Wearable Health Technology for Preoperative Risk Assessment in Elderly Patients: The WELCOME Study.

Preoperative identification of high-risk groups has been extensively studied to improve patients' outcomes. Wearable devices, which can track heart rate and physical activity data, are starting to be evaluated for patients' management. We hypothesized that commercial wearable devices (WD) may provide data associated with preoperative evaluation scales and tests, to identify patients with poor functional capacity at increased risk for complications. We conducted a prospective observational study including seventy-year-old patients undergoing two-hour surgeries under general anesthesia. Patients were asked to wear a WD for 7 days before surgery. WD data were compared to preoperatory clinical evaluation scales and with a 6-min walking test (6MWT). We enrolled 31 patients, with a mean age of 76.1 (SD ± 4.9) years. There were 11 (35%) ASA 3-4 patients. 6MWT results averaged 328.9 (SD ± 99.5) m. Daily steps and 𝑉𝑂2𝑚𝑎𝑥 as recorded using WD and were associated with 6MWT performance (R = 0.56, p = 0.001 and r = 0.58, p = 0.006, respectively) and clinical evaluation scales. This is the first study to evaluate WD as preoperative evaluation tools; we found a strong association between 6MWT, preoperative scales, and WD data. Low-cost wearable devices are a promising tool for the evaluation of cardiopulmonary fitness. Further research is needed to validate WD in this setting.

Frozen Section Analysis and Real-Time Magnetic Resonance Imaging of Surgical Specimen Oriented on 3D Printed Tongue Model to Assess Surgical Margins in Oral Tongue Carcinoma: Preliminary Results.

BACKGROUND: A surgical margin is the apparently healthy tissue around a tumor which has been removed. In oral cavity carcinoma, a negative margin is considered ≥ 5 mm, a close margin between 1 and 5 mm, and a positive margin ≤ 1 mm. Currently, the intraoperative surgical margin status is based on the visual inspection and tissue palpation by the surgeon and intraoperative histopathological assessment of the resection margins by frozen section analysis (FSA). FSA technique is limited and susceptible to sampling errors. Definitive information on the deep resection margins requires postoperative histopathological analysis. METHODS: We described a novel approach for the assessment of intraoperative surgical margins by examining a surgical specimen oriented through a 3D-printed specific patient tongue with real-time Magnetic Resonance Imaging (MRI). We reported the preliminary results of a case series of 10 patients, prospectively enrolled, with oral tongue carcinoma who underwent surgery between February 2020 and April 2021. Two radiologists with 5 and 10 years of experience, respectively, in Head and Neck radiology in consensus evaluated specimen MRI and measured the distance between the tumor and the specimen surface. We performed intraoperative bedside FSA. To compare the performance of bedside FSA and MRI in predicting definitive margin status we computed the weighted sensitivity (SE), specificity (SP), accuracy (ACC), area under the ROC curve (AUC), F1-score, Positive Predictive Value (PPV), and Negative Predictive Value (NPV). To express the concordance between FSA and ex-vivo MRI we reported the jaccard index. RESULTS: Intraoperative bedside FSA showed SE of 90%, SP of 100%, F1 of 95%, ACC of 0.9%, PPV of 100%, NPV (not a number), and jaccard of 90%, and ex-vivo MRI showed SE of 100%, SP of 100%, F1 of 100%, ACC of 100%, PPV of 100%, NPV of 100%, and jaccard of 100%. These results needed to be validated in a larger sample size of 21- 44 patients. CONCLUSION: The presented method allows a more accurate evaluation of surgical margin status, and the first clinical experiences underline the high potential of integrating FSA with ex-vivo MRI of the fresh surgical specimen.

An ultrasound-guided injection method for a syngeneic orthotopic murine model of breast cancer.

Breast cancer is the most common cancer among women worldwide. For high-risk women, contrast enhanced (CE)-magnetic resonance imaging (MRI) is recommended as supplemental screening together with mammography. The development of new MRI contrast agents is an active field of research, which requires efficacy tests on appropriate preclinical pathological models. In this work, a refined method to orthotopically induce breast cancer in BALB/c mice was developed using ultrasound (US) as a guide for the precise localisation of the tumour induction site and to improve animal welfare. The method was coupled with CE-MRI to characterise the evolution of the tumoural lesion.

Preliminary safety and imaging efficacy of the near-infrared fluorescent contrast agent DA364 during fluorescence-guided surgery in dogs with spontaneous superficial tumors.

