Aneuploid Circulating Tumor-Derived Endothelial Cell (CTEC): A Novel Versatile Player in Tumor Neovascularization and Cancer Metastasis

Abstract

Hematogenous and lymphogenous cancer metastases are significantly impacted by tumor neovascularization, which predominantly consists of blood vessel-relevant angiogenesis, vasculogenesis, vasculogenic mimicry, and lymphatic vessel-related lymphangiogenesis. Among the endothelial cells that make up the lining of tumor vasculature, a majority of them are tumor-derived endothelial cells (TECs), exhibiting cytogenetic abnormalities of aneuploid chromosomes. Aneuploid TECs are generated from "cancerization of stromal endothelial cells" and "endothelialization of carcinoma cells" in the hypoxic tumor microenvironment. Both processes crucially engage the hypoxia-triggered epithelial-to-mesenchymal transition (EMT) and endothelial-to-mesenchymal transition (EndoMT). Compared to the cancerization process, endothelialization of cancer cells, which comprises the fusion of tumor cells with endothelial cells and transdifferentiation of cancer cells into TECs, is the dominant pathway. Tumor-derived endothelial cells, possessing the dual properties of cancerous malignancy and endothelial vascularization ability, are thus the endothelialized cancer cells. Circulating tumor-derived endothelial cells (CTECs) are TECs shed into the peripheral circulation. Aneuploid CD31⁺ CTECs, together with their counterpart CD31^- circulating tumor cells (CTCs), constitute a unique pair of cellular circulating tumor biomarkers. This review discusses a proposed cascaded framework that focuses on the origins of TECs and CTECs in the hypoxic tumor microenvironment and their clinical implications for tumorigenesis, neovascularization, disease progression, and cancer metastasis. Aneuploid CTECs, harboring hybridized properties of malignancy, vascularization and motility, may serve as a unique target for developing a novel metastasis blockade cancer therapy.

Keywords: EMT; EndoMT; aneuploid CTC; aneuploid CTEC; cancerization of stromal endothelial cells; cell fusion; cellular circulating tumor biomarker; endothelialization of cancer cells; hypoxia; iFISH; transdifferentiation.

Figure 1

Schematic diagram of transdifferentiation and heterotypic cell fusion. Aneuploid tumor-derived endothelial cells (TECs) and circulating tumor-derived endothelial cells (CTECs) in carcinoma patients are primarily contributed by transdifferentiation and heterotypic cell fusion. (A) Transdifferentiation (transD). Differentiated aneuploid cancer cells and stromal cells dedifferentiate (deD) into Vimentin⁺ (Vim⁺) mesenchymal cancer stem cells (CSCs) and mesenchymal stromal cells (MSCs) via epithelial-to-mesenchymal transition (EMT) and endothelial-to-mesenchymal transition (EndoMT), respectively. The resultant undifferentiated CSCs and MSCs may transD into individual normal diploid CD31⁺ endothelial cells (ECs, top blue arrow) and/or aneuploid TECs (middle pink arrow) having either a positive expression of tumor markers (TMs) (red/green dash ring, diploid or aneuploid) or not (null, aneuploid, green). Among MSCs, some EC-derived MSCs may differentiate into the same endothelial lineage of TECs/ECs. (B) Heterotypic cell fusion. An aneuploid cancer cell fuses with an EC to generate either an individual, mononuclear synkaryon of aneuploid TEC (pink arrow) or a multinuclear heterokaryon in a cluster (microemboli, bottom blue arrow). The fusogenic hybrids may or may not (null) have a positive expression of TMs.

