Selective permeabilization of the blood-brain barrier at sites of metastasis

John J Connell, Grégoire Chatain, Bart Cornelissen, Katherine A Vallis, Alastair Hamilton, Len Seymour, Daniel C Anthony, Nicola R Sibson, John J Connell, Grégoire Chatain, Bart Cornelissen, Katherine A Vallis, Alastair Hamilton, Len Seymour, Daniel C Anthony, Nicola R Sibson

Abstract

Background: Effective chemotherapeutics for primary systemic tumors have limited access to brain metastases because of the blood-brain barrier (BBB). The aim of this study was to develop a strategy for specifically permeabilizing the BBB at sites of cerebral metastases.

Methods: BALB/c mice were injected intracardially to induce brain metastases. After metastasis induction, either tumor necrosis factor (TNF) or lymphotoxin (LT) was administered intravenously, and 2 to 24 hours later gadolinium- diethylenetriaminepentaacetic acid, horseradish peroxidase, or radiolabeled trastuzumab ((111)In-BnDTPA-Tz) was injected intravenously. BBB permeability was assessed in vivo using gadolinium-enhanced T1-weighted magnetic resonance imaging and confirmed histochemically. Brain uptake of (111)In-BnDTPA-Tz was determined using in vivo single photon emission computed tomography/computed tomography. Endothelial expression of TNF receptors was determined immunohistochemically in both mouse and human brain tissue containing metastases. Group differences were analyzed with one-way analysis of variance followed by post hoc tests, Wilcoxon signed rank test, and Kruskal-Wallis with Dunn's multiple comparison test. All statistical tests were two-sided.

Results: Localized expression of TNF receptor 1 (TNFR1) was evident on the vascular endothelium associated with brain metastases. Administration of TNF or LT permeabilized the BBB to exogenous tracers selectively at sites of brain metastasis, with peak effect at 6 hours. Metastasis-specific uptake ratio of (111)In-BnDTPA-Tz was also demonstrated after systemic TNF administration vs control (0.147±0.066 vs 0.001±0.001). Human brain metastases displayed a similar TNF receptor profile compared with the mouse model, with predominantly vascular TNFR1 expression.

Conclusions: These findings describe a new approach to selectively permeabilize the BBB at sites of brain metastases to aid in detection of micrometastases and facilitate tumor-specific access of chemotherapeutic agents. We hypothesize that this permeabilization works primarily though TNFR1 activation and has the potential for clinical translation.