Tumor-targeting contrast agents may facilitate resection of solid neoplasms during fluorescence-guided surgery. Preliminary safety and imaging efficacy of the near-infrared fluorescent probe DA364 were evaluated during surgical resection of spontaneous solid tumors in 24 dogs. Intra-operative imaging was performed in situ and on excised specimens to evaluate fluorescence intensities of tumor and adjacent tissues. After standard-of-care tumor resection, the wound bed was imaged again, and additional tissue was excised if residual fluorescence was detected. DA364 was well tolerated after intravenous administration. The median tumor-to-background ratio in situ for mammary tumors, mast cell tumors and sarcomas was 1.8 (range 1.2-3.9), 2.2 (range 1.0-5.6), and 4.2 (range 2.0-4.3), respectively. Qualitative intra-operative tumor identification was feasible in half of the cases. Remaining fluorescence was detected in four wound beds that contained residual disease, and in11 tumor-free wound beds, confirmed by histopathology. Overall, DA364 did not raise safety concerns and showed accumulation in different types of spontaneous tumors, showing potential to pinpoint residual disease. Larger clinical trials are necessary to select accurate dosing and imaging protocols for specific indications to evaluate the sensitivity and specificity of the agent.

Fluorescence-guided surgery using indocyanine green in dogs with superficial solid tumours.

BACKGROUND: Near-infrared fluorescence (NIRF) imaging is a relatively novel technique that can aid surgeons during intraoperative tumour identification. METHODS: Nine canine oncology patients (five mammary gland tumours, three mast cell tumours and one melanoma) received intravenous indocyanine green (ICG). After 24 hours, tumours were resected and fluorescence intensities of tumours and surroundings were evaluated. Additional wound bed tissue was resected if residual fluorescence was present after tumour resection. Ex vivo, fluorescence-guided dissection was performed to separate tumour from surrounding tissue. RESULTS: Intraoperative NIRF-guided tumour delineation was feasible in four out of nine dogs. Wound bed imaging after tumour removal identified nine additional fluorescent lesions, of which four contained tumour tissue. One of these four true positive in vivo lesions was missed by standard-of-care inspection. Ex vivo fluorescence-guided tumour dissection showed a sensitivity of 72 per cent and a specificity of 80 per cent in discriminating between tumour and surrounding tissue. CONCLUSION: The value of ICG for intraoperative tumour delineation seems more limited than originally thought. Although NIRF imaging using ICG did identify remaining tumour tissue in the wound bed, a high false positive rate was also observed.

Nanostructured lipid carriers accumulate in atherosclerotic plaques of ApoE-/- mice.

Nanostructured lipid carriers (NLC) might represent an interesting approach for the identification and targeting of rupture-prone atherosclerotic plaques. In this study, we evaluated the biodistribution, targeting ability and safety of 64Cu-fonctionalized NLC in atherosclerotic mice. 64Cu-chelating-NLC (51.8±3.1 nm diameter) with low dispersity index (0.066±0.016) were produced by high pressure homogenization at tens-of-grams scale. 24 h after injection of 64Cu-chelated particles in ApoE-/- mice, focal regions of the aorta showed accumulation of particles on autoradiography that colocalized with Oil Red O lipid mapping. Signal intensity was significantly greater in aortas isolated from ApoE-/- mice compared to wild type (WT) control (8.95 [7.58, 10.16]×108 vs 4.59 [3.11, 5.03]×108 QL/mm2, P < 0.05). Moreover, NLC seemed safe in relevant biocompatibility studies. NLC could constitute an interesting platform with high clinical translation potential for targeted delivery and imaging purposes in atherosclerosis.

Biodistribution of Nanostructured Lipid Carriers in Mice Atherosclerotic Model.

Atherosclerosis is a major cardiovascular disease worldwide, that could benefit from innovative nanomedicine imaging tools and treatments. In this perspective, we here studied, by fluorescence imaging in ApoE-/- mice, the biodistribution of non-functionalized and RXP470.1-targeted nanostructured lipid carriers (NLC) loaded with DiD dye. RXP470.1 specifically binds to MMP12, a metalloprotease that is over-expressed by macrophages residing in atherosclerotic plaques. Physico-chemical characterizations showed that RXP-NLC (about 105 RXP470.1 moieties/particle) displayed similar features as non-functionalized NLC in terms of particle diameter (about 60-65 nm), surface charge (about -5 - -10 mV), and colloidal stability. In vitro inhibition assays demonstrated that RXP-NLC conserved a selectivity and affinity profile, which favored MMP-12. In vivo data indicated that NLC and RXP-NLC presented prolonged blood circulation and accumulation in atherosclerotic lesions in a few hours. Twenty-four hours after injection, particle uptake in atherosclerotic plaques of the brachiocephalic artery was similar for both nanoparticles, as assessed by ex vivo imaging. This suggests that the RXP470.1 coating did not significantly induce an active targeting of the nanoparticles within the plaques. Overall, NLCs appeared to be very promising nanovectors to efficiently and specifically deliver imaging agents or drugs in atherosclerotic lesions, opening avenues for new nanomedicine strategies for cardiovascular diseases.

