Aneuploid circulating tumor endothelial cells (CTECs) affect the detection of CTC, but they are fundamentally different


 Background

Aneuploidy is an important criterion for tumor cell identification. Like CTCs (circulating tumor cells), many CTECs (circulating tumor endothelial cells) have chromosomal aneuploidy. Affected by tumor microenvironment and detection method, the only identification index CD31 may not be comprehensive enough, which greatly affects the accuracy of CTCs detection.
Methods

Here, we explored the number distribution, expression of surface tumor markers and chromosome ploidy differences of aneuploid CTECs and CTCs in a total of 98 peripheral blood from normal volunteers, benign nodules and lung cancer patients, trying to find the differences between the two and the clinical significance of CTEC.
Results

We found that CTCs were mainly dominated by triploid and hyperploid (pentaploid or above), The former was mainly small CTCs, and the latter was mainly large CTCs. While the aneuploid CTECs were mostly large tetraploid cells. Combined detection of total CTC and CTEC numbers helped identify benign and malignant nodules. Furthermore, tetraploid CTC and CTEC had more obvious advantages than other ploids in identifying benign and malignant nodules.
Conclusions

Our findings indicated that the traditional method of distinguishing tumor and non-tumor cells by aneuploidy was not accurate. The presence of aneuploidy CTECs greatly interfered with the detection of CTCs, but they were different form CTC in cell size, chromosome ploidy distribution and had certain clinical significance. Combining CTC and CTEC had better diagnostic advantages


Introduction
There are more than 700,000 new cases of lung cancer in China each year, accounting for 17% of all new cancer cases [1]. In the past 20 years, the incidence of lung cancer has continued to rise, with a five-year survival rate of about 16-18%. Lack of early diagnosis and remote metastasis without warning are important factors in the low five-year survival rate of lung cancer. Therefore, it is especially important to find accurate markers for disease discovery and disease progression.
Circulating tumor cells (CTCs) are a type of cells that are released into the blood from the primary or metastatic foci. They are scattered around the body with the blood circulation. When they encounter a suitable microenvironment, they can develop into micro-metastases. They are a very small, phenotypically and functionally diverse group of cells in the blood. The CTCs detection is now recognized as the most representative "liquid biopsy" technology, and the integrity and clinical significance of the various information it provides far exceeds other technologies. However, accurate detection of CTCs has largely limited its clinical application. A total of 98 fresh peripheral blood samples were included, including 18 healthy volunteers, 22 benign lung nodules, 34 early stage adenocarcinoma patients and 24 advanced lung adenocarcinoma.
Normal blood specimens came from healthy volunteers recruited by the thoracic surgery laboratory.
The inclusion criteria were between 45-85 years old, no precancerous lesions, no tumor history, and a family history of no tumor genetics. Specimens of benign nodules and early lung adenocarcinoma were obtained from patients undergoing surgery in thoracic surgery. Blood samples were collected for enriching and identifying for circulating cells one day before surgery. statistics on specimen categories based on postoperative pathology; Patients with advanced lung adenocarcinoma were from the Department of Oncology, and the inclusion criteria were untreated (including radiotherapy and chemotherapy) or newly diagnosed patient.
When collecting peripheral blood of patients, in order to avoid contamination of epithelial cells, the first 2 ml blood sample were discarded or blood samples were taken after routine blood sampling of patients. Blood samples were taken from ACD anticoagulated blood vessels. After collecting the blood, immediately mix the head and tail 8 times. Store at room temperature and protected it from light. Try to process within 24 hours, not more than 48 h.
The study was approved by the Ethics Committee of the Cancer Hospital of the Chinese Academy of Medical Sciences before the study. All patients were given informed consent before blood collection.

