoparticle size. In vivo biodistribution studies in two orthotopic lung tumor models showed that intrapulmonary nebulized nanoparticles accumulated in lungs, but not in the tumor nodules. In contrast, despite a significant liver capture, intravenous injection led to a better accumulation of the nanoparticles in the lung tumors compared with the surrounding healthy lung tissues. We demonstrated that the hyaluronan-based nanoparticles size plays significant role in cellular uptake and biodistribution. Small nanoparticles showed active targeting of CD44-overexpressing tumors, suggesting that they could be used as drug-delivery system. FROM THE CLINICAL EDITOR: Combating cancers remains an important goal in clinical medicine. In this study, the authors investigated the ability of two hyaluronan-based nanoparticles targeting CD44 receptors to home in on lung cancer cells in an in-vivo orthotropic model. The preferential uptake of smaller sized nanoparticles via intravenous route has further enhanced the existing knowledge of future drug designs.

Nebulized gadolinium-based nanoparticles: a theranostic approach for lung tumor imaging and radiosensitization.

Lung cancer is the most common and most fatal cancer worldwide. Thus, improving early diagnosis and therapy is necessary. Previously, gadolinium-based ultra-small rigid platforms (USRPs) were developed to serve as multimodal imaging probes and as radiosensitizing agents. In addition, it was demonstrated that USRPs can be detected in the lungs using ultrashort echo-time magnetic resonance imaging (UTE-MRI) and fluorescence imaging after intrapulmonary administration in healthy animals. The goal of the present study is to evaluate their theranostic properties in mice with bioluminescent orthotopic lung cancer, after intrapulmonary nebulization or conventional intravenous administration. It is found that lung tumors can be detected non-invasively using fluorescence tomography or UTE-MRI after nebulization of USRPs, and this is confirmed by histological analysis of the lung sections. The deposition of USRPs around the tumor nodules is sufficient to generate a radiosensitizing effect when the mice are subjected to a single dose of 10 Gy conventional radiation one day after inhalation (mean survival time of 112 days versus 77 days for irradiated mice without USRPs treatment). No apparent systemic toxicity or induction of inflammation is observed. These results demonstrate the theranostic properties of USRPs for the multimodal detection of lung tumors and improved radiotherapy after nebulization.

Tumor-associated macrophages confer resistance to chemotherapy (Trifluridine/Tipiracil) in digestive cancers by overexpressing thymidine phosphorylase.

Pyrimidine analogs are part of the first-line chemotherapy regimen for gastrointestinal cancers. Trifluridine combined with tipiracil, a specific thymidine phosphorylase inhibitor, in TAS-102 has recently emerged as a potential alternative in the face of primary or secondary chemoresistance to 5-fluorouracil. Despite its promise, we report that macrophage-specific overexpression of thymidine phosphorylase results in macrophage-induced chemoresistance to TAS-102 that is insensitive to tipiracil inhibition. Furthermore, we illustrate the human-specific nature of this mechanism, as mouse macrophages do not express substantial levels of thymidine phosphorylase, which constrains the applicability of mouse models. To study the importance of macrophages in chemoresistance to trifluridine, we developed a humanized mouse model with tumor-implanted human macrophages and demonstrated their important role in treatment resistance to pyrimidine analogs. Additionally, our findings revealed that macrophages represent a significant source of thymidine phosphorylase expression, comprising over 40 % of the expressing cells, in human colorectal cancer, thereby contributing to chemoresistance.

Engineering Radiocatalytic Nanoliposomes with Hydrophobic Gold Nanoclusters for Radiotherapy Enhancement.