Microfluidic protein isolation and sample preparation for high-resolution cryo-EM.

High-resolution structural information is essential to understand protein function. Protein-structure determination needs a considerable amount of protein, which can be challenging to produce, often involving harsh and lengthy procedures. In contrast, the several thousand to a few million protein particles required for structure determination by cryogenic electron microscopy (cryo-EM) can be provided by miniaturized systems. Here, we present a microfluidic method for the rapid isolation of a target protein and its direct preparation for cryo-EM. Less than 1 µL of cell lysate is required as starting material to solve the atomic structure of the untagged, endogenous human 20S proteasome. Our work paves the way for high-throughput structure determination of proteins from minimal amounts of cell lysate and opens more opportunities for the isolation of sensitive, endogenous protein complexes.

Array based real-time measurement of fluid viscosities and mass-densities to monitor biological filament formation.

Liquid mass density and viscosity are fundamental characteristics of fluids. Their quantification by means of classical viscosity and density meters has several drawbacks: (i) the liquid-density and the viscosity cannot be measured simultaneously, (ii) sample volumes in the mL-range are consumed, (iii) the measurements cannot be multiplexed, and, (iv) the quantifications are time-consuming (minutes). Nano-mechanical transducers promise to overcome these limitations. We use fully clamped, gold coated silicon-nitride membranes with a thickness of 200 nm to measure liquid viscosity and density of samples of 1 µL volumes residing above the membrane in a miniature well. Photo-thermal actuation is used to excite the membrane, and an optical deflection system measures the response. From the response spectra, the eigenfrequency (f) and the quality (Q) factor are extracted and used to determine liquid density and viscosity by applying a three-point calibrated, simplified lumped model. We tested the system using calibrated solutions with viscosities in the range of 1-219 mPa s and mass densities between 998 kg m-3 and 1235 kg m-3. Real-time measurements were performed that characterize the polymerization of G-actin to F-actin filaments. The method presented promises to overcome the aforementioned limitations and thereby enables the real-time characterization of sub-µL sample volumes in a multiplexed manner.

Photoacoustic imaging of integrin-overexpressing tumors using a novel ICG-based contrast agent in mice.

PhotoAcoustic Imaging (PAI) is a biomedical imaging modality currently under evaluation in preclinical and clinical settings. In this work, ICG is coupled to an integrin binding vector (ICG-RGD) to combine the good photoacoustic properties of ICG and the favourable αvß3-binding capabilities of a small RGD cyclic peptidomimetic. ICG-RGD is characterized in terms of physicochemical properties, biodistribution and imaging performance. Tumor uptake was assessed in subcutaneous xenograft mouse models of human glioblastoma (U-87MG, high αvß3 expression) and epidermoid carcinoma (A431, low αvß3 expression). ICG and ICG-RGD showed high PA signal in tumors already after 15 min post-injection. At later time points the signal of ICG rapidly decreased, while ICG-RGD showed sustained uptake in U-87MG but not in A431 tumors, likely due to the integrin-mediated retention of the probe. In conclusion, ICG-RGD is a novel targeted contrast agents for PAI with superior biodistribution, tumor uptake properties and diagnostic value compared to ICG.

Global profiling of TSEC proliferative potential by the use of a reporter mouse for proliferation.

INTRODUCTION: "Tissue-selective estrogen complex" or TSEC is a novel concept of estrogen replacement therapy for the postmenopause based on the combined use of estrogens and selective estrogen receptor modulators (SERMs). The aim of this study was to exploit the potential of a novel transgenic mouse where luciferase expression is associated with cell proliferation (the MITO-luc mouse) to investigate cell proliferation in reproductive and nonreproductive tissues in mice exposed to repetitive treatments with TSEC. MATERIAL AND METHODS: Ovariectomized MITO-Luc mice were subjected to a daily oral treatment with bazedoxifene, conjugated estrogen (CE), TSEC, or raloxifene for 21 days. During the treatment, the proliferative effects of treatments were monitored by bioluminescence-based in vivo imaging. At the end of the treatment, mice were euthanized and cell proliferation assessed in selected tissues by quantitative analysis of luciferase activity and by immunohistochemistry (IHC). RESULTS: In uterus treatment with CE, but not TSEC, induced a large increase in luciferase activity underlying the proliferative effect of the hormone. No accumulation of luciferase was observed in other organs and tissues target of estrogen action. We observed an increase of Ki67 immunoreactivity only in the uterus of mice treated with CE. CONCLUSION: Pairing of an SERM with estrogens results in a complete blockage of CE proliferative effects in uterus and the absence of any undesired proliferative effects in other organs; moreover, the MITO-Luc mouse is an efficacious tool for the global, rapid, and reliable analysis of drug-induced proliferation.