Figures

Figure 1.
Figure 1.
Histological detection of TNF receptors at sites of cerebral metastases in a mouse model. A and C) Tumor necrosis factor receptor 1 (TNFR1) (A: brown; C: red) is evident on blood vessels associated with metastases (arrows). B and D) Tumor necrosis factor receptor 2 (TNFR2) (B: brown, D: red) is not visible on vascular endothelium but appears to colocalize (arrows) with intravascular leukocytes (A) and infiltrating microglia (D). Coronal brain tissue sections are 20 μm. E and F) Neither receptor is present on nonmetastasis-associated vessels (defined as ≥500 μm from nearest metastasis). A, B, E, and F) 3, 3’-diaminobenzidine (DAB) immunohistochemistry with cresyl violet counterstain. C) Immunofluorescence for TNFR1 (red), glucose transporter 1(Glut-1) (blue), and 4T1–green fluorescent protein (GFP) (green). D) Immunofluorescence for TNFR2 (red), Glut-1 (blue), and 4T1-GFP (green). Scale bars are 100 μm (A–D) or 50 μm (E and F).
Figure 2.
Figure 2.
Histological detection of brain metastases and cytokine-induced blood–brain barrier (BBB) breakdown. Photomicrographs of brain metastases from mice injected systemically with horseradish perioxidase (HRP) and saline (A and D), 3 μg of lymphotoxin (LT) (B and E), or 3 μg of tumor necrosis factor (TNF) (C and F). Hanker–Yates histology for HRP detection (brown) reveals areas of BBB breakdown. Cresyl violet counterstain. G) Dose–response analysis of metastasis-specific BBB breakdown frequency 2 hour after systemic administration of differing doses of LT or TNF. H) Temporal analysis of metastasis-specific BBB breakdown frequency after systemic administration of 3 μg of LT or TNF. Data were analyzed blind to treatment group. Scale bars are 1mm (A–C) or 50 μm (D–F). Statistical analysis: two-sided 1-way analysis of variance with Dunnet post-hoc test vs saline group (*P < .05, **P < .01, ***P < .005). Error bars represent standard deviation.
Figure 3.
Figure 3.
Magnetic resonance imaging detection of cytokine-induced blood–brain barrier (BBB) breakdown. T1-weighted images from tumor necrosis factor (TNF)–treated, metastasis-bearing mouse brain before (A) and 5 minutes after (B) gadolinium-diethylenetriaminepentaacetic acid (Gd-DTPA) injection. Areas of high signal intensity (white) infer areas of BBB breakdown and Gd-DTPA entry. C) Photomicrograph of Hanker–Yates-stained tumor colony from brain slice corresponding to white box in (B). D) Region of interest (ROI; n = 14) signal intensities corresponding to histologically verified metastatic sites in lymphotoxin (LT)– and TNF-treated brains were statistically significantly higher than equivalent control ROIs. Signal intensity of metastasis ROI divided by signal intensity of equal ROI on contralateral hemisphere: ratio of 1.0 represents no breakdown. Wilcoxon signed rank test (two-sided): LT (n = 6; P = .03), TNF (n = 8; P = .008). Scale bars are 1mm (A and B) or 500 μm (C). Statistical analysis: two-sided 1-way analysis of variance with Dunnet post-hoc test vs saline group (*P < .05, **P < .01, ***P < .005). Error bars represent standard deviation.
Figure 4.
Figure 4.
Detection of 111-indium-benzyl diethylenetriaminepentaacetic acid-trastuzumab (111In-BnDTPA-Tz) using single photon emission computed tomography/computed tomography (SPECT/CT). Single slice SPECT/CT images showing localization of radiolabeled antibody (blue-pink) in mice treated with saline (A), 3 μg of lymphotoxin (LT) (B), or 3 μg of tumor necrosis factor (TNF) (C) (n = 3 each group). Intracerebral SPECT signal reveals accumulation of antibody at sites of BBB breakdown. Regions showing SPECT signal enhancement (arrows) were later confirmed to be sites of metastasis with cresyl violet histology (a–c). Note site of metastasis shown in (a) was not detected with SPECT in mouse injected systemically with saline (A; arrow). D) Ratio of radioactivity count from sum of intracerebral regions of interest (ROIs) compared with muscle ROI. Scale bars are 2mm (A–C) or 100 μm (a–c). Statistical analysis: two-sided Kruskal–Wallis with Dunn multiple comparison test (*P < .05). Error bars represent standard deviation.
Figure 5.
Figure 5.
Endothelial TNFR1 expression in human brain metastasis. Large insets are magnified images of boxed areas. A) Positive control section showing tumor necrosis factor receptor 1 (TNFR1) expression (brown) on a brain vessel adjacent to nonspecific inflammation. B) Normal brain tissue showing minimal TNFR1 reaction in cortical vessel. C and D) Staining for TNFR1 on brain parenchymal endothelial cells at sites of metastasis. Scale bars are 100 μm (A–D) or 20 μm (insets).

