Stromal Remodeling of the Cytokine Secretome Reveals a Chemokine Core Across Breast Cancer and Glioma 3D Cell Culture Models

Mounting evidence indicates that the tumor microenvironment plays a decisive role in cancer progression. However, the quantitative assessment of how individual stromal components contribute to cytokine secretion in 3D models remains poorly understood, especially when comparing tumours of different origins. The development of 16 groups of 3D spheroid cultures, encompassing stromal monocultures, two-component models (tumor + endothelium/glia), and three-component models (tumor + endothelium + fibroblast; glioma + astrocytes + microglia), was conducted. These models represent breast cancer and glioma cell culture models. The secretion of 32 distinct cytokines was measured using xMAP technology. Induction was defined as log2FC ≥ 1 with bootstrap-estimated 95% confidence intervals. The induced cytokines were subsequently classified into two operational categories: “emergent” (stromal monoculture level below 3D-2) or “stroma-contributing” (stromal level ≥ 3D-2). In the breast cancer model, a consensus across all cell lines identified seven cytokines (HGF, IL-10, LIF, MCP-1, MCP-3, MIP-1α, and SCGF-β). Cancer-associated fibroblasts (CAFs) were associated with a broader spectrum than normal fibroblasts (28 vs. 23), exhibiting a complete overlap in their repertoire. Triple-negative breast cancer (MDA-MB-231) showed minimal secretion (7–8 cytokines), with decreased secretion of GM-CSF and IP-10; b-FGF levels were reduced in six of eight comparisons. In the glioma model based on U87MG cells, the addition of astrocytes (CCF-STTG1) coincided with increased secretion of 13 different cytokines, whereas microglia (HMC3) were associated with 8 cytokines. The cross-tumor intersection comprised 14 cytokines (7 chemokines, 5 growth factors, and 2 immunoregulatory cytokines), with maximal production observed for LIF, IL-8, MCP-3, MIP-1α, and IL-6. These findings indicate that the addition of stromal cells is associated with both common and tissue-specific changes in the cytokine profile, and that CAFs display a broader secretory repertoire than normal fibroblasts in this model.