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Figure S1 shows fluorescence that correlates with the presence of benign lesions

Keratinocyte carcinomas, including basal and squamous cell carcinomas, are the most common human cancers worldwide. While 75% of all keratinocyte carcinoma (4 million annual cases in the United States) are treated with conventional excision, this surgical modality has much lower cure rates than Mohs micrographic surgery, likely due to the bread-loaf histopathologic assessment that visualizes ex vivo immediately following excision. “Puzzle-fit” analysis was used to correlate the fluorescent images with histology. Probe-dependent fluorescent images correlated with cancer determined by conventional histology. Point-of-care fluorescent detection of skin cancer had a clinically relevant sensitivity of 0.73 and corresponding specificity of 0.88. Importantly, clinicians were effectively trained to read fluorescent images within 15 minutes with reliability and confidence, resulting in sensitivities of 62%–78% and specificities of 92%–97%. Fluorescent imaging using 6qcNIR allows 100% tumor margin assessment by generating en face images that correlate with histology and may be used to overcome the limitations of conventional bread-loaf histology. The utility of 6qcNIR was validated in a busy real-world clinical setting, and clinicians were trained to effectively read fluorescent margins with a short guided instruction, highlighting clinical adaptability. When used in conventional excision, this approach may result in higher cure rates at a lower cost by allowing same-day reexcision when needed, reducing patient anxiety and improving compliance by expediting postsurgical specimen assessment.Significance:A fluorescent-probe-tumor-visualization platform was developed and validated in human keratinocyte carcinoma excision specimens that may provide simple, rapid, and global assessment of margins during skin cancer excision, allowing same-day reexcision when needed.

Figure S3A-C shows histology of false positive (FP) foreign body granuloma, benign inclusion cyst, and seborrheic keratosis skin samples.

Figure S2 shows histology of all false negative (FN) skin cancer samples.

Supplementary Methods include description of: 1) "Puzzle"-fit" analysis, 2) Intensity-based objective sensitivity and specificity analysis, 3) Blinded physician "Reader Study" analysis, and 4) Evaluation of reader responses regarding intensity.

Figure S3D shows histology of false positive (FP) skin samples with presence of healing area and inflammation.

Table S1 shows cancer type and histologic detection status following excision; Table S2 characterizes patients and specimens

Simple Summary: We have developed a formulation for an innovative, quenched, cathepsin-targeted, fluorescent molecular probe to enhance resection quality for several solid-tumor cancers. Unlike other formulations for imaging probes or tracers in development and entering the clinic, which require systemic administration hours before the procedure, this current formulation is applied topically into the surgical cavity immediately after a standard of care resection. Within minutes of application, the probe activates in the presence of residual cancer in the surgical wound and provides a strong fluorescent signal that precisely delineates any remaining cancer, enabling a more complete resection. Utilization of this imaging gel formulation for topical application to detect breast cancer in the surgical cavity during surgery has the potential to reduce re-excisions, with consequent savings in healthcare costs and enhancement in patient quality of life. Background: Tumor-positive surgical margins during primary breast cancer (BCa) surgery are associated with a two-fold increase in the risk of local recurrence when compared with tumor-negative margins. Pathological microscopic evaluation of the samples only assesses about 1/10 of 1% of the entire volume of the removed BCa specimens, leading to margin under-sampling and potential local recurrence in patients with pathologically clean margins, i.e., false negative margins. In the case of tumor-positive margins, patients need to undergo re-excision and/or radiation therapy, resulting in increases in complications, morbidity, and healthcare costs. Development of a simple real-time imaging technique to identify residual BCa in the surgical cavity rapidly and precisely could significantly improve the quality of care. Methods: A small-molecule, fluorescently quenched protease-substrate probe, AKRO-QC-ICG, was tested as part of a thermosensitive imaging gel formulated for topical application and imaging of the BCa surgical cavity. Results: More than forty formulations of gel mixtures were investigated to enable easy fluid application and subsequent solidification once applied, preventing dripping and pooling in the surgical cavity. The final formulation was tested using human BCa orthotopic implants in nude and NSG patient-derived xenografts (PDX) mice. This formulation of Pluronic F-127/DMSO/AKRO-QC-ICG imaging gel was found to be a good solvent for the probe, with a desirable thermo-reversible solid-gel transition and mechanical strength for distribution of AKRO-QC-ICG on the surfaces of tissue. It demonstrated excellent ability to detect BCa tissue after 10 min exposure, with a high signal-to-noise ratio both in mouse xenografts and freshly excised human lumpectomy tissue. The in vivo efficacy of the AKRO-QC-ICG imaging gel to detect BCa revealed the levels of sensitivity/specificity = 0.92/1 in 12 nude mice, which was corroborated with the sensitivity/specificity = 0.94/1 in 10 PDX mice. Conclusions: Utilization of Pluronic F-127/DMSO/AKRO-QC-ICG imaging gel for topical application to detect BCa in the surgical cavity during surgery has the potential to reduce re-excisions, with consequent savings in healthcare costs and enhancement in patient quality of life.

