Chemotherapy is a mainstay in breast cancer management, but recurrence and metastasis driven by chemoresistance remain major clinical problems. While prior work has emphasized tumor‑cell intrinsic mechanisms, there is growing evidence that cytotoxic chemotherapy also triggers host‑cell responses within the tumor microenvironment (TME) that can promote tumor progression. This study aimed to determine whether cancer‑associated fibroblasts (CAFs) exposed to paclitaxel (PTX) or doxorubicin (DOX) acquire pro‑tumorigenic properties, to identify key mediators, and to evaluate clinical relevance using integrated multi‑omics and experimental approaches.
The authors analyzed serum from 50 patients undergoing neoadjuvant chemotherapy (NAC) and paired tumor tissue from 100 patients before and after NAC; all had primary invasive ductal breast carcinoma and received PTX‑ or DOX‑based NAC. Primary CAFs and normal fibroblasts (NFs) were isolated from surgical specimens and cultured to generate conditioned medium (CM). Breast cancer cell lines (MCF‑7, MDA‑MB‑231) and patient‑derived tumor‑like cell clusters (PTCs) were used for functional assays. Data sources included GEO datasets (GSE23399, GSE268662), TCGA‑BRCA bulk RNA‑seq, and GWAS eQTL and circulating protein GWAS datasets. Approaches combined wet‑lab assays (CCK‑8, wound healing, invasion, colony formation, ELISA, qPCR, Western blot, IHC, TUNEL) with bioinformatics (BayesPrism deconvolution, scPagwas trait scoring) and Mendelian randomization (MR).
CAFs were exposed to low concentrations of PTX or DOX for 24 hours, washed, and CM collected after serum‑free culture. CM from CAFs stimulated proliferation, wound closure, and invasion of MCF‑7 and MDA‑MB‑231 cells. Importantly, CM from CAFs pretreated with PTX or DOX produced a greater increase in these pro‑tumorigenic phenotypes than CM from untreated CAFs, establishing that chemotherapy exposure enhances the tumor‑promoting activity of CAFs.
Analysis of GSE23399 and subsequent validation by qPCR and ELISA demonstrated that both PTX and DOX increased IL‑8 expression and secretion from CAFs in a dose‑ and time‑dependent manner. Immunohistochemistry on paired patient samples before and after NAC showed increased stromal IL‑8 post‑NAC, with IL‑8 predominantly localized to α‑SMA+ CAFs. CAFs produced higher IL‑8 than tumor cells or NFs; chemotherapy also elevated IL‑8 in NFs but to a much lower extent than in CAFs. Co‑culture of CAFs with tumor cells further increased IL‑8 secretion from CAFs.
To test whether IL‑8 mediates the pro‑tumorigenic effects of chemotherapy‑exposed CAFs, the IL‑8 receptor inhibitor reparixin was used. Reparixin partly reversed the enhancement of migration, invasion, and survival conferred by CM from treated CAFs, indicating that CAF‑derived IL‑8 is a key mediator of the observed tumor‑promoting activity. Additional in vitro experiments confirmed that manipulating IL‑8 expression in CAFs altered tumor cell responses to chemotherapy.
Mendelian randomization analyses using IL‑8 eQTL data and circulating IL‑8 GWAS summary statistics found that genetically predicted IL‑8 expression and plasma IL‑8 levels were significantly associated with breast cancer risk. The MR framework used the inverse‑variance weighted (IVW) method with MR‑Egger and Cochran’s Q tests to evaluate pleiotropy and heterogeneity. Deconvolution of TCGA‑BRCA bulk RNA‑seq with BayesPrism and trait scoring using scPagwas linked IL‑8+ CAFs to clinical prognosis, suggesting these CAFs may contribute to disease progression. Exact effect sizes and p‑values are reported in the original source.
Mechanistic studies indicated that CAF‑derived IL‑8 can activate the NF‑κB signaling pathway in tumor cells, modulate apoptotic regulators (increased Bcl‑2 and Bcl‑xL, altered Bax), reduce TUNEL‑positive apoptotic cells, and thereby inhibit chemotherapy‑induced apoptosis. These changes provide a plausible pathway by which chemotherapy‑exposed CAFs and their IL‑8 secretion promote chemoresistance and subsequent tumor progression.
In a xenograft model, exogenous IL‑8 administration promoted tumor growth relative to controls. Clinical correlations derived from patient samples and Kaplan‑Meier analyses indicated that higher IL‑8 expression associated with chemoresistance and poorer relapse‑free and distant metastasis‑free survival in breast cancer cohorts.
This integrated multi‑omics and experimental study identifies IL‑8+ CAFs as mediators of chemotherapy‑induced tumor progression in breast cancer. The findings support the rationale for combining chemotherapy with strategies that target IL‑8 signaling (for example, IL‑8 receptor blockade) to limit unintended pro‑tumorigenic host responses and potentially improve therapeutic outcomes.
The source provides methodological detail and reports significant associations across multiple platforms. Specific MR effect estimates, detailed statistical values for some analyses, and some experimental replicates are provided in the original article but are not reproduced here; readers should consult the published article for exact numeric results and supplementary figures referenced in the source.