Date Published: October 3, 2018
Publisher: Public Library of Science
Author(s): Philipp M. Kazmierczak, Neal C. Burton, Georg Keinrath, Heidrun Hirner-Eppeneder, Moritz J. Schneider, Ralf S. Eschbach, Maurice Heimer, Olga Solyanik, Andrei Todica, Maximilian F. Reiser, Jens Ricke, Clemens C. Cyran, Nikolas K. Haass.
To investigate αvβ3-integrin-targeted optoacoustic imaging and MRI for monitoring a BRAF/MEK inhibitor combination therapy in a murine model of human melanoma.
Human BRAF V600E-positive melanoma xenograft (A375)-bearing Balb/c nude mice (n = 10) were imaged before (day 0) and after (day 7) a BRAF/MEK inhibitor combination therapy (encorafenib, 1.3 mg/kg/d; binimetinib, 0.6 mg/kg/d, n = 5) or placebo (n = 5), respectively. Optoacoustic imaging was performed on a preclinical system unenhanced and 5 h after i. v. injection of an αvβ3-integrin-targeted fluorescent probe. The αvβ3-integrin-specific tumor signal was derived by spectral unmixing. For morphology-based tumor response assessments, T2w MRI data sets were acquired on a clinical 3 Tesla scanner. The imaging results were validated by multiparametric immunohistochemistry (ß3 –integrin expression, CD31 –microvascular density, Ki-67 –proliferation).
The αvβ3-integrin-specific tumor signal was significantly reduced under therapy, showing a unidirectional decline in all animals (from 7.98±2.22 to 1.67±1.30; p = 0.043). No significant signal change was observed in the control group (from 6.60±6.51 to 3.67±1.93; p = 0.500). Immunohistochemistry revealed a significantly lower integrin expression (ß3: 0.20±0.02 vs. 0.39±0.05; p = 0.008) and microvascular density (CD31: 119±15 vs. 292±49; p = 0.008) in the therapy group. Tumor volumes increased with no significant intergroup difference (therapy: +107±42 mm3; control +112±44mm3, p = 0.841). In vivo blocking studies with αvβ3-integrin antagonist cilengitide confirmed the target specificity of the fluorescent probe.
αvβ3-integrin-targeted optoacoustic imaging allowed for the early non-invasive monitoring of a BRAF/MEK inhibitor combination therapy in a murine model of human melanoma, adding molecular information on tumor receptor status to morphology-based tumor response criteria.
Overactivation of the mitogen-activated protein kinase (MAPK) signal pathway by b-rapidly accelerated fibrosarcoma (BRAF) gene mutations V600E/K leads to uncontrolled proliferation of human cells and is a central mechanism of oncogenesis in melanoma [1, 2]. Selective BRAF inhibitors (BRAFi) disrupt this oncogenic stimulus and demonstrate high initial tumor response rates in metastatic melanoma [3, 4]. However, intrinsic or acquired BRAFi resistance limits long-term tumor response to BRAFi monotherapies . One major mechanism of acquired BRAFi resistance is MAPK pathway activation by the mitogen-activated extracellular signal-regulated kinase (MEK), which may be overcome by selective MEK inhibitors (MEKi) . Dual targeting of the MAPK signal pathway by a BRAFi/MEKi combination therapy demonstrated significantly improved overall and progression-free survival in patients with advanced BRAF-mutant melanoma compared to BRAFi monotherapy . BRAFi/MEKi combination therapy is a first-line option in patients with BRAF-mutant metastatic melanoma (National Comprehensive Cancer Network Guidelines Version 1.2017, http://www.nccn.org).
The study was approved by the Government of Upper Bavaria Committee of Animal Research (Gz. ROB-55.2-2532.Vet_02-15-204) and conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All applicable institutional and/or national guidelines for the care and use of animals were followed. Every effort was taken to reduce animal suffering. We kept the mice in individually ventilated cages (n = 4 mice per cage, relative air humidity 65% at n = 18 room air changes/h, temperature 26°C, light-dark-cycle 12 h), nourished with water and small animal nutrition. Nest boxes and nestles ensured environmental enrichment. Daily animal monitoring including weighing and tumor growth measurement was conducted. Abnormal inactivity was considered an indicator of pain and was treated by analgesia (0.5 mg/kg buprenorphine s. c.). The experiments were performed under isoflurane anesthesia (2.5% in 1.0 L of 100% O2 per min for induction and 2.5% in 1.0 L of 100% O2 per min for maintenance). Humane endpoints leading to euthanization were: maximum tumor diameter >1.5 cm, tumor exulceration, weight loss >15%, apathy, defense reaction when palpating tumors, respiratory problems, paresis, non-physiological body posture.
In the present study, we investigated αvß3-integrin-targeted optoacoustic imaging and morphologic MRI for the non-invasive in vivo monitoring of a BRAFi/MEKi combination therapy in a murine model of human melanoma. Imaging was validated in and ex vivo: (1) In vivo blocking studies verified the αvß3-integrin specificity of the targeted fluorescent probe, (2) ex vivo planar whole-animal cryofluorescence imaging confirmed tumor-specific probe binding, and (3) ex vivo immunohistochemistry demonstrated a statistically significant suppression of ß3-integrin expression under therapy. In correlation with the ex vivo immunohistochemistry, the αvß3-integrin-targeted optoacoustic signal was statistically significantly reduced under therapy, while no statistically significant change was observed in the control group. Tumor volumes increased in both the therapy and the control group. This proof-of-principle study demonstrates the feasibility of optoacoustic imaging with a targeted fluorescent probe for the longitudinal, quantitative dual time point monitoring of a molecular cancer therapy in vivo, adding complementary molecular information on αvß3-integrin receptor status to morphology-based assessments of tumor response.