The majority of breast cancer patients is treated with breast-conserving surgery (BCS) combined with adjuvant radiation therapy. Up to 40% of patients has a tumor-positive resection margin after BCS, which necessitates re-resection or additional boost radiation. Cathepsin-targeted near-infrared fluorescence imaging during BCS could be used to detect residual cancer in the surgical cavity and guide additional resection, thereby preventing tumor-positive resection margins and associated mutilating treatments. The cysteine cathepsins are a family of proteases that play a major role in normal cellular physiology and neoplastic transformation. In breast cancer, the increased enzymatic activity and aberrant localization of many of the cysteine cathepsins drive tumor progression, proliferation, invasion, and metastasis. The upregulation of cysteine cathepsins in breast cancer cells indicates their potential as a target for intraoperative fluorescence imaging. This review provides a summary of the current knowledge on the role and expression of the most important cysteine cathepsins in breast cancer to better understand their potential as a target for fluorescence-guided surgery (FGS). In addition, it gives an overview of the cathepsin-targeted fluorescent probes that have been investigated preclinically and in breast cancer patients. The current review underscores that cysteine cathepsins are highly suitable molecular targets for FGS because of favorable expression and activity patterns in virtually all breast cancer subtypes. This is confirmed by cathepsin-targeted fluorescent probes that have been shown to facilitate in vivo breast cancer visualization and tumor resection in mouse models and breast cancer patients. These findings indicate that cathepsin-targeted FGS has potential to improve treatment outcomes in breast cancer patients.

Residual pit lakes from mining are often dangerous to sample for water quality. Thus, pit lakes may be rarely (or never) sampled. This study developed new technology in which water-sampling devices, mounted on an unmanned aerial vehicle (UAV), were used to sample three pit lakes in Nevada, USA, during 1 week in 2017. Water-quality datasets from two of the three pit lakes on public lands, Dexter and Clipper, are presented here. The current conditions of the Dexter pit lake were assessed by examining cation and anion concentration changes that have occurred over a 17-year period since the pit lake was last sampled in 2000. Data gathered during this sampling campaign assessed 2017 conditions of the Dexter and Clipper pit lakes by comparing constituent concentrations to the Nevada Division of Environmental Protection (NDEP) pit lake water-quality requirements, indicating that selenium concentrations exceeded regulatory standards. We compared our sampling data for Dexter lake to prior water-quality data from the Dexter pit lake collected in 1999 and 2000. This comparison for the Dexter pit lake indicates that evapoconcentration may have caused increasing cation and anion concentrations. This UAV sampling approach can potentially incorporate the use of additional multiparameter probes: pH, oxygen concentration, turbidity, or chlorophyll. Some limitations of this UAV water-sampling methodology are battery duration, weather conditions, and payload capacity.

Keratinocyte carcinomas, including basal and squamous cell carcinomas, are the most common human cancers worldwide. While 75% of all keratinocyte carcinoma (4 million annual cases in the United States) are treated with conventional excision, this surgical modality has much lower cure rates than Mohs micrographic surgery, likely due to the bread-loaf histopathologic assessment that visualizes Significance: A fluorescent-probe-tumor-visualization platform was developed and validated in human keratinocyte carcinoma excision specimens that may provide simple, rapid, and global assessment of margins during skin cancer excision, allowing same-day reexcision when needed.