References

    1. Lampson LA. Monoclonal antibodies in neuro-oncology: Getting past the blood-brain barrier. MAbs. 2011;3(2):153–160.
    1. Lockman PR, Mittapalli RK, Taskar KS, et al. Heterogeneous blood-tumor barrier permeability determines drug efficacy in experimental brain metastases of breast cancer. Clin Cancer Res. 2010;16(23):5664–5678.
    1. Eichler AF, Chung E, Kodack DP, Loeffler JS, Fukumura D, Jain RK. The biology of brain metastases[mdash]translation to new therapies. Nat Rev Clin Oncol. 2011;8(6):344–356.
    1. Borlongan CV, Emerich DF. Facilitation of drug entry into the CNS via transient permeation of blood brain barrier: laboratory and preliminary clinical evidence from bradykinin receptor agonist, Cereport. Brain Res Bull. 2003;60(3):297–306.
    1. Matsukado K, Inamura T, Nakano S, Fukui M, Bartus RT, Black KL. Enhanced tumor uptake of carboplatin and survival in glioma-bearing rats by intracarotid infusion of bradykinin analog, RMP-7. Neurosurgery. 1996;39(1):125–133; discussion 133–134.
    1. Prados MD, Schold SC, Jr, Fine HA, et al. A randomized, double-blind, placebo-controlled, phase 2 study of RMP-7 in combination with carboplatin administered intravenously for the treatment of recurrent malignant glioma. Neuro-oncology. 2003;5(2):96–103.
    1. Haluska M, Anthony ML. Osmotic blood-brain barrier modification for the treatment of malignant brain tumors. Clin J Oncol Nurs. 2004;8(3):263–267.
    1. Siegal T, Rubinstein R, Bokstein F, et al. In vivo assessment of the window of barrier opening after osmotic blood-brain barrier disruption in humans. J Neurosurg. 2000;92(4):599–605.
    1. Treat LH, McDannold N, Vykhodtseva N, Zhang Y, Tam K, Hynynen K. Targeted delivery of doxorubicin to the rat brain at therapeutic levels using MRI-guided focused ultrasound. Int J Cancer. 2007;121(4):901–907.
    1. Seki T, Carroll F, Illingworth S, et al. Tumor necrosis factor-alpha increases extravasation of virus particles into tumor tissue by activating the Rho A/Rho kinase pathway. J Control Release. 2011;156(3):381–389.
    1. Ferrero E, Zocchi MR, Magni E, et al. Roles of tumor necrosis factor p55 and p75 receptors in TNF-alpha-induced vascular permeability. Am J Physiol, Cell Physiol. 2001;281(4):C1173–C1179.
    1. Schnell L, Fearn S, Schwab ME, Perry VH, Anthony DC. Cytokine-induced acute inflammation in the brain and spinal cord. J Neuropathol Exp Neurol. 1999;58(3):245–254.
    1. Sibson NR, Blamire AM, Perry VH, Gauldie J, Styles P, Anthony DC. TNF-alpha reduces cerebral blood volume and disrupts tissue homeostasis via an endothelin- and TNFR2-dependent pathway. Brain. 2002;125(Pt 11):2446–2459.
    1. Carbonell WS, Ansorge O, Sibson N, Muschel R. The vascular basement membrane as “soil” in brain metastasis. PLoS ONE. 2009;4(6):e5857.
    1. Serres S, Soto MS, Hamilton A, et al. Molecular MRI enables early and sensitive detection of brain metastases. Proc Natl Acad Sci USA. 2012;109(17):6674–6679.
    1. Aslakson CJ, Miller FR. Selective events in the metastatic process defined by analysis of the sequential dissemination of subpopulations of a mouse mammary tumor. Cancer Res. 1992;52(6):1399–1405.
    1. Cornelissen B, Hu M, McLarty K, Costantini D, Reilly RM. Cellular penetration and nuclear importation properties of 111In-labeled and 123I-labeled HIV-1 tat peptide immunoconjugates in BT-474 human breast cancer cells. Nucl Med Biol. 2007;34(1):37–46.
    1. Yoneda T, Williams PJ, Hiraga T, Niewolna M, Nishimura R. A bone-seeking clone exhibits different biological properties from the MDA-MB-231 parental human breast cancer cells and a brain-seeking clone in vivo and in vitro. J Bone Miner Res. 2001;16(8):1486–1495.
    1. Valabrega G, Montemurro F, Aglietta M. Trastuzumab: mechanism of action, resistance and future perspectives in HER2-overexpressing breast cancer. Ann Oncol. 2007; 18(6):977–84.
    1. Stolpen AH, Guinan EC, Fiers W, Pober JS. Recombinant tumor necrosis factor and immune interferon act singly and in combination to reorganize human vascular endothelial cell monolayers. Am J Pathol. 1986;123(1):16–24.
    1. Blum MS, Toninelli E, Anderson JM, et al. Cytoskeletal rearrangement mediates human microvascular endothelial tight junction modulation by cytokines. Am J Physiol. 1997;273(1 Pt 2):H286–H294.
    1. Petrache I, Birukova A, Ramirez SI, Garcia JGN, Verin AD. The role of the microtubules in tumor necrosis factor-alpha-induced endothelial cell permeability. Am J Respir Cell Mol Biol. 2003;28(5):574–581.
    1. Romer LH, McLean NV, Yan HC, Daise M, Sun J, DeLisser HM. IFN-gamma and TNF-alpha induce redistribution of PECAM-1 (CD31) on human endothelial cells. J Immunol. 1995;154(12):6582–6592.
    1. Lampugnani MG, Resnati M, Raiteri M, et al. A novel endothelial-specific membrane protein is a marker of cell-cell contacts. J Cell Biol. 1992;118(6):1511–1522.
