# Establishment of a 3D Multicellular HCC Tumor Spheroid Model to Unravel Nrf2’s Influence on the Tumor Immune Microenvironment

## TL;DR

Researchers created a 3D tumor model to study how Nrf2 affects the tumor immune environment in liver cancer.

## Contribution

A novel 3D multicellular tumor spheroid model was developed to investigate Nrf2's role in hepatocellular carcinoma's immune microenvironment.

## Key findings

- 3D spheroids preserved stemness better than 2D cultures and showed Nrf2-dependent Vegf-α secretion.
- Macrophages integrated into spheroids triggered invasive growth, while Nrf2-deficient macrophages reduced tumor growth and PD-L1 expression.
- The MCT model revealed that Nrf2 signaling fosters an immunosuppressive and pro-invasive tumor environment.

## Abstract

Hepatocellular carcinoma (HCC) remains a leading cause of cancer-related death, yet adequate in vitro models mimicking the tumor immune microenvironment (TIME) are rare. Specifically, the role of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) in modulating interactions between tumor cells and tumor-associated macrophages (TAMs) is not fully understood. We established a 3D multicellular tumor spheroid (MCT) model using murine N-HCC25 cells with CRISPR/Cas9-mediated knockouts of Nrf2 and its negative regulator Kelch-like ECH-associated protein 1 (Keap1), the latter mimicking constitutive activation. N-HCC25 cells were co-cultured with bone marrow-derived macrophages (BMDMs) isolated from wild-type and Nrf2-knockout C57BL/6J mice. We compared co-culture setups (conditioned media, transwell systems, direct contact) using RT-qPCR, flow cytometry, and invasion assays. 3D spheroid systems better preserved stemness than 2D cultures and revealed functional Nrf2-dependent effects such as increased Vegf-α secretion in Keap1-deficient spheroids. Among the different co-cultivation models, the most profound effects were observed in the MCT model. Macrophages successfully integrated into the spheroids and triggered invasive outgrowth, whereas MCTs containing Nrf2-deficient macrophages displayed markedly reduced tumor spheroid growth and lower programmed cell death ligand-1 expression. These findings demonstrate that Nrf2 signaling in macrophages fosters an immunosuppressive and pro-invasive microenvironment. The established MCT model provides a suitable platform to further unravel Nrf2-dependent mechanisms in the HCC TIME.