Use of 3D Spheroid Models in Different Cancers

Version	Summary	Created by	Modification	Content Size	Created at	Operation
1		Alberto Signore	--	1595	2023-10-18 02:12:21	\|
2	layout	Jessie Wu	+ 2 word(s)	1597	2023-10-18 05:14:03	\|

This entry is adapted from the peer-reviewed paper 10.3390/cancers15194846

Advanced tissue engineering processes and regenerative medicine provide modern strategies for fabricating three-dimensional (3D) spheroids. Several different 3D cancer models are being developed to study a variety of cancers. Three-dimensional spheroids can correctly replicate some features of solid tumors (such as the secretion of soluble mediators, drug resistance mechanisms, gene expression patterns and physiological responses) better than 2D cell cultures or animal models. Tumor spheroids are also helpful for precisely reproducing the three-dimensional organization and microenvironmental factors of tumors. Because of these unique properties, the potential of 3D cell aggregates has been emphasized, and they have been utilized in in vitro models for the detection of novel anticancer drugs.

3D spheroids cancer therapy micro-environment nanotechnology nuclear medicine

1. Introduction

The intricate microenvironment in which malignant cell reside is essential for the progression of tumor growth. The biochemical as well physical properties of TME are necessary for the proliferation, metastasis, and invasion of cancer cells. Consequently, it is essential to ascertain how malignant cells interact and communicate with supporting tumor-associated cells like endothelial cells, immune cells, macrophages, and fibroblasts. Three-dimensional spheroid models are commonly used to explore the complicated mechanisms behind cancer progression because they simulate the stromal milieu and multicellular structure of an in vivo tumor. Compared to 2D systems and animal models, the 3D spheroid model delivers more accurate information regarding tumor features, drug discovery, cell–cell interactions, and the metabolic profile of cancer cells ^[1].

2. Prostate Cancer

Prostate cancer (PCa) is a leading cause of death among men worldwide. The three-dimensional (3D) cell cultures allow the functions of living tissue to be mimicked and provide essential information coded in tissue architecture. The crucial role of epithelial–mesenchymal transition (EMT) has been considered in cancer development ^[2]^[3].

Regarding the cell lines used for 3D models of prostate cancer, Xu et al. ^[4] designed a porous chitosan-alginate (CA) scaffold for tissue engineering and analyzed the impact of scaffold stiffness on 22Rv1, PC-3, and C4-2B cell lines. CA scaffold is a 3D culture technology that facilitates phenotypic expression and PCa development with long-lasting scaffold stiffness, mimicking the metastatic advancement phase. 22Rv1 and C4-2B cells (androgen receptor positive) developed multicellular spheroids, while PC-3 cells (androgen receptor negative) formed only clusters.

To design a chemotherapeutic screening tool, PCa cells were co-cultured with fibroblasts. Along this line, Fontana et al. ^[5] explored the impact of the 3D structure on the development of some primary EMT markers in cultured human DU145 and PC3 cells in 2D monolayers or 3D spheroids. Authors found that several EMT markers, like E-cadherin, are more expressed in 3D spheroids than in 2D monolayers.

This finding helps to understand the role of EMT in PCa and indicates that a 3D model of cell culture may provide further knowledge in cancer biology.

3. Liver Cancer

The use of 3D spheroid culture of hepatocellular carcinoma (HCC) cells is promising for understanding tumor–TME interactions and the mechanistic details of chemotherapeutic resistance ^[6].

Hepatic carcinoma-derived cell lines, like HepG2, C3A, HepaRG, and HuH6, are widely used due to their unlimited growth, availability, and high reproducibility of results. For a better understanding of genotoxicity, Stampar et al. ^[7] developed a HepG2 3D spheroid model and analyzed the mRNA expression profile of genes coding for cell proliferation, drug-metabolizing enzymes, transporters, and liver-specific factors. The findings showed a time-dependent reduction in cell proliferation, with cell division arrested in both the non-proliferating and proliferating phases of the cell cycle. Furthermore, the spheroids showed improved liver-specific activities as well as substantial physiological significance regarding gene expression of hepatic markers and metabolic enzymes.

The main message is that the initial cell density for spheroid formation is essential in order to produce spheroids with viable dividing cells, a prerequisite for investigating the adverse geno-/toxic effects.

In conclusion, HepG2 3D spheroid models provide a reliable assessment of the genotoxic activity of chemicals and may provide an alternative to animal models.

4. Breast Cancer

Triple-negative breast cancer is a highly aggressive form of breast cancer with few therapeutic options since it lacks estrogen and progesterone receptors as well as human epidermal growth factor receptor 2 (HER-2). Altered metabolic pathways are one of the hallmarks of breast cancer, while the concentration of nutrients plays a significant role in the metabolic process of cancer cells.

Bizjak et al. ^[8] used MDA-MB-231 breast cancer cell lines to analyze the effect of glucose, pyruvate, and glutamine on the metformin metabolic reaction in both a 2D monolayer culture model and a 3D spheroid model. The findings showed that the non-essential amino acids inhibited the effect of metformin on MDA-MB-231 cells in both the 2D culture model and the 3D spheroid model. Glutamine and pyruvate weakly diminished the effects of metformin in 2D culture. Under glucose-depleted conditions, metformin suppressed the proliferation of MDA-MB-231 cells, disintegrated tumor spheroids, and reduced cell survival.

