Direct real-time intra-operative imaging of human brain tumor vessels using intravital microscopy

doi:10.21203/rs.3.rs-5158886/v1

Our results show that HIVM can be used to study the anatomy and blood flow dynamics of the microvasculature of both non-infiltrated and infiltrated brain tissues intra-operatively in real-time, in patients with diverse pathologies ranging from non-cancerous and cancerous primary brain tumors to intracranial metastatic malignancies. Our experience shows that HIVM can detect areas of BBB integrity in glioblastomas, with clear flow through functioning vessels without noticeable leakage into the parenchyma, as well as areas of clear BBB disruption and leakage, in a direct demonstration of what was previously reported from MRI and PET studies. Further, in line with our prior reports in other cancer types, we demonstrate that a significant proportion of tumor vessels are non-functioning particularly in the most aggressive type of gliomas, glioblastomas. Interestingly, our series showed only functional vessels in the oligodendroglioma case, highlighting its different biological background and the need for further studies into its unique development. Collectively, this study is important for the brain tumor field as it demonstrates the feasibility of using HIVM in the operating room during brain tumor surgeries to assess real-time BBB integrity and vessel anatomical and dynamic assessment, which will enable future studies aimed at demonstrating the efficacy and feasibility of BBB opening with enabling technologies.

Neurosurgery

Brain cancer’s prognosis remains dismal despite significant strides in our collective understanding of its biology and numerous efforts towards developing new strategies to target its main growth and resistance engines.¹ This can be partly explained by the presence of the blood-brain barrier (BBB), which significantly limits the ability of drugs to penetrate the brain. While some regions of BBB disruption can be found in brain cancers, areas of intact BBB can be found even in the most aggressive of brain cancers, glioblastoma.^2,3 Therefore, numerous strategies have been developed aimed at producing more brain penetrant drugs with mixed results. This has motivated intense research into developing novel technologies to open the BBB to enable more effective drug delivery, such as focused ultrasound.^4,5 However, the ability to visualize the effect of these enabling technologies in real-time in the operating room has remained elusive thus far. We optimized an emerging technology, employing human intravital microscopy (HIVM) to analyze microvasculature in the non-infiltrated and tumor-infiltrated brain in real-time in the operating room during already occurring brain tumor surgeries. This enabled the visualization by the surgeon of areas of intact BBB and areas of disrupted BBB with fluorescein leakage into the parenchyma.

We performed a single center, non-randomized, prospective case series of real-time HIVM observation of grossly non-infiltrated cerebrum, cerebellum and brain tumors at the Mayo Clinic, Jacksonville between November 2023 and May 2024 (IRB 18-010370). Inclusion criteria included patients being submitted to open brain tumor surgeries, without known allergic reactions to fluorescein. Biopsies, both stereotactic and open as well as infection and traumatic cases were excluded. The feasibility of intra-operative use of HIVM had been established by our group in previous reports of other cancer types, including ovarian cancer and melanoma.^6,7 To enable real-time visualization, fluorescent dye fluorescein (AK-FLUOR) was administered intravenously to patients after exposure of the pertinent brain anatomy to enhance microvasculature imaging. Importantly, before administration of fluorescein, prick tests were performed in every patient to exclude allergic reactions, with any sign of allergic reaction representing an immediate exclusion criteria from further analysis or administration of fluorescein (no patients in the present study showed any allergic reaction to prick testing). After fluorescein was given, an ultra-high definition (UHD) probe-based confocal laser endomicroscopy device (pCLE; Gastroflex, Cellvizio System, Mauna Kea Technologies, Paris, France) was used under the neurosurgeon guidance to allow for observations at 100x magnification of the microvasculature in at least 2 non-contiguous regions of brain tumors and 2 non-contiguous regions of grossly normal brain. Analysis of the microvasculature was then performed using the Mauna Kea Technologies IC-Viewer.⁶ In total, HIVM was performed in 10 patients (3 males, 7 females, average age of 53.7 years) with different types of brain tumors, including meningiomas, brain metastases and gliomas, which encompass astrocytomas, oligodendrogliomas and glioblastomas (Table 1, Figure 1). Functional vessels in grossly non-infiltrated areas were found to have larger diameters and flow velocity (mean±SD) when compared with those within tumor areas in matched patients (27.8±30.1m vs.19.0±29.5m, p=0.0013 and 193.8±61.8m/s vs. 94.2±59.1m/s, p=0.0001, respectively). Given the potential therapeutic impact of modulating the microvasculature in gliomas,⁴ an analysis focusing on glioma patients was pursued. Indeed, the same was observed (32.3±34.4m vs.19.1±30.2m, p=0.0024 and 171.9±47.5m/s vs. 92.3±63.4m/s, p=0.0001 for diameters and velocity, respectively). In line with our previous findings in other cancers,^6,7 we also observed numerous non-functioning vessels (i.e., vessels that did not support blood flow), accounting for up to 50 % of all vessels evaluated in glioblastomas. Interestingly, despite the limited sample size, we observed that all vessels analyzed in the oligodendroglioma patient were functioning, which was higher than the proportion observed in other glioma types (100% vs 66.7%, p<0.0001).

Our results show that HIVM can be used to study the anatomy and blood flow dynamics of the microvasculature of both non-infiltrated and infiltrated brain tissues intra-operatively in real-time, in patients with diverse pathologies ranging from non-cancerous and cancerous primary brain tumors to intracranial metastatic malignancies. Our experience shows that HIVM can detect areas of BBB integrity in glioblastomas, with clear flow through functioning vessels without noticeable leakage into the parenchyma, as well as areas of clear BBB disruption and leakage,^2,3 in a direct demonstration of what was previously reported from MRI and PET studies. Further, in line with our prior reports in other cancer types,^6,7 we demonstrate that a significant proportion of tumor vessels are non-functioning particularly in the most aggressive type of gliomas, glioblastomas. Interestingly, our series showed only functional vessels in the oligodendroglioma case, highlighting its different biological background⁸ and the need for further studies into its unique development.

