Intraoperative intravital microscopy permits the study of human tumour vessels.

Patient selection Patients with a visible (>0.5 cm in maximal diameter) or palpable melanoma lesion that required excision in the OR were evaluated for entry into clinical trial I 231512 ‘A Pilot Study of Feasibility of Performing Intravital Microscopy in Melanoma Patients' at Roswell Park Cancer Institute, Buffalo, NY (ClinicalTrials.gov identifier: NCT01886235 ) approved by the Institute Review Board of Roswell Park Cancer Institute, Buffalo, NY. Inclusion and exclusion criteria are listed in Supplementary Table 1 . Eligible patients who gave informed consent were enrolled in the protocol and information was recorded including demographic data (age, gender, height and weight), tumour-specific data (tumour type, location, depth from skin and stage) as well as photographs from the surgery. Although the reported risk of anaphylactic reaction to fluorescein is low (1 in 222,000), a skin-prick test was performed on all entered patients to determine potential sensitivity 27 . At least 24 h before surgery and the planned microscopic observation, the volar aspect of the patient's non-dominant forearm or opposite side of disease was prepped with an alcohol pad. Three separate areas were demarcated for testing with a marking pen and solutions consisting of 10 μl of 10% fluorescein, histamine chloride (10 mg ml −1 ; positive control) and 50% saline solution (negative control) were placed appropriately. The skin underlying each solution was individually pricked with separate and sterile Duotip lancets (Lincoln Medical, USA). After 30 min, the appearance of a wheal >3 mm in diameter was considered a positive test result in the context of the appropriate positive and negative control. If a positive test was noted to fluorescein, the patient was no longer eligible for this study.

Microscope design

Patient tumours were observed at × 100 magnification using a highly modified Olympus microscopy system. The microscope was attached to a cantilevered arm to extend over the patient. Given the high magnification required to observe individual vessels and flow, vibrations in the microscope were dampened using a weighted marble base of over 360 kg. Fluorescein was illuminated using a 467–498-nm excitation, 513–556 emission dichronic filter set (Spectra Services, Rochester, NY) with an X-Cite 120 Led light source (Lumen Dynamics, Ontario, Canada). Images were captured using a Luca EMCCD camera (Andor Technology Ltd., Belfast, Northern Ireland) controlled through the Solis acquisition and analysis programme. Images were acquired with a minimum of a 0.05-s exposure at 20 frames a second. Offline quantification of vessel diameter and blood velocity was performed using the ImageJ software suite. Surgical technique Before definitive surgical excision, melanoma tumours were exposed in a consistent manner. The skin overlying the tumour was prepped with ChloraPrep (CareFusion Corp. San Diego, California) and sterile towels were applied for exposure of the area. A curvilinear incision within the field of planned resection was created with a scalpel in line with the longitudinal axis of the patient. With care to keep the vasculature intact, the skin was elevated bluntly and retracted laterally with the use of zero silk sutures. Electrocautery was avoided as much as possible to prevent underlying tissue damage. The sutures were anchored to a retractor affixed to the OR table on the opposite side of the planned microscopic observation. For deeper melanoma tumours, a self-retaining weitlaner retractor was used following incision of the skin and underlying subcutaneous tissue. In all instances, a saline-soaked gauze was placed over top of the tissue to prevent desiccation before microscopic interface. After exposure of the underlying tumour mass, the microscope was positioned for observation. At the completion of the observation period, the microscope was withdrawn and the skin flap was closed with a running 3-0 silk suture. The area was then re-prepped for the definitive oncologic surgery including a new skin prep, sterile towels and drapes, and new gown and gloves.

