Intraoperative intravital microscopy permits the study of human tumour vessels

Daniel T Fisher, Jason B Muhitch, Minhyung Kim, Kurt C Doyen, Paul N Bogner, Sharon S Evans, Joseph J Skitzki, Daniel T Fisher, Jason B Muhitch, Minhyung Kim, Kurt C Doyen, Paul N Bogner, Sharon S Evans, Joseph J Skitzki

Abstract

Tumour vessels have been studied extensively as they are critical sites for drug delivery, anti-angiogenic therapies and immunotherapy. As a preclinical tool, intravital microscopy (IVM) allows for in vivo real-time direct observation of vessels at the cellular level. However, to date there are no reports of intravital high-resolution imaging of human tumours in the clinical setting. Here we report the feasibility of IVM examinations of human malignant disease with an emphasis on tumour vasculature as the major site of tumour-host interactions. Consistent with preclinical observations, we show that patient tumour vessels are disorganized, tortuous and ∼50% do not support blood flow. Human tumour vessel diameters are larger than predicted from immunohistochemistry or preclinical IVM, and thereby have lower wall shear stress, which influences delivery of drugs and cellular immunotherapies. Thus, real-time clinical imaging of living human tumours is feasible and allows for detection of characteristics within the tumour microenvironment.

Conflict of interest statement

J.J.S. is an author on a portable IVM platform with a US patent—‘Clinical Intravital Microscope' serial No. 61/988,626 filed by Roswell Park Cancer Institute. K.C.D is an employee of Spectra Services. The remaining authors declare no competing financial interests.

Figures

Figure 1. Design of a portable intraoperative…
Figure 1. Design of a portable intraoperative IVM system and surgical exposure of tumour microvasculature.
(a) Schematic and (b) photograph of the intravital microscopy unit designed as a mobile system for observations in the OR. Critical systems allowing for stable epifluorescence observation of patient tumours in real-time are highlighted as follows: (1) × 10 objective lens (× 100 total including × 10 subjective lens), (2) fluorescent filter cube, (3) fine focus knob, (4) monitor to observe captured movie in real time, (5) cantilevered arm to extend the system over the patient, (6) heavy movement platform to control fine x–y motion of the system during observation, (7) solid granite base to reduce vibrations, (8) locking wheels, allowing mobility and stability, (9) integrated computer system to record observations and store data and (10) fluorescent light source. (c) Schematic and (d) representative photographs, detailing the surgical procedure to allow for observation of tumour microvasculature. Briefly, an incision is made in the skin overlying or adjacent to the tumour (red circle) to allow access for direct tumour observation. The epifluorescent light source is activated and digital movie recording commenced to observe the intravenous injection of fluorescein. Following observation the access site is closed and a standard oncologic resection performed.
Figure 2. Detection of blood flow in…
Figure 2. Detection of blood flow in patient tumour microvasculature.
(a) Representative photomicrographs of tumour microvasculature in patients following fluorescein injection. (b,c) Photomicrographs exported during movie analysis of blood flow velocity. (b) Arrow denotes the trailing edge of a bright fluorescein signal that was utilized to determine blood flow velocity in mm s−1. (c) Measurement of tumour diameters and blood vessel flow rate. White arrows indicate vessels that did not support blood flow during observation, black arrows denote functional vessels and white arrowheads show adipocytes. Brackets depict tumour diameter measurements of non-functional (white) and functional (black) vessels with wavy black lines demonstrating direction of blood flow and flow rate. (a–c) Bar is 100 μm.
Figure 3. Comparison of tumour vessel diameters…
Figure 3. Comparison of tumour vessel diameters by different methodologies.
(a) Individual measurements of tumour vessel diameters in patients. (b) Tumour vessel diameters quantified by human IVM and IHC of the same tumour tissue and murine melanoma tumours demonstrate consistently larger diameters detected by IVM. Established methods of estimating human tumour vessel diameters by IHC or mouse IVM produce similar measurements that both significantly underestimate diameters measured by human IVM. (c) The wall shear stress in tumour vessels is larger in murine tumours when compared with patient tumours. (d) Wall shear rate is lower in patient tumour in comparison to patient tumours. (a–d) *P<0.05l; NS, non significant (unpaired Student's t-test). n=9 for patients; n=10 for mice. Data is mean±s.e.m.
Figure 4. Vessel diameters are similar in…
Figure 4. Vessel diameters are similar in the periphery and core of tumours.
(a) Marking dye was applied following surgical resection to identify the imaged surface of tumour. Measurements of core (>200 μm from surface) and peripheral (<200 μm from surface) tumour blood vessels was performed on formalin-fixed tumour samples stained using haematoxylin and anti-CD31 mAb. Bar is 100 μm. (b) Comparison of tumour vessel diameters per field (minimum 5 fields) in the periphery (P) versus core (C) in humans and mice, NS, non significant (unpaired Student's t-test). n=3 for patients; n=5 for mice. Data is mean±s.e.m.

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