2014;6:1986C2011. in adjacent breast cancer cells. Thus, we propose a model by which chemotherapy-induced catabolism in healthy fibroblasts MAPT constitutes a source of energy-rich nutrients and inflammatory cytokines that would activate stemness in adjacent epithelial cells, possibly triggering new tumorigenic processes. In this context, immune cell recruitment would be also stimulated to further support malignancy. tumorigenesis . One of the most severe side-effects of cancer treatment is actually the growth of a second primary tumour, an entirely new malignancy different from metastatic growth. Second primary cancers already account for one in six new cancer diagnoses in the USA and are a significant cause of mortality amongst patients who have been cured of cancer, being the leading cause of death in Hodgkin Lymphoma AC710 survivors . Increased risk of developing a second malignancy has been reported after treatment with either radiotherapy or chemotherapy agents, including alkylating agents, topoisomerase inhibitors and anti-metabolites, and it is dose-dependent . Nevertheless, high radiation doses concentrated on a limited field are less tumorigenic than lower doses exposed to a wider area . Hence, considering the systemic nature of chemotherapeutic, the influence of therapy-damaged non-target cells on the risk of developing a subsequent malignancy becomes significant. In this study, we test the hypothesis that chemotherapy promotes the same responses in human fibroblasts as their interaction with cancer cells, the so-called catabolic tumour stroma phenotype, which would constitute an ideal environment for a new neoplastic lesion to develop. We provide evidence of treatment-induced modifications in fibroblastic cells including the expression of SMA, a glycolytic switch, an activation of the JNK/AP1, HIF1, TGF/SMAD, STAT3 and NFkB stress-induced pathways, senescence and autophagy, and a greater secretion of AC710 the inflammatory cytokine interleukin 6 (IL6). Finally AC710 we show the induction of several stemness, antioxidant and immunogenic signalling pathways in breast cancer cells when co-cultured with stromal fibroblasts in response to chemotherapy. RESULTS To study the effects of chemotherapy on stromal cell behaviour, human fibroblasts were treated with different drugs and the induction of the catabolic tumour stroma phenotype was assessed. We first hypothesised that chemotherapy would increase lactate and ketone production, as we previously showed their tumourigenic and metastatic effects . As a first screening, L-lactate and -hydroxybutyrate (-HB) concentration was measured in hTERT-BJ1 fibroblasts after treatment with 12 different commonly used drugs (Table ?(Table1)1) including azathioprine, carboplatin, cisplatin, cyclophosphamide, doxorubicin, 5-fluorouracil, gemcitabine, methotrexate, 6-mercaptopurine, mitoxantrone, 6-thioguanine and taxol, detecting an increase in their production of L-lactate or -HB compared to vehicle (Table S1-S2). Six agents were selected to proceed with the study according to their nature and their potential on increasing L-lactate and -HB production at concentrations lower than 1 mM. Azathioprine (AZA), an anti-metabolite and carboplatin (CP) and cisplatin (CIS), both alkylating-like agents, were chosen and used for further studies at a concentration of 100 M. Likewise, doxorubicin (DOX) and mitoxantrone (MTX), both topoisomerase inhibitors and taxol (TAX), a cytoskeleton drug, were selected at 100 nM. All chosen concentrations were sub-lethal and caused a decrease in cell viability lower than 50% after 72 h of treatment (Figure S1). Table 1 Chemotherapeutic agents used in the current study tumorigenesis. We hypothesised that therapy-induced stromal IL6 secretion would stimulate STAT3 in neighbouring cancer cells. Likewise, we assessed the effects of stromal cells on antioxidant (ARE), immune response (ISRE, GAS), or stem cell signalling (GLI, TCF/LEF, SMAD) in cancer cells when co-cultured with fibroblasts upon treatment. Therefore, several reporter MCF7-GFP cell lines were generated (Table ?(Table2),2), and cultured either as monolayers or co-cultured with fibroblasts, treated with chemotherapy, and assessed for luciferase activity. We first evaluated ARE-dependent signalling AC710 pathway. Azathioprine and cisplatin significantly increased luciferase signal in MCF7-GFP-ARE(luc) monocultures, whereas doxorubicin, mitoxantrone and taxol significantly decreased it (Figure ?(Figure7A).7A). ARE signalling was inhibited when fibroblasts were present in the culture. However, azathioprine, mitoxantrone and taxol co-culture treatments did not inhibit luciferase activity compared to vehicle, showing a reactivation of ARE signalling.