Intravital microscopic observations in patients

The microscope was grossly moved into position by the surgeon over the field of interest and locked into place. Fine adjustments in the x and y axis were performed by using the built-in motor controls. Once the microscope was directly overlying the exposed tumour tissue, the fluorescent light source and image acquisition software were activated. Vertical control of the microscope was performed manually by the surgeon until tumour vessels came into view on the digital monitor. Tumour vessels were clearly recognized by their bizarre architecture, coiling, and branching patterns and observations focused on these areas. To facilitate stabilization of the images, respirations were temporarily held by anaesthesia for a maximum of 30 s. When a stable view of the vessels was achieved, 0.5 ml of 25% (250 mg ml −1 ) fluorescein (Akorn Inc., Lake Forest, Illinois) was injected via a peripheral intravenous catheter. An observation was completed when fluorescein was noted to have extravasated into the tumour tissue and the vessels no longer manifested a detectable fluorescent signal. The microscope was then re-positioned with fine motor controls to observe a different tumour area. The microscopic observations were finalized when the 2 ml of fluorescein was exhausted. IVM in murine tumours For intraoperative IVM experiments 10 6 B16/F10 melanoma cells 12 , established from mycoplasma-free frozen stocks, were injected subcutaneously in the hind leg of 8–10-week-old female C57BL/6 mice (National Cancer Institute). Experimental analysis was performed 2–3 weeks post-implantation. The skin overlying the tumour was incised and retracted with care to keep the vasculature intact. Vessels were visualized and movie recorded by the same epifluorescent intravital microscope platform used in humans. Lumenal cross-sectional diameter ( D ) of vessels was measured in offline observations of five mice. 4T-1 mammary carcinoma cells and B16/F10 melanoma cells were implanted in C57BL/6 mice (National Cancer Institute; 8–10 weeks) and EMT6 mammary carcinoma cells and CT26 colon carcinomas in BALB/c mice (National Cancer Institute; 8–10 weeks) for correlative window chamber studies as reported 12 . Animal protocols were approved by the Roswell Park Cancer Institute Institutional Animal Care and Use Committee, Buffalo, NY.

Post hoc analyses

Vessels were defined morphologically and a single vessel was described as beginning at a branch point continuing to the next branch point. To be measured, vessels had to be 100 μm in length with no branch points. Vessel density was established by enumerating vessels within a field of observation. ImageJ software was used to measure vessel diameter ( D ) and radius ( r ) at the vessel's largest width. Blood flow velocity ( v ) was evaluated by determining the time Δ T distinct features in the fluorescent dye would take to travel a known distance Δ S and then averaging ΔS /Δ T for at least 10 points per vessel. Per cent functionality was determined as mean±s.e.m. on a per field basis. Wall shear stress was computed as ( τ =32 ηQ /[ πD 3 ], where η is blood viscosity (assumed to be 2.2 centipoise 31 )) and Q is the blood flow rate ([ Q = v * πr 2 ] with r being the radius of the tumour vessel). Wall shear rate was determined using the formula ( w = v *8/ D ).

Tissue processing

Histologic sections were stained with standard haematoxylin and eosin and for CD31 (Human—1:50 dilution, Clone JC70A, Dako, Carpinteria, CA, Murine—1:20 dilution, Clone SZ31, Dianova, Hamburg, Germany). Stained sections were scanned with an Aperio Scanscope XT (Leica Microsystems Inc., Buffalo Grove, IL) and evaluated using Aperio Spectrum software. Vessel density was assessed using standard ‘hot spot' methodology 50 . For each tumour, vessels in five separate hot spot fields were counted at × 200 total magnification. In these same fields, the maximum cross-sectional diameter was measured in all vessels with an identifiable lumen. Measurement utilized the Spectrum software toolset. To distinguish vessels in the periphery versus core, a distance of 200 μm was measured from tumour edge to define a boundary and vessels measured in this region consisted of the periphery.

Statistical analyses Unpaired

Student's t -tests were utilized to examine differences between vessel diameters measured by human IVM, IHC and murine IVM, IHC, comparing each set separately using Graph Pad Prism following testing for data normality with the D'Agostino & Pearson omnibus normality test. No statistical method was used to predetermine sample size. Statistical significance for all comparisons was accepted at P