Paclitaxel enhances therapeutic efficacy of the F8-IL2 immunocytokine to EDA-fibronectin-positive metastatic human melanoma xenografts.

The selective delivery of bioactive agents to tumors reduces toxicity and enhances the efficacy of anticancer therapies. In this study, we show that the antibody F8, which recognizes perivascular and stromal EDA-fibronectin (EDA-Fn), when conjugated to interleukin-2 (F8-IL2) can effectively inhibit the growth of EDA-Fn-expressing melanomas in combination with paclitaxel. We obtained curative effects with paclitaxel administered before the immunocytokine. Coadministration of paclitaxel increased the uptake of F8 in xenografted melanomas, enhancing tumor perfusion and permeability. Paclitaxel also boosted the recruitment of F8-IL2-induced natural killer (NK) cells to the tumor, suggesting a host response as part of the observed therapeutic benefit. In support of this likelihood, NK cell depletion impaired the antitumor effect of paclitaxel plus F8-IL2. Importantly, this combination reduced both the tumor burden and the number of pulmonary metastatic nodules. The combination did not cause cumulative toxicity. Together, our findings offer a preclinical proof that by acting on the tumor stroma paclitaxel potentiates the antitumor activity elicited by a targeted delivery of IL2, thereby supporting the use of immunochemotherapy in the treatment of metastatic melanoma.

E-3810 is a potent dual inhibitor of VEGFR and FGFR that exerts antitumor activity in multiple preclinical models.

Tumor angiogenesis is a degenerate process regulated by a complex network of proangiogenic factors. Existing antiangiogenic drugs used in clinic are characterized by selectivity for specific factors. Antiangiogenic properties might be improved in drugs that target multiple factors and thereby address the inherent mechanistic degeneracy in angiogenesis. Vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) family members and their cognate receptors are key players in promoting tumor angiogenesis. Here we report the pharmacologic profile of E-3810, a novel dual inhibitor of the VEGF and FGF receptors. E-3810 potently and selectively inhibited VEGF receptor (VEGFR)-1, -2, and -3 and FGF receptor (FGFR)-1 and -2 kinases in the nanomolar range. Ligand-dependent phosphorylation of VEGFR-2 and FGFR-1 was suppressed along with human vascular endothelial cell growth at nanomolar concentrations. In contrast, E-3810 lacked cytotoxic effects on cancer cell lines under millimolar concentrations. In a variety of tumor xenograft models, including early- or late-stage subcutaneous and orthotopic models, E-3810 exhibited striking antitumor properties at well-tolerated oral doses administered daily. We found that E-3810 remained active in tumors rendered nonresponsive to the general kinase inhibitor sunitinib resulting from a previous cycle of sunitinib treatment. In Matrigel plug assays performed in nude mice, E-3810 inhibited basic FGF-induced angiogenesis and reduced blood vessel density as assessed by histologic analysis. Dynamic contrast-enhanced magnetic resonance imaging analysis confirmed that E-3810 reduced the distribution of angiogenesis-sensitive contrast agents after only 5 days of treatment. Taken together, our findings identify E-3810 as a potent antiangiogenic small molecule with a favorable pharmacokinetic profile and broad spectrum antitumor activity, providing a strong rationale for its clinical evaluation.

Protease-activated receptor-1 (PAR-1) promotes the motility of human melanomas and is associated to their metastatic phenotype.

Protease-activated receptor-1 (PAR-1) is a unique G-protein-coupled receptor belonging to the protease-activated receptor family. Its activation leads to downstream signaling events that launch a variety of cellular responses related to tumor progression. PAR-1 expression has been associated to a variety of human cancers, and our previous studies reveal a high PAR-1 expression in melanoma specimens as compared to common nevi. In the present study, we investigated the contribution of PAR-1 to the malignant phenotype of human melanoma cell lines obtained from cutaneous primary lesions, capable of different metastatic behaviors in the patients from which they have been derived. We found that melanoma cells isolated from lesions giving rise to metastases in patients (WM115, WM278A, WM1361A, WM983A), had higher PAR-1 mRNA and protein expression, as compared to those obtained from lesions that did not develop metastatic disease (WM793, WM35). The cells isolated from metastatic primary lesions were able to colonize the lungs of immunodeficient SCID mice while those isolated from non-metastatic lesions were not. Additionally, cells expressing elevated PAR-1 had higher migratory and invasive abilities than those holding minimal PAR-1 expression. The migration and invasion capabilities of the melanoma cells expressing high PAR-1 were hampered by genetic and pharmacological interventions. The reduction of PAR-1 expression by siRNA and the inhibition of PAR-1 function by the SCH79797 specific antagonist significantly decreased the melanoma cell motility and invasiveness, down to an extent similar to that of the non-metastatic and low PAR-1 expressing cells. Our results provide strong evidence supporting the implication of PAR-1 in the malignant progression of human melanoma.