Regulators often require mining companies to monitor the water quality of pit lakes during closure. Aerial drones, or unmanned aerial vehicles (UAVs), are poised to revolutionise pit lake monitoring and management by: (i) reducing risks associated with water sampling, (ii) lowering costs associated with sampling, and (iii) increasing the frequency of data acquisition. This paper demonstrates how in situ profiles of temperature and specific conductance collected by aerial drones in advance of water sampling can be used to select optimal sampling depths and to inform samplers of the physical state of the pit lake. We provide case studies of drone water sampling at two pit lakes located 295 km apart in the northwest United States. These pit lakes have similar maximum depths, latitudes, and surface elevations, and both require drone water sampling. The Montana Tunnels Pit Lake near Jefferson City, Montana is inaccessible to both foot and vehicle traffic due to previous pit wall failures. The Thompson Creek Pit Lake near Clayton, Idaho has unstable pit walls that as recently as 2016 generated a large landslide that entered the pit lake and produced a tsunami. The health and safety risks associated with future tsunamis have suspended boat-based water sampling. Both pit lakes were sampled during a three-week period in autumn 2018 when most temperate-zone lakes in North America undergo complete top-to-bottom circulation, called ‘turnover’. The aerial drone first suspended a conductivity-temperature-depth (CTD) probe capable of measuring in situ parameters to a depth of 100 m, and then suspended a water sampling device capable of collecting 2 L water samples up to 120 m deep. On 23 October 2018, in situ profiles collected in the Montana Tunnels Pit Lake showed that complete turnover had occurred and informed samplers that a minimum number of water samples would be sufficient to characterise the geochemistry of the water column. The sampling team collected three water samples from 0, 28 and 56 m depths, and subsequent lab results confirmed homogeneous conditions. State and federal regulators observed the sampling event and accepted the water samples for compliance purposes. In contrast, on 13 November at the Thompson Creek Pit Lake, in situ profiles indicated variable water chemistry with depth and the persistence of summer stratification. As a result of this complexity, samplers collected eight water samples from 3, 8, 15, 17, 36, 40, 55, and 83 m depths. In both studies, the aerial drone methods presented herein provided pit lake managers with important information about pit lake behaviour and water quality which could not have been obtained with boat-based methods owing to access and health and safety risks. These studies highlight the potential for future aerial drone water sampling applications during closure.

In an effort to increase the efficiency and cure rate of nonmelanoma skin cancer (NMSC) excisions, we have developed a point-of-care method of imaging and evaluation of skin cancer margins. We evaluate the skin surgical specimens using a smart, near-infrared probe (6qcNIR) that fluoresces in the presence of cathepsin proteases overexpressed in NMSC. Imaging is done with an inverted, flying-spot fluorescence scanner that reduces scatter, giving a 70% improved step response as compared to a conventional imaging system. We develop a scheme for careful comparison of fluorescent signals to histological annotation, which involves image segmentation, fiducial-based registration, and nonrigid free-form deformation on fluorescence images, corresponding color images, “bread-loafed” tissue images, hematoxylin and eosin (H&E)-stained slides, and pathological annotations. From epidermal landmarks, spatial accuracy in the bulk of the sample is [Formula: see text] , which when extrapolated with a linear stretch model, suggests an error at the margin of [Formula: see text] , within clinical reporting standards. Cancer annotations on H&E slides are transformed and superimposed on the fluorescence images to generate the final results. Using this methodology, fluorescence cancer signals are generally found to correspond spatially with histological annotations. This method will allow us to accurately analyze molecular probes for imaging skin cancer margins.

Background Mohs micrographic surgery examines all margins of the resected sample and has a 99% cure rate. However, many nonmelanoma skin cancers (NMSCs) are not readily amenable to Mohs micrographic surgery. This defines an unmet clinical need to assess the completeness of non-Mohs micrographic surgery resections during surgery to prevent re-excision/recurrence. Objective We sought to examine the utility of quenched activity-based probe imaging to discriminate cancerous versus normal-appearing skin tissue. Methods The quenched activity-based probe GB119 was applied to NMSC excised from 68 patients. We validated activation of the probe for hematoxylin-eosin–confirmed cancerous tissue versus normal-appearing skin tissue. Results Topical application of the probe differentiated basal cell carcinoma and squamous cell carcinoma from normal-appearing skin with overall estimated sensitivity and specificity of 0.989 (95% confidence interval 0.940-1.00) and 0.894 (95% confidence interval 0.769-0.965), respectively. Probe activation accurately defined peripheral margins of NMSC as compared with conventional hematoxylin-eosin–based pathology. Limitations This study only examined NMSC debulking excision specimens. The sensitivity and specificity for this approach using final NMSC excision margins will be clinically important. Conclusions These findings merit further studies to determine whether quenched activity-based probe technology may enable cost-effective increased cure rates for patients with NMSC by reducing re-excision and recurrence rates with a rapid and easily interpretable technological advance.