Monitoring cancer spheroid growth and cytotoxicity with Omni™

 

Key Findings

 

>> Effects of doxorubicin on C6 spheroids can be evaluated with brightfield and fluorescence imaging using the Omni live-cell analysis platform.

>> C6 spheroid formation occurs in a two-phase process, aggregation and growth.

>> Doxorubicin has a dose-dependent effect on C6 spheroids.

 

Abstract

Given the high number of cancer-related deaths, new therapies are urgently needed. Traditionally, treatment studies have relied on monolayer cell cultures, a well-established but oversimplified model that can miss complex interactions between cellular and extracellular environments. Three-dimensional (3D) cell cultures such as spheroids and organoids can overcome those limitations as they serve as a more representative model for tumors and the interaction with the microenvironment. Here, the Omni live-cell imaging platform was used to monitor and analyze spheroid formation for multiple cell types. Additionally, the Omni was used to monitor the uptake and impact of the chemotherapy drug doxorubicin on the spheroids, demonstrating its use as a valuable tool for evaluating drug impact on 3D models.

 

Introduction

Cancer accounts for a significant number of deaths worldwide and continues to be the second leading cause of mortality1. The invasiveness of the type of cancer plays a major role in determining treatment options and survival. Highly invasive, difficult-to-treat cancer types include glioblastoma and other high-grade malignant brain tumors, non-small cell lung cancer, and triple-negative breast cancer2.

These tumors are characterized by genetic mutations in oncogenes and tumor suppressor genes. In combination with their highly invasive nature, these mutations pose significant challenges for effective treatment3-5.

Traditionally, two-dimensional (2D) monolayer cell cultures have been used to investigate the antitumor activity of a therapeutic compound. Those culture methods are relatively straightforward, cost-effective, and ideal for high-throughput screening using well-established assays. However, monolayer cultures present a simplified version of tumors that may insufficiently capture essential cellular organization and interactions found in vivo6,7. To overcome these limitations, three-dimensional (3D) cell culture models such as spheroids and organoids are increasingly developed in research.

3D cultures have emerged as powerful tools in tumor research, primarily due to their ability to better mimic the natural microenvironment8,9. Spheroids, which are self-assembled cell aggregates, exhibit more complex cell-cell interactions as well as a nutrient and oxygen gradient, making them promising models for drug treatment and drug development studies, especially in oncology research10,11.

Despite their advantages over 2D cultures, 3D models also have limitations, including higher cost and variability in spheroid formation efficiency. These challenges highlight the necessity to optimize culture methods. Additionally, assays traditionally designed for 2D setups may pose challenges when adapted to suit the complexities of 3D cultures12,13. Live-cell imaging presents a valuable tool in oncology research using spheroids, as it enables continuous monitoring of spheroid development in size and morphology. This method will facilitate evaluation of various culture methods and supports drug treatment analysis by providing kinetic data on drug uptake and cytotoxicity.

In this study, the Omni live-cell imaging platform was used to monitor the effect of doxorubicin (DOX) on cancer spheroids to demonstrate the benefits of live-cell imaging in analyzing drug studies with 3D cell culture models. First, formation and growth of various cancer spheroids was characterized. C6 cells (rat glial cells), A549 cells (adenocarcinomic human alveolar basal epithelial cells), and MDA-MB-231 cells (human epithelial mammary adenocarcinoma cells) were used as they serve as a valuable model for glioblastoma, lung cancer, and triple-negative breast cancer, respectively14-16. DOX, an anthracycline drug, is widely used as an antitumor agent in the treatment of various types of cancer, including breast, lung, ovarian, and brain tumors17. Therefore, after the characterization of spheroid formation, C6 spheroids were exposed to various concentrations of DOX to assess its effect on their growth.

 

Materials and Methods

Cells and reagents

C6 cells (ATCC, Cat. CCL-107) were maintained in DMEM medium (Gibco, Cat. 41965039) supplemented with 10% fetal bovine serum (FBS; Gibco, Cat. 16000044) and 1% penicillin/ streptomycin (Gibco, Cat. 15140122).

