Application of surface-enhanced Raman spectroscopy in colorectal cancer liquid biopsy analysis

Colorectal cancer (CRC) is one of the most common cancers worldwide. Since epidermal growth factor receptor (EGFR) plays an important role in the tumorigenesis and progression of CRC, the emergence of two anti-EGFR targeted monoclonal antibodies, cetuximab and panitumumab, makes an era forward to the personalized treatment of metastatic CRC (mCRC). However, the presence of activating mutations on Kirsten rat sarcoma viral oncogene (KRAS), which is the downstream effector on EGFR signaling pathway, has been identified as a strong negative predictor for response to EGFR-targeted therapy. Thus, testing of KRAS mutation status has become crucial prior to the personalized anti-EGFR therapy. In addition, comparison of the similarity and difference of cells phenotypes in response to EGFR-targeted therapy for KRAS mutant and wild-type cells will provide significant insights on the drug treatment and resistance, as well as targeting drug discovery. 

Liquid biopsy, involving the use of cell-free circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs), may offer a promising noninvasive alternative for diagnosis and for real-time monitoring of tumor evolution and therapeutic response compared to traditional tissue biopsy. However, due to the limited trace amounts of the circulating biomarkers in biofluids, the analysis of cancer biomarkers in liquid biopsy poses technical challenges to be routinely used as clinical tools. Current techniques used in the liquid biopsy include polymerase chain reaction (PCR), or next generation sequencing for analyzing nucleic acids; enzyme-linked immunosorbent assay or western blot for protein detection, and fluorescence-assisted cell sorting for circulating tumor cells analysis, none of which can meet the requirement of the sensitivity and specificity of liquid biopsy in practical detection. 

Thus, the overall aim of this thesis is to design, develop and establish new strategies for detecting the CRC-related liquid biopsy biomarkers including ctDNA mutations and CTCs by employing the surface-enhanced Raman scattering/spectroscopy (SERS), which is a vibrational spectroscopic technique for probing molecules on or near the nanoscale surface of metallic substrates. 

Firstly, we demonstrated a strategy by integrating PCR with SERS (PCR/SERS) for multiplex detection of clinically important CRC mutations (KRAS G12V, KRAS G13D and BRAF V600E) from patient plasma. The multiplex PCR/SERS assay interrogated three mutation targets per tube. The approach meets the criteria of being facile, sensitive and specific for multiplex detection of ctDNA mutations, and the results were consistent with findings from droplet digital PCR. Thus, we believe this PCR/SERS strategy is a competitive candidate for multiplex detection of ctDNA mutations in both research and clinical diagnostics. Nevertheless, the PCR/SERS assay is highly reliant upon the design and optimization of allele-specific primers and the fidelity of polymerase. 

To further improve the PCR/SERS assay for detecting single nucleotide polymorphisms (SNPs) which locate on the same or very close points on the DNA sequence (e.g. KRAS G12V and KRAS G12D), we developed a new strategy by integrating asymmetric PCR with SERS (Asy-PCR/SERS) for highly specific detection of SNPs for KRAS G12V in a simple, sensitive and specific manner. This strategy was achieved by hybridization of Raman reporter-coded and allele-specific oligonucleotide-functionalized AuNPs (SERS nanotags) with the target mutant single-stranded DNA (ssDNA) from Asy-PCR. The Asy-PCR/SERS strategy showed high specificity and sensitivity for identifying as low as 0.1% KRAS G12V mutation from non-target CRC cell line (LS174T, KRAS G12D). 

Since SERS has enabled the rapid, sensitive and non-destructive detection of the target molecules with characteristic spectra via localized surface plasmon resonances, we further analyzed the change of expression levels of CRC cell surface biomarkers in response to anti-EGFR treatment using antibody-conjugated SERS nanotags. Interestingly, we found that cetuximab-conjugated gold nanoparticles (cetuximab-AuNPs) not only improved the cytotoxicity of cancer cells, but also introduced expression change of the related biomarkers on cancer cell surface. The size-dependent cytotoxicity of cetuximab-AuNPs to CRC cell line (HT-29) indicated that AuNPs with the size of 60 nm showed the highest impact for cell cytotoxicity, which was tested by cell counting kit-8 (CCK-8) assay. Three cell surface biomarkers including epithelial cell adhesion molecule (EpCAM), melanoma cell adhesion molecule (MCAM), and human epidermal growth factor receptor 3 (HER3) were found to be expressed at higher heterogeneity when cetuximab was conjugated to AuNPs for cells treatment. 

Built on the preliminary study for analyzing the expression level changes of cell surface biomarkers (EpCAM, MCAM and HER3) of HT-29 cell line in response to cetuximab treatment, the phenotypic evolution of multiple cell surface markers (EpCAM, EGFR, HER2 and HER3) on KRAS mutant cell line (SW480) and KRAS wild-type cell line (SW48) were conducted before and during drug treatment using SERS assay and validated by flow cytometry. The results have shown SW48 cells (KRAS wild-type) are more sensitive to EGFR-targeted therapy with more significant decrease of HER2 and HER3 expression levels.  In summary, this thesis reports the application of SERS nanotags and assays for detecting CRC-related ctDNA mutations and recording the phenotypic changes (relative expression level of CRC cell surface biomarkers) in response to drug treatment. The proposed PCR/SERS assay shows great potential in multiplex, specific and sensitive detection of different ctDNA mutations in patient plasma. While, the adopting of Asy-PCR with SERS further enabled the highly specific detection of SNPs located at the same point on KRAS oncogene. Furthermore, the cytotoxicity of cetuximab-AuNPs as well the phenotypic changes of KRAS mutant and wild-type cells upon the drug treatment were investigated with multiplex SERS assay, which allows for dynamic monitoring of the phenotypic changes to indicate the cell heterogeneity and resistance, thus helps timely cease/adjust drug usage. Though the presented works are still in the early stages as proof-of-concept demonstrations, it is anticipated this thesis will send the message of developing clinically relevant SERS-based assays and highlighting the applications into liquid biopsy analysis for precision oncology.