The key message is that glucose is probably the major carbon source to sustain the proliferation of metformin-treated cells. As a result, it is reasonable to believe that MDA-MB-231 cells treated with metformin rely on glutamine metabolism only to a limited extent.

According to the above findings, researchers should examine the source of nutrients when analyzing the effectiveness of metformin in 2D culture and biologically more relevant 3D tumor spheroids.

4. Pancreatic Cancer

Pancreatic ductal adenocarcinomas (PDACs) are considered morphologically and functionally heterogeneous. Genetic, transcriptional, and morphological abnormalities have been reported, while researchers found that epithelial or mesenchymal features were more enhanced in 3D cancer models than in 2D models.

Minami et al. ^[9] investigated the morphological and functional differences between eight PDAC cell lines in 2D and 3D cultures.

They found, in 2D cultures, that most PDAC cells exhibited comparable pleomorphic morphologies. PDAC cells with high E-cadherin and low vimentin expression levels (epithelial) formed small round spheres surrounded by flat-lining cells in 3D culture, whereas those with high vimentin and low E-cadherin expression levels (mesenchymal) formed large grape-like spheres without lining cells and were highly proliferative.

In conclusion, the 3D-culture method can be used to investigate the diversity of PDAC cell lines and may play a significant role in developing customized early detection methods and anticancer drugs for PDAC.

5. Thyroid Cancer

Thyroid cancer incidence has increased globally in recent years because of the high population awareness of screening programs, increased laboratory testing and identification in imaging examination, and more accurate diagnostic methods ^[10].

Oh et al. ^[11] studied the expression of thyroid differentiation proteins related to iodide-metabolizing pathways in thyroid cancer cells under various culture conditions. One cell line from the thyroid follicular epithelium (Nthy-Ori 3-1) and four (BCPAP, BHP10-3SCp, K1, and TPC-1) from thyroid cancer were grown on agarose-coated plates in 2D adherent cell culture and 3D spheroid culture.

They found that the proliferation in 3D spheroids was significantly reduced, whereas hypoxia-inducible factor-1 (HIF-1) was upregulated. Moreover, 3D spheroids with thyroid cancers exhibited diminished thyroid differentiation markers, whereas thyroid follicular epithelial cells exhibited either a stable or significant decline in protein expression.

Due to cellular proliferation, hypoxia, ECM, morphology, viability, thyroid differentiation, and cytoskeleton changes, researchers confirmed that the 3D spheroid culture environment could mimic in vivo environments.

6. Lung Cancer

An estimated 1.6 million deaths/year from lung cancer have been recorded globally, with a 10% survival rate in the last five years. Among this, more than 80% of cases are from non-small-cell lung cancer (NSCLC).

Chauhan et al. ^[12] investigated the in vitro efficacy of inhaled erlotinib nanoemulsion in the NSCLC A549 cell line.

In this research, the IC50 for the erlotinib-loaded nanoemulsion was 2.8 times lower than that of the erlotinib-free solution. In addition, ex vivo experiments utilizing a 3D spheroid model demonstrated that erlotinib-loaded nanoemulsion is more effective against NSCLC.

Therefore, synthesized nanoemulsion has the potential to be a promising therapy against NSCLC that can be nebulized locally into the lungs.

7. Ovarian Cancer

Ovarian cancer (OC) is a significant issue, with a five-year survival rate of about 40%. This is due to the lack of evident and consistent symptoms at the beginning of the disease, which causes more than 80% of patients to be detected at severe stages. More relevant in vitro models that mimic the complexity of the OC microenvironment and the dynamics of the OC cell population are needed to understand OC pathophysiology better and improve drug screening. Recent advances in 3D cell culture and microfluidics have enabled the development of highly novel models capable of bridging the gap between pathophysiology and mechanical models for clinical research ^[13].

Fiegl et al. ^[14] analyzed OPRM1 expression, the main receptor and action site of methadone, in OC cell lines and OC tissues. They also investigated pro-angiogenetic, cytotoxic, and apoptotic effects of D,L-methadone in OC cell lines (A2780 A2780Cis, HTB77, OVCAR3, SKOV6, and HOC7) and four patient-derived tumor-spheroid models.

Only OVCAR3 showed OPRM1 expression out of eight at the mRNA and protein level, whilst, in 69% of the analyzed OC tissues, OPRM1-mRNA was detected at a very low level without protein expression. Irrespective of OPRM1 expression, D, L methadone treatment dramatically reduced cell viability in five OC cell lines (SKOV6, OVCAR3, A2780, A2780 Cis, and M019i). D, L-methadone, alone or in combination with cisplatin, had no effect on apoptosis or VEGF secretion in cell lines. There was a significant increase in cell proliferation in two of the four spheroid models after prolonged exposure to D L-methadone, while inhibitory effects of cisplatin in three spheroid models were observed after the addition of D,L-methadone.

In conclusion, the expression of OPRM1 is not necessary for D,L-methadone function in all OC samples. As a result, D,L-methadone may also have negative consequences by promoting the proliferation of certain OC-cells and countering the therapeutic effects of cisplatin.

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