Figure 2

In situ phenotypic and karyotypic characterization of aneuploid CTECs and disseminated tumor-derived ECs (DTECs) detected in varieties of cancer patients by the integrated immunostaining-fluorescence in situ hybridization (iFISH). Aneuploid TECs in peripheral blood (CTECs) and bone marrow (BM; DTECs) of a variety of carcinoma patients were enriched by subtraction enrichment (SE), followed by 6-channel iFISH to simultaneously co-characterize aneuploidy of chromosome 8 and TM expression in CD31⁺/CD45^– CTECs and DTECs. (A) An aneuploid hepatocellular carcinoma (HCC) CTEC enriched from blood had plasma membrane staining of the stemness marker EpCAM and cytoplasmic vesicular staining of secretory α-fetoprotein (AFP). (B) A multinuclear mesenchymal (Vimentin⁺) and EpCAM⁺ DTEC fusion cluster enriched from bone marrow in a non-small cell lung cancer (NSCLC) patient (DTEC microemboli). The cell cluster in BM had dual phenotypes of both epithelium and mesenchyme. (C) A multinuclear mesenchymal (Vimentin⁺) and stemness marker CD44v6⁺ DTEC fusogenic microembolus in BM of a lung cancer patient. The cell cluster in BM was composed of disseminated tumor-derived endothelial stem cells (DTESCs) with a mesenchymal phenotype. (D) An aneuploid mesenchymal (Vimentin⁺) NSCLC CTEC revealed a granule-like staining of PD-L1, suggesting that PD-L1 may localize in the secretory granules. (E) An aneuploid ovarian cancer CTEC showed a positive staining of cytoplasmic vesicle-like CA 125 and human epididymis protein 4 (HE4). (F) An aneuploid prostate cancer CTEC had a positive plasma membrane and cytoplasmic staining of the prostate-specific membrane antigen (PSMA), but negative for the oval cell marker OV-6 staining. (G) A diploid mesenchymal (Vimentin⁺) and PD-L1⁺ sarcoma CTEC. Fluorescence dyes conjugated to diverse antibodies (Cytelligen, USA): Alexa Fluor 488 (green), AF594 (red), Cyanine 5 (yellow), and Cy7 (purple).

Figure 3

Hypoxia-induced tumor neovascularization cascade generates CTECs in cancer patients. Tumor neovascularization in carcinoma patients is contributed by endothelium-dependent angiogenesis, vasculogenesis, lymphangiogenesis, and endothelium-independent cancer cell-derived VM channels. The lining of blood vessels (BVs) in tumor vasculature is constituted primarily by CD31⁺ tumor-derived ECs. Aneuploid TECs, the endothelialized cancer cells in essence, are derived from both cancerization of stromal cells (such as ECs) and endothelialization of malignant tumor cells, of which the latter consists of the heterotypic cell fusion of cancer cells with ECs and the transdifferentiation (transD) of carcinoma cells via CSCs. In the tumor microenvironment (TME), in addition to participating in channel formation by vasculogenic mimicry (VM), undifferentiated tumorigenic CSCs differentiate (D) into cancer cells, and some of these differentiated cancer cells are able to inversely dedifferentiate (deD) back into mesenchymal CSCs through EMT. Cancerization-relevant MSCs are derived from induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs). The dedifferentiation (deD) of ECs and other stromal cells via EndoMT provides an extra origin of MSCs. Subsequent in vivo transD of CSCs and MSCs into TECs/ECs is also observed in vitro. Besides transD, EC-derived MSCs are also able to conversely differentiate (D) into the same endothelial lineage of TECs/ECs in the hypoxic TME. Obtained normal ECs may, in turn, fuse with cancer cells to turn into TECs. Compared to stromal cell cancerization, most TECs actually originate from the endothelialization of neoplastic cells. Vasculogenesis, a process that involves differentiation (D) of bone marrow (BM)-derived epithelial progenitor cells (EPCs) and pericyte progenitor cells (PPCs) into ECs, is an additional source of ECs for heterotypic cell fusion. BVs and lymphatic vessels (LVs) share the same pathways to generate ECs and TECs. CTECs and DTECs are aneuploid TECs that are respectively shed into the peripheral blood and BM. During the formation of TECs and CTECs, both EMT and EndoMT, which have identical transcription pathways and the same core set of EMT-inducing transcription factors (EMT-TFs: Twist1, Snail, Slug, ZEB1, ZEB2, and Notch), are critically involved in the formation processes. In the illustrated CTEC formation cascade, hypoxia is a vital inducer for every step, EMT and EndoMT are the crucial hubs, CSCs and MSCs are the central nodes, TECs are the essential contributors, and mobile CTECs and DTECs are the critical versatile players. D, differentiation; deD, dedifferentiation; transD, transdifferentiation.

Figure 4

Participation of CTECs and TECs in cancer metastases. TECs constitute a significant proportion of endothelial composition in lymphatic and blood vessels (BVs) of tumor vasculature. Following shedding into peripheral circulation or lymph flow, TECs turn into CTECs. CTECs actively participate in hematogenous and lymphogenous cancer metastases. Lymphogenous metastatic cancer cells and CTECs in the lymph eventually converge into the peripheral blood of hematogenous distant metastasis.