Phenotypic diversity of CTCs and tdEVs in liquid biopsies of tumour-draining veins is linked to poor prognosis in colorectal cancer

Abstract

Background: Circulating tumour cells (CTCs) and tumour-derived extracellular vesicles (tdEVs) have great potential for monitoring therapy response and early detection of tumour relapse, facilitating personalized adjuvant therapeutic strategies. However, their low abundance in peripheral blood limits their informative value. In this study, we explored the presence of CTCs and tdEVs collected intraoperatively from a tumour-draining vein (DV) and via a central venous catheter (CVC) prior to tumour resection.

Methods: CellSearch analyses of 395 blood samples from 306 patients with gastrointestinal tumours and 93 blood samples from healthy donors were used to establish and validate gates for the automated detection of CTCs and tdEVs with ACCEPT software and R scripts. The selected gate settings were applied to 227 samples of 142 patients with colorectal cancer (CRC) from two independent collectives. Phenotypic features were obtained via numeric analysis of their fluorescence signals (e.g. size, shape, and intensity) and were used for calculating diversity using Shannon index (SI) of clusters generated via the k-means algorithm after Uniform Manifold Approximation and Projection (UMAP) pre-processing, and standard deviation (SD).

Results: CTCs and tdEVs were more abundant in the DV samples compared to CVC samples (p < 0.05). tdEVs were detected in higher numbers than CTCs in both compartments. Importantly, tdEVs in CVCs were associated with tumor spread, whereas CTCs in DVs were linked to tumor size. In both compartments, the prognostic value of tdEVs for overall survival (OS) surpassed that of CTCs, as demonstrated by univariate, multivariate, and Kaplan-Meier analyses. CTCs and tdEVs in DVs were phenotypically distinct, being larger, more eccentric, and displaying stronger cytokeratin intensities (p < 0.05) compared to those in CVC samples. Furthermore, increased diversity in CTC and tdEV phenotypes was significantly associated with shorter survival, validating the prognostic relevance of the SD-diversity metric.

Conclusion: Our study demonstrates that DV sampling significantly enhances the detection of prognostically relevant CTCs and tdEVs in CRC patients, underscoring the superior prognostic significance of tdEVs compared to CTCs. Importantly, the combined phenotypic diversity of both markers emerges as a more powerful biomarker than their enumeration alone. These findings suggest that comprehensive, automated analysis of CTCs and tdEVs in DVs may open new avenues for tailoring individualized therapies in CRC patients.

Keywords: CRC; CTCs; CellSearch; Circulating tumour cells; Colorectal cancer; Diversity; Intraoperative blood sampling; Single cell analysis; Tumour-derived extracellular vesicles; Tumour-draining vein; tdEVs.

Fig. 1

Identification of CTCs and tdEVs with the CellSearch system. (A) Representative circulating tumour cells (CTCs), tumour-derived extracellular vesicles (tdEVs), white blood cells (WBCs), lymphocyte-derived extracellular vesicles (ldEVs) and bare nuclei identified using the ACCEPT tool. For every object, a thumbnail image overlay of the three fluorescent channels (CD45, DAPI, PE) and each channel separately is shown. The red contour indicates the detected boundary of each object. The scale bar represents 10 pixels equivalent to 6.4 μm. (B) UMAP visualization of the different types of events identified with ACCEPT in samples from CRC R0 patients from the combined DU + HE cohort (N = 93 CVCs; N = 134 DVs). All detected CTCs (N = 5870) are represented, while for purposes of better visualization, for all other populations, we randomly selected N = 5870 objects. (C) Heatmap representation of chromosomal copy number alterations (CNAs) of 20 single CTCs obtained by low-pass NGS after single-cell whole-genome amplification. (D) Detection rates (positivity rates) and counts of CTCs and (E) tdEVs in samples from CRC R0 patients from the combined DU + HE cohort (N = 93 CVCs; N = 134 DVs). The horizontal lines represent the median. *p < 0.05

Fig. 2

Prognostic value of CS-tdEVs detected in DV samples of CRC R0 M0 patients. (A) Receiver operating characteristic (ROC) curves for CTCs and tdEVs and the respective area under the curve (AUC) values calculated for CTCs and tdEVs detected in DV samples of patients from the DU-cohort (N = 53). (B) Ten most relevant tdEV cut-offs identified in the DV samples from CRC R0 M0 patients of the DU-cohort (N = 53), and validation of the 8 tdEV cut-off in the HE (N = 58) and DU + HE (N = 111) cohorts. (C) Kaplan-Meier estimates of overall survival for patients dichotomized on the basis of the 8 tdEV cut-off in the DU (N = 53), HE (N = 58), and DU + HE (N = 111) cohorts of patients. (D) Univariate analysis of clinicopathological factors (including the ≥ 8 tdEV cut-off) in the DU + HE cohort of patients (N = 111). (E) Multivariate analysis of clinicopathological factors, including the ≥ 8 tdEV cut-off, in the DU + HE cohort of patients (N = 111)

Fig. 3

Prognostic value of diversity in DV samples of CRC R0 M0 patients. (A) Kaplan‒Meier estimates of overall survival for patients dichotomized on the basis of the SD-diversity cut-off of 0.6389 in the DU (N = 53), HE (N = 58), and DU + HE (N = 111) cohorts of patients. (B) Multivariate analysis was performed including variables that were found to be significant in the univariate model. (C) SD-diversity index calculated for each of the N = 111 patients of the DU + HE cohort showing the positive predictive value (PPV) and negative predictive value (NPV) of the cutoff 0.6389 as a biomarker. In red are indicated the patients that died