Chemoradiation therapy is on the forefront of pancreatic cancer care, and there is a continued effort to improve its safety and efficacy. Liposomes are widely used to improve chemotherapy safety, and may accurately deliver high-Z element- radiocatalytic nanomaterials to cancer tissues. In this study, the interaction between X-rays and long-circulating nanoliposome formulations loaded with gold nanoclusters is explored in the context of oxaliplatin chemotherapy for desmoplastic pancreatic cancer. Hydrophobic gold nanoclusters stabilized with dodecanethiol (AuDDT) are efficiently incorporated in nanoliposomal bilayers. AuDDT-nanoliposomes significantly augmented radiation-induced •OH production, which is most effective with monochromatic X-rays at energies that exceed the K-shell electron binding energy of Au (81.7 keV). Cargo release assays reveal that AuDDT-nanoliposomes can permeabilize lipid bilayers in an X-ray dose- and formulation-dependent manner. The radiocatalytic effect of AuDDT-nanoliposomes significantly augments radiotherapy and oxaliplatin-chemoradiotherapy outcomes in 3D pancreatic microtumors. The PEGylated AuDDT-nanoliposomes display high tumor accumulation in an orthotopic mouse model of pancreatic cancer, showing promise for nanoliposomes as carriers for radiocatalytic nanomaterials. Altogether, compelling proof for chemo-radiation dose-enhancement using AuDDT-nanoliposomes is presented. Further improving the nanoliposomal loading of high-Z elements will advance the safety, efficacy, and translatability of such chemoradiation dose-enhancement approaches.

Stimuli-Responsive Polymer Nanoprobes Intended for Fluorescence-Guided Surgery of Malignant Head-and-Neck Tumors and Metastases.

Nano-sized carriers are widely studied as suitable candidates for the advanced delivery of various bioactive molecules such as drugs and diagnostics. Herein, the development of long-circulating stimuli-responsive polymer nanoprobes tailored for the fluorescently-guided surgery of solid tumors is reported. Nanoprobes are designed as long-circulating nanosystems preferably accumulated in solid tumors due to the Enhanced permeability and retention effect, so they act as a tumor microenvironment-sensitive activatable diagnostic. This study designs polymer probes differing in the structure of the spacer between the polymer carrier and Cy7 by employing pH-sensitive spacers, oligopeptide spacers susceptible to cathepsin B-catalyzed enzymatic hydrolysis, and non-degradable control spacer. Increased accumulation of the nanoprobes in the tumor tissue coupled with stimuli-sensitive release behavior and subsequent activation of the fluorescent signal upon dye release facilitated favorable tumor-to-background ratio, a key feature for fluorescence-guided surgery. The probes show excellent diagnostic potential for the surgical removal of intraperitoneal metastasis and orthotopic head and neck tumors with very high efficacy and accuracy. In addition, the combination of macroscopic resection followed by fluorescence-guided surgery using developed probes enable the identification and resection of most of the CAL33 intraperitoneal metastases with total tumor burden reduced to 97.2%.

Optimization of spatial resolution and scattering effects for biomedical fluorescence imaging by using sub-regions of the shortwave infrared spectrum.

We evaluated the impact of light-scattering effects on spatial resolution in different shortwave infrared (SWIR) sub-regions by analyzing two SWIR emissive phantoms made of polydimethylsiloxane (PDMS)-gold nanoclusters (Au NCs) composite covered with mice skin, or capillary tubes filled with Au NCs or IRDye 800CW at different depth in intralipids and finally, after administration of the Au NCs intravenously in mice. Our findings highlighted the benefit of working at the highest tested spectral range of the SWIR region with a 50% enhancement of spatial resolution measured in artificial model when moving from NIR-II (1000-1300 nm) to NIR-IIa (1300-1450 nm) region, and a 25% reduction of the scattering from the skin determined by point spread function analysis from the NIR-II to NIR-IIb region (1500-1700 nm). We also confirmed that a series of Monte Carlo restoration of images significantly improved the spatial resolution in vivo in mice in deep tissues both in the NIR-II and NIR-IIa spectral windows.

Chromophore reconstruction at depth in bilayered media: a method for quantification.

We report a method for deriving the absolute value of absorption coefficients at depth in bilayered media. The method was simplified from that of time-resolved diffuse optical tomography (TR-DOT) into one dimension to validate and set up the main parameters with the help of simulations, and to test it in an easy preclinical model. The method was applied to buried flaps as used in reconstructive surgery, and absolute chromophore concentrations in the flap and in the upper (skin and fat) layer were derived. The encouraging results obtained lay a foundation for developing more complex multidimensional models.

Noninvasive Monitoring of Deep Tissue Oxygenation in Buried Flaps by Time-Resolved Near-Infrared Spectroscopy in Pigs.