A549 cells (Innoprot, Cat. P20118) were maintained in Ham’s F-12K medium (Gibco, Cat. 21127022) supplemented with 10% FBS (Gibco, Cat. 16000044) and 250 µg/mL G418 (Gibco, Cat. 10131035). MDA-MB-231 cells (Innoprot, Cat. P20317) were maintained in RPMI medium (Gibco, Cat. 11875093), 10% FBS (Gibco, Cat. 16000044), 1% penicillin/streptomycin (Gibco, Cat. 15140122) and 10 µg/mL puromycin dihydrochloride (Gibco, Cat. A1113803). Cells were passaged every 3 days when confluence reached 80%.

Cancer spheroid formation

First, a 96-well U-bottom plate (VWR, Cat. 734- 2782) was prepared by adding 100 µL Anti-Adherence Rinsing Solution (StemCell Technologies, Cat. 07010) to each well that was used. The plate was incubated for 5 minutes at room temperature, followed by aspiration of the Anti-Adherence Rinsing Solution. The wells were washed by adding 100 µL PBS, incubating for 5 minutes, and aspirating the PBS.

C6 cells, A549 cells, and MDA-MB-231 cells were lifted from flasks and dissociated via trypsinization and subsequently counted using the Exact FLTM. Next, the cells were centrifuged and resuspended in appropriate medium to the desired working concentrations. Cells were introduced to the pre-treated U-bottom plate by adding 100 µL of cell suspension per well, resulting in the respective final cell densities of 1,000, 1,250, 2,500, 5,000, and 10,000 cells per well. The plate was centrifuged at 1000 rpm for 4 minutes. The well plate was placed on the Omni inside an incubator at 37°C and 5% CO2. Spheroid formation was monitored simultaneously across all cell types over 72 hours using the Omni platform with a time interval of 3 hours. During brightfield acquisition, the camera scans the sample stage while acquiring a series of sequential images that are stitched together to create a total surface area image. This allows for the assessment of all spheroids simultaneously. Data analysis was performed using the Axion portal.

Doxorubicin dose-response assay

C6 spheroids were prepared as described before using a final cell density of 2,500 cells per well. DOX (Sigma, CAS-no: 25316-40-9) was diluted to 2x the desired final concentration from the stock solution using the appropriate cell culture medium. After 72 hours of spheroid formation, 100 µL of DOX solution was added to each well containing the spheroid in 100 µL of medium, yielding final concentrations of 0, 5, 10, 20 and 32 µM. The well plate was placed on the Omni inside an incubator at 37°C and 5% CO2. DOX uptake was monitored over 72 hours by imaging every hour (Figure 1).

 

Figure 1
Figure 1: Experimental workflow of spheroid formation in a pre-treated 96-well U-bottom plate followed by treatment with DOX after 3 days. The entire process is monitored using the Omni imaging platform.

 

As DOX is inherently red fluorescent, an increase in red fluorescence indicates DOX uptake. The size of the spheroid and the red fluorescence intensity within spheroids were analyzed using a third party analysis software. To determine the growth of the spheroids, the area was normalized using the following equation:

 

Normalized Growth Formula

 

where A(t) represents the area at time t and A(0) represents the area of the spheroid when the DOX was added (day 3).

 

Results

Real-time monitoring of cancer spheroid formation and growth

To investigate the effect of DOX on cancer spheroids, the optimal seeding density and spheroid culture time had to be determined first. Various seeding densities were investigated for a 72-hours period (Figure 2). The brightfield images (Figure 2, top) showed that the different cell types form varying types of spheroids. C6 and A549 cells both form compact spheroids, whereas MDA-MB-231 form loose clusters of cells instead of a compact spheroid. The A549 spheroids are more irregularly shaped compared to the C6 spheroids. As C6 cells form the most round and compact spheroids, C6 cells were used for the remaining experiments.