Subtraction Enrichment Of Human Peripheral Blood
The CTC enrichment kit was from Cytointelligen, and the procedure was performed according to the procedures specified in the kit. After collecting the blood sample, the blood collection tube was leveled, and the head and tail were mixed upside down, and then centrifuged at room temperature for 15 minutes (200 × g). Discard the supernatant to 5 mm above the brown-red precipitate and add CRC buffer to the blood vessel label to fill the tube to dilute the blood cells. Next, 3 ml of the sample density separation liquid was added to a 50 ml centrifuge tube, and the diluted blood cells were added to the upper layer of the sample density separation solution, and after trimming, the mixture was centrifuged at room temperature for 6 minutes (350 × g). After centrifugation, the sample was divided into three layers, and the intermediate transparent or yellow liquid and the upper yellow liquid were taken up to another new 50 ml centrifuge tube. 300 ul of the washed magnetic beads were added to the portion of the liquid and incubated on a shaker at room temperature for 20 minutes. Finally, the magnetic beads are removed by adsorption of a magnetic frame, and non-bloodderived cells (including tumor cells and endothelial cells) are enriched in the remaining liquid.
Tumor Marker Immunostaining-chromosome Fluorescence In Situ Hybridization (TM-iFISH) The following reagents are derived from Cytointelligen. Take 100 ul of the enriched cell solution, add 2 ul of antigen repair buffer, mix gently and shake, and let stand for 10 minutes at room temperature.
At the same time, the staining solution was configured. According to each slide, 200 ul of blood cell analysis dilution + 1 ul CD45 staining solution + 1 ul CD31 staining solution + 1 ul vimentin staining solution; Use a sampler to gently mix and mix 10 times to fully mix the antibody. All the prepared mixture was added to the antigen-repaired cell sample and incubated for 20 minutes at room temperature. The stained cell sample was then washed by adding a CRC washing solution, centrifuged for 5 minutes (500 × g), and the supernatant was discarded to 100 ul. The cells were initially fixed by adding an equal volume of tissue fixative. Then dry overnight. The cells on the slide were fixed again the next day, and the fish probe was added for hybridization (hybridization conditions: denaturation at 76℃for 10 minutes; hybridization at 37℃ for 3 hours). After the hybridization was completed, the coverslip was slowly removed with a buffer, and the sample washing area was added to wash the sample area again to reduce specific staining. Dry the cleaned slide specimen frame with a hair dryer, and add 10 ul of DAPI dye solution to the center of the specimen frame, and cover the cover glass so that the dye solution was fully immersed in the entire specimen area. Immediately observe under a fluorescence microscope or preserve at 2-8 ℃ in the dark.

Statistical Analyses
Statistical analysis was done with Prism GraphPad software v8.0. All data are presented descriptively as means, medians, or proportions. Two-tailed Student's t-test was used for statistical comparison between groups; Pearson correlation was used to compare the correlation between the number of CTCs and CTECs. Statistical significance was defined as p < 0.05.

Isolation and identification of aneuploid CTCs and CTECs
Firstly, we removed the blood-derived cells such as red blood cells and white blood cells by centrifugation and phase-enrichment to achieve higher CTC enrichment efficiency, and then we borrowed the iFISH platform, which combine the immunofluorescence staining of tumor proteins  Table 1). When screening aneuploid CTCs under a fluorescence microscope, we also found a large number of aneuploid endothelial cells (CTECs). These cells had the same degree of aneuploidy as tumor cells, but the endothelial cell marker CD31 was strongly positive (Fig. 1B)

Distribution of total CTC and CTEC numbers in patients by stage
We used the iFISH platform to enrich and identify CTCs and CTECs in 17 healthy volunteers, 22 benign pulmonary nodules, 34 early lung adenocarcinoma patients, and 22 advanced lung adenocarcinoma patients. We found that CTCs could be detected in the very early stages of lung cancer. Of the 34 early stage lung cancer patients, 29 cases detected the presence of CTCs, of which 17 cases had more than three CTCs. A certain number of CTCs could also be isolated in normal and benign nodule patients. This part of the results indicated that there was a certain background pollution in normal people and benign nodules (non-tumor cells of aging or apoptosis also had a certain aneuploidy, which was mistaken for tumor cells), but the number was mostly less than three, which indicated that CTCs still had certain advantages in identifying benign nodules and early stage lung cancer(P = 0.0119) ( Fig. 2A). The number of CTCs in patients with advanced lung cancer was significantly increased. At the same time, the number of peripheral blood CTCs in patients with advanced lung adenocarcinoma was significantly higher than that in early lung cancer, benign nodules and normal controls, the difference was statistically significant (P < 0.0001) ( Fig. 2A). In addition, we found some CTC-like aneuploidy circulating tumor endothelial cells (CTECs) during the experiment. Their quantitative changes in normal human, benign nodules and early and advanced lung cancer were consistent with CTCs (Fig. 2B). The number of CTECs in advanced patients was significantly higher than that of normal, benign nodules, and early stage lung cancer patients, and it had the same early diagnostic value (P = 0.0287) and tumor staging diagnostic value(P = 0.0009) as CTCs. Since there are still normal diploid endothelial cells (CECs) in peripheral blood, this part of the cells is reported to have a certain staged diagnostic significance [2][3][4][5][6], and closely related to survival [7] and relapse [8] of cancer patient. We also counted the number of diploid CECs and found the number of diploid CECs in peripheral blood was very few, therefore we combined diploid CECs and aneuploid CTECs to observe their dynamic changes. The results showed that the total CEC score is not as good as the aneuploid CTECs in identifying early and late lung adenocarcinoma (Fig. 2C).Unexpectedly, we found that there was a certain correlation between CTCs and aneuploid CTECs in the peripheral blood, and there was a statistically significant difference(

