A β-Camera Integrated with a Microfluidic Chip for Radioassays Based on Real-Time Imaging of Glycolysis in Small Cell Populations

Molecular imaging tools such as PET can provide in vivo measurements of biochemical processes in tissue to reveal the status and monitor the therapeutic response of disease, for example, cancer (1). However, complicating factors such as tissue microenvironment (2), body clearance, cell heterogeneity, and technologic limitations in sensitivity and spatial resolution prohibit accurate measurements of biochemical processes in subpopulations and single cells. Alternatively, in vitro radioassays can provide a better connection to more specific cellular functions, such as glycolysis (3), which can be correlated with physiologic states of therapeutic responses. Changes in cellular metabolic state—for example, the many types of cancer cells that exhibit increased glycolysis rates, compared with normal cells—can be linked to several diseases (4,5). Current technologies for in vitro radioassays can provide high sensitivity for detection of radiotracers; however, they rely on macroscopic systems, thereby limiting the level of control for small populations or single-cell cultures (6). The use of microfluidic technologies can provide a platform for integrated, digital control of small volumes of reagents and samples suitable for bioassays of small cell populations. Recent microfluidic bioassays have demonstrated the ability to measure concentrations of multiple-signal proteins in single cells among heterogeneous populations (7), low-copy-number proteins in single cells (8), and intracellular calcium ion concentrations in single cells (9). Although many techniques are available for measuring biochemical functions in microfluidic systems, the use of radiometric methods can provide high sensitivity for small amounts of radiotracers. Furthermore, radiolabeled probes that adhere to the composition and structure of the target molecule can be readily translated to clinical applications. Thus, a microfluidic radioassay platform for measuring cellular 18F-FDG uptake can complement conventional clinical techniques such as 18F-FDG PET and enable monitoring of glycolysis in response to novel clinical therapies. Oncogenic mutations in cancer profoundly affect cellular metabolism with the activation of the Warburg effect (10), whereas oncogene inhibition with novel therapies can alter the metabolic signatures. This effect could be particularly important for the monitoring of antitumor effects of novel treatments in cancer histologies with high 18F-FDG uptake, as has been demonstrated with mutations in the mitogen-activated protein kinase pathway (11). The B-RafV600E oncogenic mutation is present in 60%–70% of melanomas and leads to uncontrolled cell growth (12,13) and increased cellular glucose metabolism (11). There are several B-Raf inhibitors in clinical development with evidence of inducing response rates in over 70% of patients with melanoma harboring the B-RafV600E mutation (14). Patients with metastatic melanoma restricted to tumors with the B-Raf oncogene have a high rate of tumor response. This was predicted in preclinical models, and the data in humans closely corroborate prior experiences in cell lines and tumor xenograph studies in mice (15–18). Patients without a response to this targeted therapy do not show a decrease in 18F-FDG uptake. Therefore, the successful implementation of these targeted therapies in patients with metastatic melanoma is critically dependent on patient stratification and monitoring of treatment course, because only patients with the mutation respond. However, current approaches based on invasive surgical biopsies are not suited for sequential target sampling and analysis. It is infrequent that patients with cancer undergo more than 1 tumor biopsy with any given treatment. Repeated tumor sampling is feasible with fine-needle aspirates, which provide single-cell suspensions amenable to ex vivo analysis using sensitive detection systems. In addition, clinical 18F-FDG PET can provide early prediction of treatment response (19). However, PET scans can be performed only every 8–12 wk in routine practice given the limitations of radiation exposure and costs. Advanced microfluid-based technologies sensitive to metabolic changes in small populations of cells obtained from fine-needle aspirates could provide a means to the sequential sampling of tumors from patients. Compared with imaging systems that rely on the detection of penetrating high-energy photons (20), charged-particle imaging (e.g., with 18F positrons) can achieve much higher detection sensitivity and spatial resolution in a compact form factor suitable for radioassays of small cell populations (21,22). Charged-particle imaging systems have typically been dedicated for imaging ex vivo tissue sections, such as in autoradiography (23–25). Less common are systems designed for in vitro applications. One system, developed by the Medipix group, used a silicon pixel array detector for in vitro imaging of 14C-l-leucine amino acid uptake in Octopus vulgaris eggs (26). Phosphor imaging plates have also been used to detect charged particles from radiolabeled peptides in microfluidic channels; however, the system required several hours of continuous exposure to produce a single image frame (27). Recent studies have used systems with a charge-coupled device camera to detect light emitted from charged particles interacting with ultra-thin phosphors (28) and from Cerenkov radiation (29). The latter work used Cerenkov radiation to image radiolabeled probes inside a microfluidic chip; however, the low sensitivity of the system and the requirement of using a light-tight box make it difficult to perform radioassays in small cell populations. This paper describes an integrated, miniaturized, in vitro radiometric imaging system, capable of measuring the glucose utilization of a small population (1–200) of cells in a real-time fashion. The radioassay system consists of a microfluidic chip for maintaining and controlling arrays of cells integrated with a β-camera for real-time imaging of charged particles emitted from radioactive sources in vitro (Fig. 1). The uptake of 18F-FDG in melanoma cell lines and primary cells in response to specific drug therapies was monitored in a controlled in vitro microfluidic environment using the β-camera, with which simultaneous measurements can be obtained from radioactive sources confined within the microfluidic chambers. The advantages of the integrated β-camera and microfluidic chip are 2-fold. The system allows for in vitro imaging of cells in a controlled microfluidic platform without major disturbance or removal of the cell cultures—in contrast to conventional radiometric methods that use well-type γ-counters or liquid scintillation counters. In addition, the integrated system is an exquisitely sensitive technology with low background, providing a significant improvement over conventional well-type γ-counters (30). FIGURE 1 Integrated β-camera and microfluidic chip for real-time radioassay imaging of glycolysis in small cell populations. (A) Schematic cross-section of β-camera integrated with microfluidic chip. (B) Micrograph of microfluidic chip loaded with ...