Rademaekers Max, Johansson Emil Oliver, Johansson Ellen, Roberg Karin, Wiechec Emilia
Division of Cell Biology, Department of Biomedical and Clinical Sciences, Linköping University, Linköping, Sweden.
Department of Otorhinolaryngology, Region Östergötland, Linköping, Sweden.
Cancer Cell Int. 2024 May 31;24(1):190. doi: 10.1186/s12935-024-03388-0.
Cancer-associated fibroblasts (CAFs) are the major cellular component of the tumor microenvironment and are known to affect tumor growth and response to various treatments. This study was undertaken to investigate the crosstalk between tumor-matched or unmatched CAFs and head and neck squamous cell carcinoma (HNSCC) cells regarding tumor growth and treatment response.
Three HNSCC cell lines (LK0412, LK0902 and LK0923), were cocultured in 2D or in 3D with their tumor-matched CAFs, site matched CAFs from other tumors or normal oral fibroblasts (NOFs). Cell proliferation was assessed as the amount of Ki67 positive cells/ spheroid area in formalin-fixed- paraffin-embedded 3D spheroids stained with Ki67 antibody. Viability after seven days of cisplatin treatment was measured with CellTiter-Glo 3D Viability Assay. The mRNA expression of CAF-associated markers (ACTA2, COL1A2, FAP, PDGFRα, PDGFRβ, PDPN, POSTN and S100A4) in CAFs before and after coculture with tumor cells as well as mRNA expression of CAF-induced genes (MMP1, MMP9 and FMOD) in tumor cells separated from CAFs after co-culture was measured with RT-qPCR. The expression of selected protein biomarkers was validated with immunohistochemistry based on previous mRNA expression results.
The proliferation of the LK0412 and LK0902 tumor spheroids varied significantly when cocultured with different CAFs and NOFs as shown by Ki-67 positive cells. RT‒qPCR analysis revealed different molecular profile of the analyzed HNSCC-derived CAFs concerning the expression of CAF-associated markers. The interaction between CAFs and HNSCC cells was more pronounced after coculture with unmatched CAFs as shown by changes in mRNA expression pattern of CAF-specific markers. Additionally, the unmatched CAFs significantly upregulated the mRNA expression of MMP1, MMP9 and FMOD in tumor cells compared to tumor-matched CAFs.
Our results indicate that tumor-matched CAFs are unique for each tumor and affect the proliferation and the gene/protein expression of tumor cells in a distinct manner. The interaction between tumor unmatched CAFs and HNSCC cells in the tumor spheroids is associated with significant changes in the mRNA expression of CAF-specific markers and significant increases in FMOD and MMP9 in tumor cells compared to when cocultured with tumor-matched CAFs. Taken together, our results show how important the selection of CAFs is to get a reliable in vitro model that mimics the patients' tumor.
癌症相关成纤维细胞（CAFs）是肿瘤微环境的主要细胞成分，已知其会影响肿瘤生长及对各种治疗的反应。本研究旨在探究肿瘤匹配或不匹配的CAFs与头颈部鳞状细胞癌（HNSCC）细胞之间在肿瘤生长和治疗反应方面的相互作用。
将三种HNSCC细胞系（LK0412、LK0902和LK0923）与它们肿瘤匹配的CAFs、来自其他肿瘤的部位匹配的CAFs或正常口腔成纤维细胞（NOFs）进行二维或三维共培养。通过用Ki67抗体对福尔马林固定石蜡包埋的三维球体进行染色，将细胞增殖评估为Ki67阳性细胞数量/球体面积。用CellTiter - Glo 3D活力测定法测量顺铂处理七天后的活力。用RT - qPCR测量共培养前后CAFs中CAF相关标志物（ACTA2、COL1A2、FAP、PDGFRα、PDGFRβ、PDPN、POSTN和S100A4）的mRNA表达，以及共培养后从CAFs分离的肿瘤细胞中CAF诱导基因（MMP1、MMP9和FMOD）的mRNA表达。基于先前的mRNA表达结果，用免疫组织化学验证所选蛋白质生物标志物的表达。
如Ki - 67阳性细胞所示，当LK0412和LK0902肿瘤球体与不同的CAFs和NOFs共培养时，其增殖有显著差异。RT - qPCR分析显示，所分析的源自HNSCC的CAFs在CAF相关标志物表达方面具有不同的分子特征。如CAF特异性标志物mRNA表达模式的变化所示，与不匹配的CAFs共培养后，CAFs与HNSCC细胞之间的相互作用更为明显。此外，与肿瘤匹配的CAFs相比，不匹配的CAFs显著上调肿瘤细胞中MMP1、MMP9和FMOD的mRNA表达。
我们的结果表明，肿瘤匹配的CAFs对每个肿瘤都是独特的，并以独特的方式影响肿瘤细胞的增殖以及基因/蛋白质表达。与与肿瘤匹配的CAFs共培养时相比，肿瘤球体中肿瘤不匹配的CAFs与HNSCC细胞之间的相互作用与CAF特异性标志物的mRNA表达显著变化以及肿瘤细胞中FMOD和MMP9的显著增加相关。综上所述，我们的结果表明选择CAFs对于获得模拟患者肿瘤的可靠体外模型是多么重要。
