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Aug 29, 2022

Summary

Molecular imaging technologies facilitate cancer diagnosis and disease management and can potentially improve treatment efficacy. Among these, optical imaging technologies are predominantly used in preclinical settings and in vitro, although this approach is gaining popularity in the clinic. Optical biosensors are frequently designed using fluorochromes, as exemplified by fluorescently labeled affibodies. Nevertheless, bioluminescent biosensors may provide high sensitivity to benefit cancer diagnosis. Recently, Rodriguez de la Fuente et al. have developed a novel biosensor, which leverages the stability, small size, and high bioluminescence activity of a new luciferase from Metridia lucens copepod (MlLuc).

Background

Receptor tyrosine-protein kinase erbB-2 (HER2), a growth factor receptor family member, is commonly overexpressed or amplified in breast cancer. A therapeutic strategy in early and metastatic breast cancer leverages monoclonal antibodies for specific targeting of HER2 to inhibit downstream signaling. For instance, monoclonal antibodies such as Trastuzumab and Pertuzumab target the extracellular domain of HER2 and inhibit signaling by different mechanisms (Oh and Bang, 2020). HER2 is involved in 20-30% of breast cancers, making this protein target attractive from a therapeutic and diagnostic standpoint. Currently, HER2 expression is commonly monitored as a biomarker to evaluate the effectiveness of HER2-targeted immunotherapies in breast cancer (Rodriguez de la Fuente et al. 2022).

Molecular imaging, defined by the Society of Nuclear Medicine and Molecular Imaging as “the visualization, characterization, and measurement of biological processes at the molecular and cellular levels in humans and other living systems,” provides excellent benefits to personalized cancer therapies (Rowe and Pomper, 2021). Specifically, the ability to evaluate relevant biomarkers associated with metastatic cancer spread, such as in colorectal, brain, skin, and breast tumors, has great potential to guide therapy decisions, improve treatment efficacy, and lead to better clinical outcomes.

Non-invasive molecular imaging strategies often rely on radiolabeled tracers. Nevertheless, new approaches continue to emerge that leverage optical techniques, such as bioluminescence, fluorescence, and chemiluminescence. Optical imaging has been extensively used in preclinical models to evaluate cellular function, enable cell tracking, and for tumor targeting in vivo, among others. Applications of optical imaging techniques in humans include the use of near-infrared fluorescence and Raman spectroscopy for real-time surgical guidance in cancer patients. Nevertheless, some limitations to the widespread use of optical techniques at the clinic include the tracer’s access to tissues of interest, overall rates of uptake and clearance from tissues, and the potential for adverse effects. Additionally, the strength of the emitted signal may present limitations to optimal tracer detection (Rowe and Pomper, 2021). To tackle some of these shortcomings, Rodriguez de la Fuente et al. have developed a novel biosensor that combines affibodies' desirable properties and those of a new luciferase from Metridia lucens copepod (MlLuc) (Rodriguez de la Fuente et al. 2022).

Affibodies are highly specific, and their small molecular size (below 10 kDa) favors tissue infiltration. Rodriguez de la Fuente and colleagues reasoned that in combination with the small luciferase MlLuc, which is characterized by strong bioluminescence activity, the novel MlLuc-HER2 affibody (MlLuc-aff) could serve as a sensitive and specific tracer for HER2 detection.

Experiment

To first evaluate the activity and stability of MlLuc, Rodriguez de la Fuente et al. leveraged a pcDNA3.1(+)-MlLuc1.1 plasmid constructed by GenScript to transfect the human Uppsala 87 Malignant Glioma cell line (U87MG). Secretion of MlLuc by U87MG into the culture media enabled assaying luminescent activity over time after adding the luciferase substrate, coelenterazine. Besides function, the thermostability and pH sensitivity of MlLuc were also determined.

Next, the construct fusing the anti-HER2 affibody (ZHER2:4) and MlLuc luciferase sequences into a pET-17b vector was generated by GenScript for expression in E. coli. The fused MlLuc-ZHER2:4 or MlLuc-aff protein having a His6-tag tail was recovered from lysed cultures, reconstituted, and purified. Last, the specific binding of MlLuc-aff and associated bioluminescence were evaluated in vitro, using breast adenocarcinoma SK-BR-3 and triple-negative breast MDA-MB-231 cancer cell lines

Result

Expression of MlLuc in U87MG cells led to luciferase secretion resulting in increased bioluminescence over the period analyzed, between 15 to 75 hours following initial cell culture. Rodriguez de la Fuente et al. found that emitted light intensity was susceptible to pH conditions and peaked at pH 8.0. Additionally, the MlLuc protein was thermostable at a wide range of temperatures, from 4 to 90 ^oC.

Following expression of the fused MlLuc-affibody, Rodriguez de la Fuente et al. demonstrated its functionality by measuring bioluminescence activity throughout the purification process. Binding assays confirmed specific and high-affinity binding to HER2 receptors by leveraging high- and low- HER2 expressing cell lines, SK-BR-3, and MDA-MB-231, respectively. The authors propose that the newly developed MlLuc-ZHER2:4 meets properties desirable in an optical biosensor with potential for cancer diagnosis applications.

Reference

Rodríguez de la Fuente, L. et al. Development of a biosensor based on a new marine luciferase fused to an affibody to assess Her2 expression in living cells. Analytica Chimica Acta (2022) https://doi.org/10.1016/j.aca.2022.340084.

Oh, D. Y. & Bang, Y. J. HER2-targeted therapies — a role beyond breast cancer. Nature Reviews Clinical Oncology (2020) doi:10.1038/s41571-019-0268-3.

Rowe, S. P. & Pomper, M. G. Molecular imaging in oncology: Current impact and future directions. CA. Cancer J. Clin. (2021) doi:10.3322/caac.21713.