# Optimising the method to develop spheroids from MDA-MB-468 human triple negative breast cancer cells

## TL;DR

This study optimizes methods to grow 3D spheroids from a breast cancer cell line, finding that normoxic conditions and specific techniques produce the most consistent and biologically relevant models.

## Contribution

A novel workflow combining hanging-drop pre-aggregation with ULA-plates under normal oxygen yields compact, structurally stable spheroids with reliable gene expression.

## Key findings

- Spheroids grown under normoxic conditions showed better structural integrity and gene expression patterns.
- Hanging-drop pre-aggregation with ULA-plates produced the most compact and resilient spheroids.
- HIF1A and VEGFA were more highly expressed under normoxic conditions, indicating pseudo-hypoxic signaling.

## Abstract

Three-dimensional (3D) spheroid models are increasingly used to emulate the tumour microenvironment for preclinical drug screening. This study aimed to optimise and assess spheroid formation from MDA-MB-468 triple-negative breast cancer (TNBC) cells using hanging drop, liquid overlay, and rigid scaffold methods under normal oxygen (NOC) and low oxygen (LOC) culture conditions.

Spheroids were generated and characterised using bright-field microscopy with AnaSP morphometrics (sphericity, solidity, and perimeter). Gene expression of Epithelial-Mesenchymal Transition (EMT), stemness, and hypoxia/angiogenesis markers (CD44, HIF1A, VEGFA, TWIST1, SNAI1, and NES) was quantified using qPCR. The optimised model was further evaluated using field-emission scanning electron microscopy (FE-SEM) and Hoechst fluorescence.

A workflow combining hanging-drop pre-aggregation with ultra-low attachment (ULA) or agarose-coated plates under NOC produced consistent, compact spheroids. Scaffold cultures formed rapidly but showed size variability under NOC and LOC. Across methods, spheroids were less compact, and gene expression patterns deviated from expected hypoxic responses. HIF1A and VEGFA were more highly expressed under NOC, suggesting pseudo-hypoxic signalling and activation of angiogenesis-related pathways. CD44 and TWIST1 were upregulated in most spheroid types, whereas SNAI1 and NES exhibited condition- and method-specific variability.

Spheroids cultivated under normoxic conditions demonstrated enhanced structural integrity and transcriptional fidelity. Nonetheless, the study identified that the most compact and resilient spheroids were achieved through the use of hanging-drop pre-aggregation combined with ULA-plates under NOC. The enhanced structural integrity and transcriptional fidelity observed in these spheroids make them valuable models for studying cancer biology and drug responses.

The online version contains supplementary material available at 10.1007/s11033-026-11451-4.