    1. Megyeri P, Abrahám CS, Temesvári P, Kovács J, Vas T, Speer CP. Recombinant human tumor necrosis factor alpha constricts pial arterioles and increases blood-brain barrier permeability in newborn piglets. Neurosci Lett. 1992;148(1–2):137–140.
    1. Saija A, Princi P, Lanza M, Scalese M, Aramnejad E, De Sarro A. Systemic cytokine administration can affect blood-brain barrier permeability in the rat. Life Sci. 1995;56(10):775–784.
    1. Kerkar S, Williams M, Blocksom JM, Wilson RF, Tyburski JG, Steffes CP. TNF-alpha and IL-1beta increase pericyte/endothelial cell co-culture permeability. J Surg Res. 2006;132(1):40–45.
    1. Friedl J, Puhlmann M, Bartlett DL, et al. Induction of permeability across endothelial cell monolayers by tumor necrosis factor (TNF) occurs via a tissue factor-dependent mechanism: relationship between the procoagulant and permeability effects of TNF. Blood. 2002;100(4):1334–1339.
    1. Wajant H, Pfizenmaier K, Scheurich P. Tumor necrosis factor signaling. Cell Death Differ. 2003;10(1):45–65.
    1. Grell M, Douni E, Wajant H, et al. The transmembrane form of tumor necrosis factor is the prime activating ligand of the 80kDa tumor necrosis factor receptor. Cell. 1995;83(5):793–802.
    1. Blond D, Campbell SJ, Butchart AG, Perry VH, Anthony DC. Differential induction of interleukin-1beta and tumor necrosis factor-alpha may account for specific patterns of leukocyte recruitment in the brain. Brain Res. 2002;958(1):89–99.
    1. Lucas R, Garcia I, Donati YRA, et al. Both TNF receptors are required for direct TNF-mediated cytotoxicity in microvascular endothelial cells. Eur J Immunol. 1998;28(11):3577–3586.
    1. Grell M, Wajant H, Zimmermann G, Scheurich P. The type 1 receptor (CD120a) is the high-affinity receptor for soluble tumor necrosis factor. Proc Natl Acad Sci U S A. 1998;95(2):570–575.
    1. Katakami N, Inaba Y, Sugata S, et al. Magnetic resonance evaluation of brain metastases from systemic malignances with two doses of gadobutrol 1.0 m compared with gadoteridol: a multicenter, phase ii/iii study in patients with known or suspected brain metastases. Invest Radiol. 2011;46(7):411–418.
    1. Nomoto Y, Miyamoto T, Yamaguchi Y. Brain metastasis of small cell lung carcinoma: comparison of Gd-DTPA enhanced magnetic resonance imaging and enhanced computerized tomography. Jpn J Clin Oncol. 1994;24(5):258–262.
    1. Vogel CL, Cobleigh MA, Tripathy D, et al. Efficacy and safety of trastuzumab as a single agent in first-line treatment of HER2-overexpressing metastatic breast cancer. J Clin Oncol. 2002;20(3):719–726.
    1. Valabrega G, Montemurro F, Aglietta M. Trastuzumab: mechanism of action, resistance and future perspectives in HER2-overexpressing breast cancer. Ann Oncol. 2007;18(6):977–984.
    1. Pestalozzi BC, Brignoli S. Trastuzumab in CSF. J Clin Oncol. 2000;18(11):2349–2351.
    1. Stemmler H-J, Schmitt M, Willems A, Bernhard H, Harbeck N, Heinemann V. Ratio of trastuzumab levels in serum and cerebrospinal fluid is altered in HER2-positive breast cancer patients with brain metastases and impairment of blood-brain barrier. Anticancer Drugs. 2007;18(1):23–28.
    1. Bartus RT, Elliott PJ, Dean RL, et al. Controlled modulation of BBB permeability using the bradykinin agonist, RMP-7. Exp Neurol. 1996;142(1):14–28.
    1. Borlongan CV, Emerich DF, Hoffer BJ, Bartus RT. Bradykinin receptor agonist facilitates low-dose cyclosporine-A protection against 6-hydroxydopamine neurotoxicity. Brain Res. 2002;956(2):211–220.
    1. Qin L-J, Gu Y-T, Zhang H, Xue Y-X. Bradykinin-induced blood-tumor barrier opening is mediated by tumor necrosis factor-alpha. Neurosci Lett. 2009;450(2):172–175.
    1. Abbruzzese JL, Levin B, Ajani JA, et al. Phase I trial of recombinant human gamma-interferon and recombinant human tumor necrosis factor in patients with advanced gastrointestinal cancer. Cancer Res. 1989;49(14):4057–4061.
    1. Qin Z, van Tits LJ, Buurman WA, Blankenstein T. Human lymphotoxin has at least equal antitumor activity in comparison to human tumor necrosis factor but is less toxic in mice. Blood. 1995;85(10):2779–2785.
    1. Tsutsumi Y, Kihira T, Tsunoda S, et al. Molecular design of hybrid tumor necrosis factor-alpha III: polyethylene glycol-modified tumor necrosis factor-alpha has markedly enhanced antitumor potency due to longer plasma half-life and higher tumor accumulation. J Pharmacol Exp Ther. 1996;278(3):1006–1011.
    1. Ceran C, Cokol M, Cingoz S, Tasan I, Ozturk M, Yagci T. Novel anti-HER2 monoclonal antibodies: synergy and antagonism with tumor necrosis factor-α. BMC Cancer. 2012;12:450.
    1. Menart V, Fonda I, Kenig M, Porekar VG. Increased in vitro cytotoxicity of TNF-alpha analog LK-805 is based on the interaction with cell surface heparan sulfate proteoglycan. Ann N Y Acad Sci. 2002;973(11):194–206.

Source: PubMed

スポンサーと協力者

医学的状態

薬物療法

3
購読する