Aneuploidy analysis of CTCs and CTECs
Our study found that both CTCs and CTECs exhibited varying degrees of chromosome aneuploidy, namely, haploid, triploid, tetraploid, pentaploid and above, standard images are shown in Fig. 3A. The clinical relevance of different types of aneuploid CTCs has been demonstrated, for example, highploid CTCs has been shown to be associated with tumor resistance and relapse [9,10]. However, the clinical significance of aneuploid CTECs are still unclear.
We found that these sorted CTC chromosomes 8 had a certain pattern. In all normal human, benign nodules, patients with early-stage or late-stage lung cancer, chromosomal abnormalities were predominantly polyploid (the most common are triploid and pentaploid), and only a few are haploid( Fig. 3B and 3D) (due to the very small number of haploids, there was no statistical subtype), which was in line with the literature reports that the polyploid genetic material was relatively abundant, conducive to the development of malignant biological behavior of cells. the vast majority of CTCs in patients with benign nodules were triploid, but in patients with cancer, especially in patients with advanced stages (Fig. 3B and 3D), the pentaploid begun to increase, explaining that the increase in genetic material was likely to increase the degree of malignancy. Although CTECs were also dominated by triploid and pentaploid, pentaploid account for the vast majority of patients, especially in advanced patients ( Fig. 3C and 3E).

Distribution of CTC and CTEC with different size in patients
Peripheral blood CTCs and CTECs vary in size. The definition of large and small cells is generally based on white blood cells (5um), cells larger than 5um are defined as large cells, and cells smaller than 5 cm are defined as small cells, and the standard image is shown in Fig. 4A. The biological and clinical significance of CTCs are different. For example, small CTCs has been reported to be significantly associated with recurrence and disease-free survival (DFS) of liver cancer [10]. However, the correlation between the size of CTECs and its clinical significance is still unclear. Therefore, we separately calculated the distribution of size of CTCs and CTECs in patients with different stages of cancer. On average, except for small CTECs, both large and small CTCs and large CTECs had the effect of distinguishing between benign and malignant nodules and staging ( Fig. 4B-4E). But compared to other subtypes, large CTCs had an advantage in identifying early and late lungs( Since chromosomal ploidy of CTCs and CTECs subclasses with different sizes have certain clinical significance, we next analyzed the chromosomal ploidy differences of large and small cells. We observed a significant difference in the cell size of different ploidy numbers (Fig. 5A-5D). According to the standard of 5um in diameter, CTCs were divided into large cells and small cells, and small cells were mainly triploid, while large cells mostly were pentaploid. The number of tetraploid was between that of triploid and pentaploid, and its size (large or small) was not much different ( Fig. 5A and 5B), but the proportion of tetraploid in large CTECs was significantly higher than that in small CTECs ( Fig. 5C and 5D). However, as shown in Fig. 4, the large cells were more significant than small cells in staging diagnosis between normal humans, benign nodules, and lung cancer.

