Novel applications of liquid Biopsy: Comprehensive methodology for circulating biomarker exploration in peripheral blood

Abstract

The liquid biopsy (LB) represents a minimally invasive method for cancer screening that has been introduced in clinical practice for over a decade and that can accelerate treatment response assessment. LB allows the analysis of tumor cells or tumor-derived products (e.g. cell-free circulating nucleic acids, extracellular vesicles, and proteins) released from primary or metastatic tumor lesions into blood or other body fluids. In the era of immune-oncology, recent evidence indicates that tumor-specific immune responses can be detected in peripheral immune cells. The improvement of knowledge and the standardization of the isolation methods of these techniques will allow the detection and characterization of circulating tumor and immune biomarkers at an early stage as innovative tools to predict response to therapies. Nowadays, the analysis of peripheral blood mononuclear cells (PBMCs), circulating tumor cells (CTCs), peripheral blood-derived extracellular vesicles (EVs) and circulating tumor RNA (ctRNA) remains under-developed even if these non-invasive techniques can provide the complete genetic landscape of tumors and allow systematic tracking of cancer evolution. In addition, the evaluation of blood circulating cytokines, and early dynamics changes in the PBMCs of patients with solid tumors represent a promising area of research. Here, we present a comprehensive methodological framework for the evaluation of innovative peripheral blood-derived biomarkers. We also address the current challenges in isolation methods and analysis of PBMC, CTC, EVs and TEPs which are crucial for structuring the large amount of comprehensive information obtained from such samples, with the aim of advancing the translational cancer field.

Keywords: Circulating tumor cells; Exosomes; Liquid biopsy; Peripheral blood immune cells; Tumor-educated platelets.

Graphical abstract

Fig. 1

(A) Cell viability of the PBMCs from naive, post-chemotherapy, and post-immunotherapy patients after 24 h of culture in a serum-free medium or in a medium containing different percentages (1 %, 5 % and 10 %) of FBS or HS. Data are expressed as mean ± SD derived from n = 4 technical. One-tailed unpaired Student's t-test with CI = 95 %, ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001. (B) FACS analysis of SCLC patients-derived PBMCs subset. (C) Real time PCR analysis of in vitro IL8, IL12, IL6, IL10, IL1β, IL2 and IL4 mRNA expression in SCLC patients-derived PBMCs collector before (T0) and after (T1) receiving chemotherapy treatment. Changes in mRNA levels were normalized to the expression of housekeeping genes (18s). Data are expressed as means ± SEM derived from n = 2 technically calculated by the comparative method 2-ΔΔCt. One-tailed unpaired Student's t-test with CI = 95 %, ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗∗p < 0.05.

Fig. 2

(A) Representative immunofluorescence images of CTCs with different extraction methods. Co-localization of STING (green, Alexa Fluor 488) and vimentin (red, Alexa Fluor 647). Nuclei were stained with DAPI (blue). Magnification 63X. (B) Representative SEM images of the isolated exosomes (scale bar = 2 μm). (C) Western blot of exosomal markers CD81 and CD63. The isolated exosomes with different protein concentrations (EXO#1 = 40 μg and EXO#2 = 20 μg) were successfully obtained from the culture supernatants of PBMCs from SCLC patients. Red line indicated the band of each protein on the gel. Original western blots are presented in File S1. (D) Stacked bar plots showing the log10-transformed positive control read counts across the samples. (E) Violin plot displaying the distribution of the log of the un-normalized gene counts for all the samples.

Diagrammatic Representation 1

PBMCs isolation workflow using the density gradients method. Created with BioRender Tool. The graphical scheme was produced by the authors using the BioRender platform (https://www.biorender.com/) (basic licence terms).

Diagrammatic Representation 2

Protocol workflow for CTC isolation from whole blood using (A) RosetteSep™ CTC Enrichment Cocktail (B) MACS® Cell Separation technology (C) the Parsortix™ cell separation system. The graphical scheme was produced by the authors using the BioRender platform (https://www.biorender.com/) (basic licence terms).

Diagrammatic Representation 3

(A) Protocol workflow for EVs derived from PBMCs isolation using ultracentrifugation method. (B) Protocol workflow for RNA isolation from TEPs and analysis of gene expression by nCounter Analysis System. This figure was produced using the BioRender platform (https://www.biorender.com/) (basic licence terms).