Comprehensive Atlas of Circulating Rare Cells Detected by SE-iFISH and Image Scanning Platform in Patients With Various Diseases

Abstract

Objective: Circulating rare cells (CRCs) are known as a crucial nucleated cellular response to pathological conditions, yet the landscape of cell types across a wide variety of diseases lacks comprehensive understanding. This study aimed at detecting and presenting a full spectrum of highly heterogeneous CRCs in clinical practice and further explored the characterization of CRC subtypes in distinct biomarker combinations and aneuploid chromosomes among various disease groups.

Methods: Peripheral blood was obtained from 2,360 patients with different cancers and non-neoplastic diseases. CRC capture and identification were accomplished using a novel platform integrating subtraction enrichment and immunostaining-fluorescence in situ hybridization (SE-iFISH) strategy with a high-throughput automated image scanning system, on which hemocyte, tumor, epithelial, endothelial, mesenchymal, and stemness biomarkers were immunostained and displayed simultaneously. Double chromosome enumeration probe (CEP8 and CEP12) co-detection was performed on isolated CRCs from an extended trial for two chromosome ploidy patterns.

Results: A comprehensive atlas categorizing the diverse CRCs into 71 subtypes outlining was mapped out. The presence of epithelial-mesenchymal transition (EMT) or endothelial-mesenchymal transition (EndoMT), the cells with progenitor property, hematologic CRCs expressing multiple biomarkers, CRCs at "naked nuclei" status, and the rarely reported aneuploid mesenchymal epithelial-endothelial fusion cluster were described. Circulating tumor cells (CTCs) were detected in 2,157 (91.4%) patients; the total numbers of CTCs and circulating tumor-derived endothelial cells (CTECs) were relatively higher in several digestive system cancer types and non-neoplastic infectious diseases (p < 0.05). Co-detection combining CEP8 and CEP12 showed a higher diagnostic specificity on account of 57.27% false negativity of CRC detection through a single probe of CEP8.

Conclusions: The alternative biomarkers and chromosomes to be targeted by SE-iFISH and the image scanning platform, along with the comprehensive atlas, offer insight into the heterogeneity of CRCs and reveal potential contributions to specific disease diagnosis and therapeutic target cell discovery.

Keywords: SE-iFISH; cellular biomarkers; circulating rare cells; comprehensive atlas; double probes; image scanning; various diseases.

Figure 1

Schematic diagram of CRC analysis procedure through SE-iFISH and image scanning platform and the representative cellular images. Distinguished from WBC by hemocyte biomarker (red arrow represents WBC (CD45⁺) whereas white arrow represents the CD45^- non-hematologic cell) and cell size discrimination (red arrow represents the large cell whereas white arrow represents the small cell), karyotypic FISH carried out using an enumeration probe of chromosome 8 and in situ phenotypic immunostaining of multiple biomarker proteins are successively performed on the identical enriched target cell.

Figure 2

The comprehensive atlas categorizing CRC subtypes. Diverse CRC subtypes were categorized by cell size, biomarker expression, and chromosome 8 ploidy. For small cells (≤WBC size), distinguishing from WBC by hemocyte marker (CD45) took priority over all other features. For large cells (>WBC size), the chromosome 8 ploidy characterization comes to the first of subtypes classification.

Figure 3

In situ phenotypic and karyotypic characterization of CRC subtypes in varieties of patients by SE-iFISH and image scanning platform. CRCs in peripheral blood were enriched and identified by 6-channel iFISH to co-characterize aneuploidy of chromosome 8 and a series of biomarker expression including hemocyte, tumor, and endothelial and mesenchymal markers. (A) An aneuploid hepatocellular carcinoma CTEC revealed a granule-like staining of secretory α-fetoprotein (AFP) from a liver cancer patient with local recurrence before interventional chemotherapy. (B) An aneuploid small colorectal cancer CTC had dual phenotypes of both epithelium (EpCAM) and mesenchyme (Vimentin) from a colorectal cancer patient during the tumor progression period after postoperative chemotherapy. (C) An aneuploid mesenchymal CTEC (CD45^-EpCAM^-CD31⁺Vim⁺) in a breast cancer patient during neoadjuvant endocrine therapy before surgery showing a strong intracellular distribution of CD31 and nuclear localization of Vimentin. (D) A diploid CRC in a lung cancer patient sampled after radical radiation therapy reveals positive expression of CK18, CD31, and Vimentin simultaneously (white arrow). An adjacent WBC (CD45⁺) is indicated by a red arrow. (E) A large aneuploid mesenchymal endothelial-epithelial fusion cluster (CD45^-EpCAM⁺CD31⁺Vim⁺) in a breast cancer patient sampled when distant metastasis was detected after postoperative chemotherapy displayed a scattered vesicle-like staining of EpCAM in the nuclei and a positive staining of both CD31 and Vimentin. An attached mesenchymal WBC (CD45⁺Vim⁺) is indicated by a red arrow. (F) A CTM in a lung cancer patient with systemic multiple metastasis after 4 routine chemotherapy containing several aneuploid CTCs with nuclear localization of PD-L1. The adjacent WBC (CD45⁺PD-L1^-) is indicated by a red arrow. (G) A null CTM from a non-neoplastic infectious patient had no biomarker expression. (H) An enriched aneuploid circulating stem-like cell displayed a strong expression of CD133 from a lung cancer patient sampled before radical surgery. (I) An endogenous cluster containing two aneuploid CTECs co-expressed CD133, showing CD45-/CD133+/CD31+/Vim- phenotype from peripheral blood in a lung cancer patient before radical surgery. (J) A CD45⁺ cell cluster consisting of two haploid cells with CK18, CD31, and Vimentin expression simultaneously enriched from a laryngeal cancer patient upon completion of radical surgery. (K) A large aneuploid CD45⁺ endothelial cell showed a strong expression of nuclear located CK18 in a renal cancer patient sampled when systemic multiple metastasis was detected after radical surgery. Fluorescence dyes conjugated to diverse antibodies (Cytelligen, USA): Alexa Fluor 488 (green), AF594 (red), Cyanine 5 (yellow), and Cy7 (pearl blue). Scale bar, 5 μm.

Figure 4

The landscape of total numbers of CTCs and CTECs identified for each disease type. CTC and CTEC counts were obtained using blood samples from patients in 31 groups of cancer and non-neoplastic diseases (n = 2,360). Statistical analysis was performed with the Kruskal–Wallis H-test. Box boundaries indicate the interquartile range; center lines: medians; whiskers: values within 1.5 interquartile ranges of the median.

Figure 5

Characterization of double-probe (CEP8 and CEP12) co-detection of aneuploid CRCs. (A) Total numbers of CTCs and CTECs with chromosome polyploidy co-detected by CEP8 and CEP12 in a cohort of varieties of patients (n = 346). Statistical analysis was performed with a two-tailed t-test; **p < 0.01. (B) In situ phenotypic and karyotypic characterization of aneuploid CTCs and CTECs through a double-probe (CEP8 and CEP12) co-detection method. Ploidy combination of different patterns of two chromosomes (Chr) was visualized and described as aneuploidy Chr 8 and diploidy Chr 12 (Patient 1), diploid Chr 8 and aneuploidy Chr 12 (Patient 2), multiploidy Chr 8 and Chr 12 (Patient 3), aneuploidy Chr 8 and Chr 12 with CD31⁺ (Patient 4), and aneuploidy Chr 8 and Chr 12 with EpCAM⁺ (Patient 5); the WBC (CD45⁺) is indicated by a red arrow. Scale bar, 5 μm.