Euiheon Chung

The biology of brain metastases—translation to new therapies

Brain metastases are a serious obstacle in the treatment of patients with solid tumors and contribute to the morbidity and mortality of these cancers. It is speculated that the frequency of brain metastasis is increasing for several reasons, including improved systemic therapy and survival, and detection of metastases in asymptomatic patients. The lack of preclinical models that recapitulate the clinical setting and the exclusion of patients with brain metastases from most clinical trials have slowed progress. Molecular factors contributing to brain metastases are being elucidated, such as genes involved in cell adhesion, extravasation, metabolism, and cellular signaling. Furthermore, the role of the unique brain microenvironment is beginning to be explored. Although the presence and function of the blood–brain barrier in metastatic tumors is still poorly understood, it is likely that some tumor cells are protected from therapeutics by the blood–tumor barrier, creating a sanctuary site. This Review discusses what is known about the biology of brain metastases, what preclinical models are available to study the disease, and which novel therapeutic strategies are being studied in patients.

BIM expression in treatment-naïve cancers predicts responsiveness to kinase inhibitors

Cancers with specific genetic mutations are susceptible to selective kinase inhibitors. However, there is a wide spectrum of benefit among cancers harboring the same sensitizing genetic mutations. Herein, we measured apoptotic rates among cell lines sharing the same driver oncogene following treatment with the corresponding kinase inhibitor. There was a wide range of kinase inhibitor-induced apoptosis despite comparable inhibition of the target and associated downstream signaling pathways. Surprisingly, pretreatment RNA levels of the BH3-only pro-apoptotic BIM strongly predicted the capacity of EGFR, HER2, and PI3K inhibitors to induce apoptosis in EGFR-mutant, HER2-amplified, and PIK3CA-mutant cancers, respectively, but BIM levels did not predict responsiveness to standard chemotherapies. Furthermore, BIM RNA levels in EGFR-mutant lung cancer specimens predicted response and duration of clinical benefit from EGFR inhibitors. These findings suggest assessment of BIM levels in treatment-naïve tumor biopsies may indicate the degree of benefit from single-agent kinase inhibitors in multiple oncogene-addiction paradigms.

Targeting placental growth factor/neuropilin 1 pathway inhibits growth and spread of medulloblastoma

Medulloblastoma is the most common pediatric malignant brain tumor. Although current therapies improve survival, these regimens are highly toxic and are associated with significant morbidity. Here, we report that placental growth factor (PlGF) is expressed in the majority of medulloblastomas, independent of their subtype. Moreover, high expression of PlGF receptor neuropilin 1 (Nrp1) correlates with poor overall survival in patients. We demonstrate that PlGF and Nrp1 are required for the growth and spread of medulloblastoma: PlGF/Nrp1 blockade results in direct antitumor effects in vivo, resulting in medulloblastoma regression, decreased metastasis, and increased mouse survival. We reveal that PlGF is produced in the cerebellar stroma via tumor-derived Sonic hedgehog (Shh) and show that PlGF acts through Nrp1-and not vascular endothelial growth factor receptor 1-to promote tumor cell survival. This critical tumor-stroma interaction-mediated by Shh, PlGF, and Nrp1 across medulloblastoma subtypes-supports the development of therapies targeting PlGF/Nrp1 pathway.

In vivo wide-area cellular imaging by side-view endomicroscopy

In vivo imaging of small animals offers several possibilities for studying normal and disease biology, but visualizing organs with single-cell resolution is challenging. We describe rotational side-view confocal endomicroscopy, which enables cellular imaging of gastrointestinal and respiratory tracts in mice and may be extensible to imaging organ parenchyma such as cerebral cortex. We monitored cell infiltration, vascular changes and tumor progression during inflammation and tumorigenesis in colon over several months.

Combined targeting of HER2 and VEGFR2 for effective treatment of HER2-amplified breast cancer brain metastases.

Brain metastases are a serious obstacle in the treatment of patients with human epidermal growth factor receptor-2 (HER2)–amplified breast cancer. Although extracranial disease is controlled with HER2 inhibitors in the majority of patients, brain metastases often develop. Because these brain metastases do not respond to therapy, they are frequently the reason for treatment failure. We developed a mouse model of HER2-amplified breast cancer brain metastasis using an orthotopic xenograft of BT474 cells. As seen in patients, the HER2 inhibitors trastuzumab and lapatinib controlled tumor progression in the breast but failed to contain tumor growth in the brain. We observed that the combination of a HER2 inhibitor with an anti–VEGF receptor-2 (VEGFR2) antibody significantly slows tumor growth in the brain, resulting in a striking survival benefit. This benefit appears largely due to an enhanced antiangiogenic effect: Combination therapy reduced both the total and functional microvascular density in the brain xenografts. In addition, the combination therapy led to a marked increase in necrosis of the brain lesions. Moreover, we observed even better antitumor activity after combining both trastuzumab and lapatinib with the anti-VEGFR2 antibody. This triple-drug combination prolonged the median overall survival fivefold compared with the control-treated group and twofold compared with either two-drug regimen. These findings support the clinical development of this three-drug regimen for the treatment of HER2-amplified breast cancer brain metastases.