Subclass analysis of CTCs and CTECs based on chromosomal ploidy and cell size combination
To further understand the significance of the various subtypes of CTCs and CTECs in the early diagnosis and staging of lung cancer, we divided CTCs and CTECs respectively into 24 subgroups based on cell size, chromosome ploidy and four stages (normal, benign, early, and late) of tumor development for separate analysis (Fig. 6A-6D).
Among the large CTCs, all the triploid, tetraploid and pentaploid could distinguish between early and late patients(P < 0.05), but only the pentaploid could identify benign nodules and early patients ( Fig. 6A). And compared to the other two ploidy, the ability of the pentaploid to distinguish between early and late patients was stronger(P < 0.001). For small CTCs, tetraploid had obvious early diagnostic significance(P < 0.001), while pentaploid had no advantage in both early diagnosis and staged diagnosis. However, because tetraploids accounted for only a small proportion of the entire CTC population, it might affect the sensitivity of its clinical application. When we combined the large CTCs and the small CTCs to reduce the subgroups, the sub-categories obtained after the analysis were similar in the distribution of the large CTCs in the different stages ( Fig. 6A and 6E). Current reports on CTC ploidy tend to combine tetraploid and pentaploid into a subgroup, collectively referred to as polyploid. Compared to triploids, polyploids do find some clinical significance, such as related to metastasis, recurrence and drug resistance [9]. Therefore, we also tried to combine triploid and tetraploid into a subclass, namely, polyploid, and then compare the significance of this new subclass in the diagnosis of tumor staging. It was found that for large CTCs, the polyploid subclass after the combination had no obvious advantage in distinguishing between benign and malignant nodules and distinguishing between early and late patients compared with the single subclass before the merger (Fig. S1A). Moreover, once merged, the ability of small CTCs in identifying benign and malignant nodules was lower than before (Fig. S1B).
For CTECs, we did the same analysis. Large pentaploid CTECs could simultaneously identify benign and malignant nodules and classify for patients with early and late stage tumors (Fig. 6B). Small triploid CTECs could distinguish between early and late patients, but could not identify benign nodules and early patients (Fig. 6D). But after the combination of large and small CTECs, both tetraploid and pentaploid had certain clinical significance. The pentaploid was only meaningful for early and late stage patients, and tetraploid could distinguish between benign nodules and early lung cancer patients (Fig. 6F). When we combined the tetraploid and pentaploid of CTECs into a polyploid subclass, small polyploid CTECs could not stage clinical patients (Fig. S1D). And large CTECs had the same effect as before the merger, that is, only pentaploid was meaningful, and could distinguish between benign nodules and early patient or between early and late patients (Fig. S1C).
Based on the results of CTCs and CTECs, we found that for CTCs, the most advantageous one for identifying benign nodules and early patients was tetraploid small CTCs; the most appropriate for identifying early and late patients was the pentaploid large CTCs; For CTECs, the sub-class of best identifying benign nodules and early patients was the combination of large and small tetraploid cells or large polyploid cells. But the best choice for distinguishing between early and late patients was large pentaploid CTECs or large polyploid (tetraploid and pentaploid) CTECs. Finally, comparing CTCs and CTECs as a whole, whether in differentiating of benign and malignant nodules or of early and late lung cancer, CTCs were slightly more advantageous than CTECs.
CTEC was not a senescent or apoptotic cell, but an active cell population In principle, endothelial cells are non-tumor cells, and the genetic material is relatively stable, while CTEC chromosomes exhibit different degrees of copy number variation, similar to the malignant transformation process of tumor cells. However, because these cells are strongly positive for CD31, although they have many similarities with tumor cells, they are classified as a column of endothelial cells. We suspected that these CTECs that entered the blood were all aging or impending apoptosis, therefore they showed chromosomal variation. However, we had discovered some new phenomena, such as the phenomenon of cell division in CTECs isolated from peripheral blood (Fig. 7A), and CTECs could be clustered like tumor cells (Fig. 7B) or grouping with other types of cells such as white blood cells (Fig. 7C) and tumor cells, in line with the literature reports [11]. The binding of CTCs to leukocytes can promote their own malignant phenotype [12], but the clinical significance of CTECs and leukocyte or tumor cell agglomeration is still unclear. At the same time, some CTECs could also express tumor markers such as vimentin (Fig. 7D). These phenomena together indicated that aneuploid CTECs could indeed exist as a separate active cell population like a tumor cell, rather than simply shed cells that fall into the blood after aging or death. But whether CTECs have potential malignant behavior and its true biological differences with CTCs requires further cellular and molecular experiments to validate, and the future will help to understand these issues with single-cell sequencing.