 

Figure 2
Figure 2: (Top) Images of spheroids from C6, A549, and MDA-MB-231 cells (top to bottom) using cell densities between 1,000 and 10,000 cells per well (left to right). The scale bar represents 200 µm. (Bottom) Quantitative assessment of spheroid size for the varying conditions 72 hours after seeding (n=3). The error bars represent standard deviation.

 

Figure 2 displays the size of the different types of spheroids for the varying seeding densities. According to existing literature, spheroids exceeding 500 µm in diameter tend to develop necrotic regions18. To address this, 2,500 cells per well was chosen as the optimal seeding density. This condition ensures the formation of stable midsized spheroids without necrotic regions and facilitates ongoing growth for the duration of the drug treatment experiments. This seeding density was used to further characterize the growth pattern of C6 spheroids.

As shown in Figure 3, the formation and growth behavior of C6 spheroids at a seeding density of 2,500 cells per well can be divided into two phases: the formation phase and the growth phase. During the first phase, dispersed cells aggregate to form the spheroid, resulting in an initial decrease in size. The duration of this phase can vary based on cell type and seeding density. For C6 cells with a seeding density of 2,500 cells per well, it takes approximately 9 hours. Following this initial phase, the second phase commenced, during which the spheroids expand.

 

Figure 3
Figure 3: (Top) Images acquired by the Omni of C6 spheroids cultured over time. The bottom images show the Organoid Analysis module overlay. The scale bar represents 200 µm. (Bottom) Quantitative assessment of spheroid size over time (n=3). The error bars represent standard deviation.

 

Doxorubicin inhibits spheroid growth

The effect of increasing concentrations of DOX on C6 spheroids is shown in Figure 4.

 

Figure 4
Figure 4: Brightfield, red fluorescence, and composite images of the spheroid over time exposed to 5 µM of doxorubicin. The scale bar represents 200 µm.

 

The fluorescence images (Figure 4) demonstrate the progressive uptake of DOX by the spheroids over time.

For more insight into the effect of DOX uptake, the brightfield images were used to analyze the change in spheroid size over time (Figure 5). During the first 12 hours, a similar size change is observed across all concentrations. After this, a significantly different response is observed when comparing 5 µM and 32 µM. While the normalized growth for 32 µM stabilizes during the remainder of the experiment, the spheroids treated with 5 µM of DOX start to shrink, as indicated by the decrease in normalized growth followed by an increase in spheroid size. No significant difference was observed between the other concentrations. After 72 hours, the spheroids treated with 32 µM of DOX showed an increased size compared to 5 µM. This dose-dependent response is supported by the brightfield images (Figure 5, bottom) as different concentrations result in a different spheroid morphology. The group treated with 5 µM displayed a more loose spheroid with rough edges as individual cells seems to move out of the spheroid. The spheroids treated with 32 µM of DOX stay compact and become dark indicating dead cells inside the spheroid. This suggests higher concentrations of DOX have a different effect on spheroids. However, more extensive research is required to confirm this.

 

Figure 5
Figure 5: (Top) Spheroid size over 72 hours after exposure to concentrations of 0, 5, 10, 20 and 32 µM of DOX normalized to the size of the spheroid when DOX was added (n=2). (Middle) Normalized spheroid growth at 72 hours (n=2) with significant differences observed be-tween all groups and the control group (p<0.001) and between 5 µM and 32 µM (p<0.05). The error bars represent standard deviation. (Bottom) Brightfield images of spheroids at 72 hours after exposure to 0 µM, 5 µM and 32 µM of DOX. The scale bar represents 200 µm.

 

 

Conclusion

This study demonstrates the advantages of using the Omni live-cell imaging platform to track cancer spheroid formation and assess the effect of antitumor compounds on complex 3D cell culture models. We demonstrated a simple, automated approach that can support evaluation of spheroid culture by tracking spheroid size. Furthermore, the Omni allowed for efficient drug response assessment in both brightfield and fluorescence.

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Authors

Nathalie Opdam – van de Laar, Inge Thijssen – van Loosdregt
Axion BioSystems, Atlanta, GA, USA