Secreted Gaussia Luciferase as a Biomarker for Monitoring Tumor Progression and Treatment Response of Systemic Metastases

Background Currently, only few techniques are available for quantifying systemic metastases in preclinical model. Thus techniques that can sensitively detect metastatic colonization and assess treatment response in real-time are urgently needed. To this end, we engineered tumor cells to express a naturally secreted Gaussia luciferase (Gluc), and investigated its use as a circulating biomarker for monitoring viable metastatic or primary tumor growth and their treatment responses. Methodology/Principal Findings We first developed orthotopic primary and metastatic breast tumors with derivative of MDA-MB-231 cells expressing Gluc. We then correlated tumor burden with Gluc activity in the blood and urine along with bioluminescent imaging (BLI). Second, we utilized blood Gluc assay to monitor treatment response to lapatinib in an experimental model of systemic metastasis. We observed good correlation between the primary tumor volume and Gluc concentration in blood (R2 = 0.84) and urine (R2 = 0.55) in the breast tumor model. The correlation deviated as a primary tumor grew due to a reduction in viable tumor fraction. This was also supported by our mathematical models for tumor growth to compare the total and viable tumor burden in our model. In the experimental metastasis model, we found numerous brain metastases as well as systemic metastases including bone and lungs. Importantly, blood Gluc assay revealed early growth of metastatic tumors before BLI could visualize their presence. Using secreted Gluc, we localized systemic metastases by BLI and quantitatively monitored the total viable metastatic tumor burden by blood Gluc assay during the course of treatment with lapatinib, a dual tyrosine kinase inhibitor of EGFR and HER2. Conclusion/Significance We demonstrated secreted Gluc assay accurately reflects the amount of viable cancer cells in primary and metastatic tumors. Blood Gluc activity not only tracks metastatic tumor progression but also serves as a longitudinal biomarker for tumor response to treatments.

Extended resolution wide-field optical imaging: objective-launched standing-wave total internal reflection fluorescence microscopy

Standing-wave total-internal-reflection fluorescence (SW-TIRF) microscopy uses a super-diffraction-limited standing evanescent wave to extract the high-spatial-frequency content of an object through a diffraction-limited optical imaging system. The effective point-spread function is better than a quarter of the emission wavelength. With a 1.45 numerical aperture objective and 532 nm excitation wavelength, a Rayleigh resolution of approximately 100 nm can be achieved, which is better than twice the resolution of conventional TIRF microscopy. This first experimental realization of SW-TIRF in an objective-launched geometry demonstrates the potential for extended resolution imaging at high speed by using wide-field microscopy.

Toward a miniature endomicroscope: pixelation-free and diffraction-limited imaging through a fiber bundle

A fiber bundle is widely used for endoscopic imaging due to its direct image delivery capability. However, there exists an inevitable pixelation artifact, which limits spatial resolution to the diameter of individual fibers. In this Letter, we present a method that can eliminate this artifact and achieve diffraction-limited spatial resolution. We exploited the binary control of a digital micromirror device to measure a transmission matrix of a fiber bundle and to subsequently control mode mixing among individual fibers. In doing so, we achieved a 22 kHz scanning rate of a diffraction-limited focused spot and obtained fluorescence endoscope imaging (58 μm × 58 μm) with near video-rate (10.3 Hz) acquisition. Our study lays a foundation for developing an ultrathin and high-resolution microendoscope.

Investigation of the point spread function of surface plasmon-coupled emission microscopy.

Surface plasmon-coupled emission microscopy (SPCEM) was proposed as a high sensitivity technique that makes use of a thin layer of metal deposited on glass slides to efficiently excite fluorophores and to collect the emission light. However, the theoretical aspect of SPCEM imaging has not been well studied. We propose a model for SPCEM and show, through theoretical analysis and empirical results, that the point spread function of SPCEM is irregular and has an annular-like structure, significantly different from the familiar point spread function of the conventional wide-field microscopy. This result is due to the highly polarized and anisotropic emission caused by the metal layer.

Spraying Quantum Dot Conjugates in the Colon of Live Animals Enabled Rapid and Multiplex Cancer Diagnosis Using Endoscopy

The detection of colon cancer using endoscopy is widely used, but the interpretation of the diagnosis is based on the clinicians naked eye. This is subjective and can lead to false detection. Here we developed a rapid and accurate molecular fluorescence imaging technique using antibody-coated quantum dots (Ab-QDs) sprayed and washed simultaneously on colon tumor tissues inside live animals, subsequently excited and imaged by endoscopy. QDs were conjugated to matrix metalloproteinases (MMP) 9, MMP 14, or carcinoembryonic antigen (CEA) Abs with zwitterionic surface coating to reduce nonspecific bindings. The Ab-QD probes can diagnose tumors on sectioned mouse tissues, fresh mouse colons stained ex vivo and also in vivo as well as fresh human colon adenoma tissues in 30 min and can be imaged with a depth of 100 μm. The probes successfully detected not only cancers that are readily discernible by bare eyes but also hyperplasia and adenoma regions. Sum and cross signal operations provided postprocessed images that can show complementary information or regions of high priority. This multiplexed quantum dot, spray-and-wash, and endoscopy approach provides a significant advantage for detecting small or flat tumors that may be missed by conventional endoscopic examinations and bestows a strategy for the improvement of cancer diagnosis.