Engineering surface-enhanced Raman scattering strategies for liquid biopsy analyses: a step towards precision medicine in cancer management

Abstract

Precision medicine is poised to have an impact on cancer management by guiding treatment decisions based on molecular landscapes of tumours. Normally, the molecular profile of a tumour is established from a surgically-obtained biopsy tissue sample. As tumours are highly heterogeneous and dynamic, tissue-based molecular profiling is subject to temporal and/or spatial sampling bias. Additionally, biopsies from some tumour sites remain difficult, which could result in an inadequate amount of tissue for subsequent testing. To fully enable precision medicine, it is desirable to have an easily accessible, minimally invasive way to determine and monitor the molecular make-up of tumour subclones in real-time.Liquid biopsies are such an approach, from which a number of circulating cancer-associated biomolecules can be non-invasively isolated for subsequent analyses to better reflect the overall molecular make-ups of primary and metastatic tumours. These circulating biomolecules released by tumours including circulating tumour cells (CTCs), extracellular vesicles (EVs), circulating soluble cancer proteins, and circulating nucleic acids, are extremely rare, thus posing challenges in downstream analyses. Recent developments in microfluidic chips and nanotechnologies have significantly advanced liquid biopsy analyses. Nevertheless, these techniques are still limited by either insufficient sensitivity, low multiplexing capacity, or high-cost reagents. To enable liquid biopsy applications in precision medicine, there is thus a clear need for a highly sensitive and cost-effective technology with high multiplexing capability.The application of surface-enhanced Raman spectroscopy (SERS) as an analytical tool for liquid biopsy analyses is gaining ground due to its ultrasensitivity and multiplexing capacity. Principally, SERS refers to amplified Raman signals of molecules on or near the surface of SERS substrates (mainly plasmonic nanomaterials). According to this concept, liquid biopsy biomarkers could be identified based on their unique molecular structures or by SERS nanotags (i.e., plasmonic nanomaterials functionalised with Raman reporters and/or target-specific ligands). Majority of current SERS techniques in liquid biopsies focus on the technique development; and their clinical translation is rarely attempted/explored. The research described in this thesis includes the design and clinical evaluation of cutting-edge SERS strategies that enable comprehensive characterisation of various circulating biomolecules.This thesis first investigates the stability of SERS nanotags in biological systems by differential centrifugal sedimentation (DCS), a high-resolution particle sizing technique, given that the accuracy of SERS measurements is highly dependent on the former. Both functionalisation of antibodies to the surface of SERS nanotags and inclusion of proteins in detection environments improved the SERS nanotag stability to enable accurate, reproducible SERS measurements. This protocol to achieve optimal nanotag stability was applied in subsequent studies described in the thesis.To explore the potential of CTCs as a biomarker for treatment monitoring, a multiplex SERS strategy has been designed for CTC phenotypic analyses without the need for prior isolation. This strategy was utilised to investigate the phenotypic evolution of melanoma patient-derived cell lines and CTCs from ten stage-IV melanoma patients during treatment. The drug-resistant subclones having different CTC phenotypes of potential clinical value have been discovered, demonstrating that this CTC SERS strategy may be a promising means for treatment monitoring.As tumour-derived EVs are relatively abundant compared to CTCs in circulation and carry molecular cargoes (e.g., proteins and RNA) representative of their parental cells, an EV phenotype analyser chip was developed as an alternative treatment monitoring tool. This system encompassed a nanomixing-enhanced miniaturised EV isolation chip and the multiplex SERS readout system that offered a streamlined plasma EV phenotype analysis without any pre-isolation step. This strategy not only discriminated melanoma patients from healthy individuals but also predicted patients’ treatment responses.In addition to membrane proteins, many circulating soluble cancer proteins have shown their potential for cancer diagnosis and treatment monitoring. Thus, a new-generation SERS assay with advanced plasmonic nanomaterials and cost-efficient antibody alternatives has been invented for simultaneous, picogram level detection of multiple circulating soluble cancer protein biomarkers. This sensing platform may be adopted to further advance the strategies for CTC and EV phenotypic analyses.Lastly, a label-free SERS strategy for urinary RNA detection has been described to delineate the genetic profile of tumours, which could be complemented with those obtained through analyses of CTCs, tumour-derived EVs, and circulating soluble cancer proteins. This SERS strategy detected a panel of clinical-proven RNA biomarkers enriched separately by reverse transcription-recombinase polymerase amplification. A comprehensive clinical evaluation was performed by comparing this technique with gold standard tests in a large patient cohort to determine its clinical feasibility in prostate cancer subtyping and risk prediction.In summary, this thesis proposes novel, robust SERS strategies to address critical challenges in liquid biopsies and also provides guidance for translating liquid biopsy SERS strategies towards the clinics. It is envisioned that liquid biopsy SERS detection could progress the field of treatment monitoring, as well as cancer subtyping and risk prediction in the clinic, thus providing a more personalised treatment strategy.