Circulating tumor cells isolated from colorectal cancer patients can form spheroids spontaneously: a small study in Ciptomangunkusumo Hospital, Indonesia

Introduction

Circulating Tumour Cells (CTCs) are commonly defined as tumour cells that enter circulating systems such as the blood or lymphatic vessels Grover et al., 2014). CTCs are cells that shed from either primary tumors or from metastatic sites (Wang et al., 2018). Recently, CTCs have been found to initiate tumorigenesis in Patient-Derived Xenograft models (Rodrigues and Vanharanta, 2019). Therefore, CTC might play a key role in understanding such metastatic processes (Castro-Giner and Aceto, 2020).

The tumorigenic capacity of CTC may be demonstrated using xenograft models as previously described (Rodrigues and Vanharanta, 2019) or by tumor spheroid formations in in vitro assay (Hu et al., 2021). However, xenograft models and 3D culture using Matrigel cell culture matrix is expensive, in particular for developing countries such as Indonesia. In this study, we report the interesting findings from our pilot study that show CTCs were able to be cultured and that they can form spheroid aggregations spontaneously. Therefore, such methods may be promising and should be further investigated, in particular in developing an economical assay to test the tumorigenesis ability of CTCs. This pilot study is a part of a larger study that aims to assess CTC as a diagnostic tool for colorectal cancer (CRC).

Methods

Results

In this study, we identified two patients with similar clinical profiles which rectal adenocarcinoma and metastatic cancer in the liver. The demographic information of the two patients is shown in Table 1. At first glance, we wanted to optimize the culture conditions for CTC of the metastatic cancer patients in the short term for approximately 3-7 days using standard culture media. The first question we wished to address was how the CTC culture had grown in vitro under standard culture conditions. Our suggestion was to increase the chance of cell-to-cell contact using 3D cultures.

In this study, we were able to identify two cultures from two patients, namely ID 001 and ID 002 after three days of incubation. Surprisingly, we found that ID 001 was able to form aggregation of cells (Figure 1A) while the CTC 002 did not have aggregation formation (Figure 1B). The ID 002 displayed clustered grape-like morphology. Therefore, we suspected that the aggregation formation may vary between samples and may depend on intrinsic factors between samples such as homophilic interactions of Cluster of Differentiation (CD)-44/CD44 antigen/CD44 that induced multicellular aggregations.

Figure 1. Spheroid culture of circulating tumor cells (CTCs) from patients with similar metastasis status after three days culture.

(A) The CTC with ID001 was able to spontaneously aggregate at the bottom of the well, forming a spheroid aggregation. (B) The CTC with ID 002 was unable to form uniform aggregation.

Since tumors are known for their ability to form aggregations, we chose an aggressive solid type of tumor, namely breast cancer as a control for the study. Breast cancer was a type of solid tumor with a known lobular structure and estimated behold potency to form aggregation in the 3D culture platform. We tried to seed these primary cells directly to the same type of plates used for the CTCs, for the duration of three days. We found that this produced a more rigid structure of spheroid formation (as shown in Figure 2).

Figure 2. Breast cancer primary cultures derived from biopsy.

The primary breast cancer cultures derived from primary tumor were able to form spontaneous spheroid formations after three days culture. This is one of the characteristics of tumor cells.

Discussion

In this study, we wanted to know the characteristics of isolated CTC cells by observing the ability of isolates to form spheroids. Based on this study, it was shown that CTC can form spheroids spontaneously. We recommend using an u-plate for the test to allow the aggregation process at the bottom of the plate. After that, it is recommended that the sample be left for three days. Our results showed that, surprisingly, CTC ID 001 had the ability to spontaneously form spheroids within three days, whereas CTC ID 002 did not spontaneously form spheroids (Figure 1).

The spheroid formation has been found as in vitro assay to evaluate the tumorigenic capacity of the cells (Hu et al., 2021) In this study, we found facts that CTC was able to form spheroids and aggregations. Therefore we suspected that CTCs might have some tumorigenic capacity, which may be displayed by the aggregations. We confirmed these aggregation processes in primary breast cancer cells using the same methods and observed a more dense and darker spheroid formation (Figure 2). Thus, the aggregations may also be related to tumorigenesis of the CTCs or at least some similar features, as those primary cells were able to form spheroids.

Previously, the tumorigenicity of CTCs has been reported using Patient-Derived Xenograft (PDX) models (Rodrigues and Vanharanta, 2019). From our perspective, such conditions of spheroid formation may be related to the tumorigenic potential of CTC. At least, these CTC cultures were isolated from two metastatic colorectal cancer patients (shown in Table 1). Our results suggested that the CTCs have the ability to mimic primary tumors in their ability to spontaneously aggregate in vitro. The capacity of the CTC to form aggregation may vary depending on intrinsic factors such as expressions of CD44 in the CTC’s that could not be identified in this study. A previous study by Liu et al. showed that homophilic interactions between CD44 increase the chance of cell to cell contact, leading to multicellular aggregations of the patient-derived breast cancer models (Liu et al., 2019). In the future, we suggest that our findings regarding spheroid aggregation assay may be used as a potential alternative to predict either patient metastasis or tumorigenic capacity of CTC in a larger study.

Data availability

Open Science Framework: Circulating Tumor Cells from CRC CIptoMangunkusumo. https://doi.org/10.17605/OSF.IO/7CGYA (Abdullah, 2021).

This project contains the following underlying data:

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).