Collectively, this study is important for the brain tumor field as it demonstrates the feasibility of using HIVM in the operating room during brain tumor surgeries to assess real-time BBB integrity and vessel anatomical and dynamic assessment, which will enable future studies aimed at demonstrating the efficacy and feasibility of BBB opening with enabling technologies.

Our study was reviewed and approved by the Mayo Clinic IRB.

De Biase G, Garcia DP, Bohnen A, Quinones-Hinojosa A. Perioperative Management of Patients with Glioblastoma. Neurosurg Clin N Am. Jan 2021;32(1):1-8. doi:10.1016/j.nec.2020.09.005
Pramanik PP, Parmar HA, Mammoser AG, et al. Hypercellularity Components of Glioblastoma Identified by High b-Value Diffusion-Weighted Imaging. Int J Radiat Oncol Biol Phys. Jul 15 2015;92(4):811-9. doi:10.1016/j.ijrobp.2015.02.058
Pafundi DH, Laack NN, Youland RS, et al. Biopsy validation of 18F-DOPA PET and biodistribution in gliomas for neurosurgical planning and radiotherapy target delineation: results of a prospective pilot study. Neuro Oncol. Aug 2013;15(8):1058-67. doi:10.1093/neuonc/not002
Sonabend AM, Gould A, Amidei C, et al. Repeated blood-brain barrier opening with an implantable ultrasound device for delivery of albumin-bound paclitaxel in patients with recurrent glioblastoma: a phase 1 trial. Lancet Oncol. May 2023;24(5):509-522. doi:10.1016/S1470-2045(23)00112-2
Yuan J, Xu L, Chien CY, et al. First-in-human prospective trial of sonobiopsy in high-grade glioma patients using neuronavigation-guided focused ultrasound. NPJ Precis Oncol. Sep 16 2023;7(1):92. doi:10.1038/s41698-023-00448-y
Gabriel EM, Sukniam K, Popp K, et al. Human tumour vessel heterogeneity in ovarian cancer and its association with response to neoadjuvant chemotherapy. Clin Transl Med. Apr 2024;14(4):e1633. doi:10.1002/ctm2.1633
Fisher DT, Muhitch JB, Kim M, et al. Intraoperative intravital microscopy permits the study of human tumour vessels. Nat Commun. Feb 17 2016;7:10684. doi:10.1038/ncomms10684
Venteicher AS, Tirosh I, Hebert C, et al. Decoupling genetics, lineages, and microenvironment in IDH-mutant gliomas by single-cell RNA-seq. Science. Mar 31 2017;355(6332)doi:10.1126/science.aai8478

Table 1: Characteristics of patients undergoing brain tumor surgery with intra-operative HIVM analysis.

				Control	Tumour	Control	Tumour
ID	Age, Gender	Pathology	WHO	FV diameter – mean (SD)^a		Flow velocity – mean (SD)^a
Patient 1	60, F	GBM	IV	44.3 (39.3)	13.3 (7.2)	148.1 (23.5)	64.3 (9.7)
Patient 2	39, M	Astrocytoma	II	37.4 (54.5)	18 (5.2)	99.6 (15)	22.1 (2.6)
Patient 3	73, F	GBM	IV	25.8 (23.5)	13.5 (11.6)	182.2 (28.4)	77.3 (11.2)
Patient 4	35, M	Astrocytoma	II	41.5 (35.3)	5.9 (1.4)	229.3 (36.3)	66.9 (44)
Patient 5	30, M	Oligodendroglioma	III	34 (33.9)	24 (39.2)	166.2 (57)	225.9 (44)
Patient 6	83, F	Meningioma	II	14.4 (17.2)	n.a.	230 (40.8)	n.a.
Patient 7	30, F	Metastatic Melanoma	n.a.	24.7 (20.2)	26.6 (17)	189.6 (40.5)	66.9 (15.7)
Patient 8	60, F	GBM	IV	30.2 (26)	38.1 (28.3)	203.3 (31.5)	105.4 (18.6)
Patient 9	55, F	Meningioma	I	26 (21.6)	9.4 (2.9)	315.1 (20.3)	135 (36.2)
Patient 10	72, F	GBM	IV	12.6 (4.5)	9.6 (0.4)	174.5 (11.1)	84 (22.5)

WHO: World Health Organization; FV: Functional vessels; SD: Standard deviation; GBM: Glioblastoma; n.a.: not applicable; F: Female; M: Male; ^aThe average of functional vessel diameter was observed to be significantly higher in non-infiltrated tissues at mean (SD) diameter of 27.8m (30.1), when compared with brain tumors 19.0 m (29.5) (p=0.0013), with the same result being observed when restricting to only gliomas. Flow velocity was also found to be significantly higher in non-infiltrated tissues at an average of 193.8 m/s (61.8), when compared with brain tumor tissues at 94.2 m/s (59.1) (p=0.0001). The same results were also observed when restricting to only glioma cases.

The authors declare no competing interests.

Direct real-time intra-operative imaging of human brain tumor vessels using intravital microscopy

Status:

Version 1

Abstract

Figures

Letter

Conclusion

Declarations

References

Table

Additional Declarations

Status:

Version 1