BACKGROUND: Flap monitoring in reconstructive surgery is particularly important because flap failure is a dramatic event for the patient and for the medical team. Noninvasive deep tissue oxygenation monitoring is a challenge. The aim of this experimental study was to assess the performance of time-resolved near-infrared spectroscopy compared with continuous-wave near-infrared spectroscopy and with invasive oxygen partial pressure measurement in pigs. METHODS: Thirty fasciocutaneous flaps based on the superficial epigastric inferior pedicle were harvested and buried under the transcutaneous dorsal muscle (approximately 1 cm thick). An optical probe was placed on the skin above each buried flap. For each pig, two buried flaps were performed, one submitted to arterial occlusion and one to venous occlusion. Oxyhemoglobin and deoxyhemoglobin concentrations were observed for over 40 minutes before clamping, almost 20 minutes during clamping and during a period of release of approximately 20 minutes. Variations in time-resolved near-infrared spectroscopy were compared to the oxygen partial pressure and continuous-wave near-infrared spectroscopy variations. RESULTS: All vascular events were detected by the time-resolved near-infrared spectroscopy. During arterial clamping, oxyhemoglobin decreased rapidly, whereas deoxyhemoglobin increased moderately. The divergence of oxyhemoglobin and deoxyhemoglobin curves indicated arterial occlusion. During venous clamping, deoxyhemoglobin increased, whereas oxyhemoglobin increased briefly then remained stable or decreased moderately. The initial increases in the oxyhemoglobin and deoxyhemoglobin curves indicated venous occlusion. Oxygen partial pressure failed to detect vascular events in three cases. Continuous-wave near-infrared spectroscopy could not clearly identify vascular occlusions. CONCLUSIONS: Thus, the authors demonstrated the relevance of time-resolved near-infrared spectroscopy to buried flap monitoring. Time-resolved near-infrared spectroscopy could differentiate between arterial occlusion and venous occlusion.

Noninvasive monitoring of liver metastasis development via combined multispectral photoacoustic imaging and fluorescence diffuse optical tomography.

Rationale:In vivo molecular imaging in preclinical animal models is a tool of choice for understanding the pathophysiological mechanisms involved in cancer development and for conducting drug development research. Moreover, combining several imaging modalities can provide multifaceted, complementary and cross-validated information. Photoacoustic imaging (PAI) is a promising imaging modality that can reflect blood vasculature and tissue oxygenation as well as detect exogenous molecules, but one shortcoming of PAI is a lack of organic photoacoustic contrast agents capable of providing tumor contrast. Methods: In the present study, we designed an animal model of liver metastases from colon cancer and monitored metastasis development by in vivo bioluminescence and X-ray microcomputed tomography. Contrast-agent-free PAI was used to detect the respective amounts of oxy- and deoxyhemoglobin and, thus, liver tissue oxygenation. two contrast agents, Angiostamp800 and indocyanin green (ICG), respectively with and without tumor targeting specificity, were then evaluated for their dual fluorescence and photoacoustic detectability and were then used for combined PAI and fluorescence diffuse optical tomography (fDOT) at various disease development stages. Findings: Contrast-agent-free PAI reflected tumor angiogenesis and gradual hypoxia during metastasis development. Multispectral PAI enabled noninvasive real-time monitoring of ICG blood pharmacokinetics, which demonstrated tumor-related liver dysfunction. Both PAI and fluorescence ICG signals were clearly modified in metastasis-bearing livers but did not allow for differentiation between different disease stages. In contrast, there was a significant improvement achieved by using the tumor-specific marker Angiostamp800, which provided gradually increasing PAI and fDOT signals during metastasis development. Conclusion: We demonstrated for the first time the value of using Angiostamp800 as a bimodal tumor-targeting contrast agent for combined PAI and fluorescence imaging of liver metastasis progression in vivo.

High-Resolution Shortwave Infrared Imaging of Vascular Disorders Using Gold Nanoclusters.