Discussion
In this article, we explored the biological characteristics of aneuploid CTCs and CTECs and their dynamic changes at different stages of lung cancer development based on chromosome 8 mutatio.
We found that there were some similarities between the distribution and chromosomal changes of CTCs and aneuploidy CTECs. In terms of quantitative changes, both CTCs and aneuploid CTECs could be used as good indicators of lung adenocarcinoma staging; In chromosome ploidy variation, tetraploid small CTCs had advantages in identifying early and benign nodules, for tetraploid Small CTCs only occurred in patients with tumors; While pentaploid large CTCs could better distinguish early patient and late patient. CTECs were mainly composed of pentaploid large cells. The main advantage lied in the identification of early and advanced lung cancer.
Human blood is rich in biological information, and liquid biopsy can capture cancerous information at an early stage and prevent tumor progression as early as possible. Common liquid biopsy indicators are cfDNA, RNA, miRNA [13], and proteins (free proteins or proteins contained in CTCs, exosomes [14] or platelets [15]. cfDNA is commonly used to detect gene mutations, and is suitable for drug resistance and efficacy evaluation, but the blood contains a high abundance of non-tumor-derived cfDNA [16]; RNA is low in blood and not stable; Protein-based markers generally respond to disease progression stages depending on the level of content, but provide limited information that does not reflect changes in gene levels and tumor heterogeneity. Unlike fragmented nucleic acid and protein markers, aneuploid CTCs and CTECs together form a pair of "cell-type circulating tumor markers" that are biologically active and rich in a range of tumor markers and complete genetic information. The significantly; They also validated that the copy number of chromosome 8 was closely related to chemotherapy efficacy and drug resistance, during which tetraploid CTCs were most sensitive to chemotherapy, while triploid CTCs were most relevant to drug resistance [17]. Polyploid chromosome 8 is also associated with tumor resistance and recurrence in breast cancer, gastric cancer [18], and liver cancer. Therefore, a more accurate sub-class analysis of CTCs based on chromosome 8 aneuploid brings new breakthroughs in the diagnosis and treatment of tumors.
Another important focus of the article is the aneuploid CTECs. Since 2004, the Japanese Kyoko Hida team has discovered in mouse tissue for the first time the phenomenon of DNA abnormalities in endothelial cells in tumor tissues [19]. Then in 2009, they found that in human kidney cancer tissues, approximately 22-58% of endothelial cells had aneuploidy of chromosomes 7 and 8 [20]. Moreover, unlike normal stable diploid cells, the degree of karyotype variation of this aneuploid endothelial cell increased with the number of passages in vitro, that is, in the tumor microenvironment, tumor endothelial cells can obtain cytogenetic abnormalities [20]. They later did research to speculate that the production of aneuploid endothelial cells was likely caused by hypoxic conditions in the tumor microenvironment [21]. However, the discovery of aneuploid CTECs in the peripheral blood circulation was only unexpectedly discovered during the detection of CTCs after the rise of iFISH technology.
First reported in 2017 [11]. In our study, aneuploid CTEC does have obvious enrichment in peripheral blood. At different stages of lung cancer development and progression, its number shows dynamic changes, and it is obviously enriched in advanced patients, which has certain clinical reference significance, similar to the previous researches exploring the distribution trend of circulating endothelial cells [22,23]. However, previous studies were mainly focused on total CEC and did not distinguish between aneuploid and normal diploids in detail. We separately analyzed aneuploid cells and diploid cells and found that the number of aneuploid CECs was much higher than that of normal Moreover, we observed aneuploidy CTECs rarely expressed the interstitial marker vimentin, while this indicator has a strong positive rate in the blood vessels. Then whether this classification method is reasonable or whether there may be some connection between the two kinds of cells in the source? It is probably a kind of transformation of cells just in the process of entering the blood. This conjecture needs further verification.
This study also has some shortcomings, for example, a small amount of CTCs and aneuploid CTECs were also found in normal and benign nodules. These two types of cells need to be further expanded in the early diagnosis of lung cancer to find a suitable cut off value. CTCs and aneuploid CTECs found in normal and benign nodules may be cells of chromosomal variation caused by inflammation and aging. This part of CTCs can be classified as "non-authentic" CTCs, and the degree of biological malignancy may be far less than the "true" CTCs, it may disappear by the autoimmune system at any time, so it is especially important to dynamically detect the number of such cells. The rise of future single-cell sequencing technology promises to find a marker that truly distinguishes between "truth" and "false" CTCs, making detection more precise.

Conclusions
There are significant differences in the number, size, surface markers and chromosomal aneuploidy between aneuploid and CTCs and CTECs in patients with lung adenocarcinoma at different stages.
Both types of cells can play a certain role in the diagnosis and treatment of lung cancer. The future direction of development will be based on these biological characteristics for sub-class analysis of aneuploid CTCs and CTECs, guiding in-depth mechanism research and targeted therapy.