We synthesized a generation of water-soluble, atomically precise gold nanoclusters (Au NCs) with anisotropic surface containing a short dithiol pegylated chain (AuMHA/TDT). The AuMHA/TDT exhibit a high brightness (QY ∼ 6%) in the shortwave infrared (SWIR) spectrum with a detection above 1250 nm. Furthermore, they show an extended half-life in blood (t1/2ß = 19.54 ± 0.05 h) and a very weak accumulation in organs. We also developed a non-invasive, whole-body vascular imaging system in the SWIR window with high-resolution, benefiting from a series of Monte Carlo image processing. The imaging process enabled to improve contrast by 1 order of magnitude and enhance the spatial resolution by 59%. After systemic administration of these nanoprobes in mice, we can quantify vessel complexity in depth (>4 mm), allowing to detect very subtle vascular disorders non-invasively in bone morphogenetic protein 9 (Bmp9)-deficient mice. The combination of these anisotropic surface charged Au NCs plus an improved SWIR imaging device allows a precise mapping at high-resolution and an in depth understanding of the organization of the vascular network in live animals.

The pyrrolopyrimidine colchicine-binding site agent PP-13 reduces the metastatic dissemination of invasive cancer cells in vitro and in vivo.

Standard chemotherapies that interfere with microtubule dynamics are a chemotherapeutic option used for the patients with advanced malignancies that invariably relapse after targeted therapies. However, major efforts are needed to reduce their toxicity, optimize their efficacy, and reduce cancer chemoresistance to these agents. We previously identified a pyrrolo[2,3d]pyrimidine-based microtubule-depolymerizing agent (PP-13) that binds to the colchicine site of ß-tubulin and exhibits anticancer properties in solid human cancer cells, including chemoresistant subtypes. Here, we investigated the therapeutic potential of PP-13 in vitro and in vivo. PP-13 induced a mitotic blockade and apoptosis in several cancer cells cultured in two-dimensions or three-dimensions spheroids, in conjunction with reduced cell proliferation. Capillary-like tube formation assays using HUVECs showed that PP-13 displayed antiangiogenic properties. It also inhibited cancer cell motility and invasion, in in vitro wound-healing and transwell migration assays. Low concentration PP-13 (130 nmol.L-1) treatment significantly reduced the metastatic invasiveness of human cancer cells engrafts on chicken chorioallantoic membrane. In nude mice, 0.5 or 1 mg.kg-1 PP-13 intraperitoneally administered three-times a week reduced the sizes of paclitaxel-refractory orthotopic breast tumors, delayed the progression of metastasis, and decreased the global metastatic load compared to 0.5 mg.kg-1 paclitaxel or vehicle alone. PP-13 did not show any apparent early adverse effect in vivo. These data suggest that PP-13 is a promising alternative to standard chemotherapy in antimitotic drug-refractory tumors, especially through its impact on metastasis.

Verteporfin-Loaded Lipid Nanoparticles Improve Ovarian Cancer Photodynamic Therapy In Vitro and In Vivo.

Advanced ovarian cancer is the most lethal gynecological cancer, with a high rate of chemoresistance and relapse. Photodynamic therapy offers new prospects for ovarian cancer treatment, but current photosensitizers lack tumor specificity, resulting in low efficacy and significant side-effects. In the present work, the clinically approved photosensitizer verteporfin was encapsulated within nanostructured lipid carriers (NLC) for targeted photodynamic therapy of ovarian cancer. Cellular uptake and phototoxicity of free verteporfin and NLC-verteporfin were studied in vitro in human ovarian cancer cell lines cultured in 2D and 3D-spheroids, and biodistribution and photodynamic therapy were evaluated in vivo in mice. Both molecules were internalized in ovarian cancer cells and strongly inhibited tumor cells viability when exposed to laser light only. In vivo biodistribution and pharmacokinetic studies evidenced a long circulation time of NLC associated with efficient tumor uptake. Administration of 2 mg.kg-1 free verteporfin induced severe phototoxic adverse effects leading to the death of 5 out of 8 mice. In contrast, laser light exposure of tumors after intravenous administration of NLC-verteporfin (8 mg.kg-1) significantly inhibited tumor growth without visible toxicity. NLC-verteporfin thus led to efficient verteporfin vectorization to the tumor site and protection from side-effects, providing promising therapeutic prospects for photodynamic therapy of cancer.