Qianyi Huang1,2, Joscha Kraske1,2, Frederik Glatting1,2, Li Li1,2, Thomas Walle1, Bingwen Zou1,2, Alexandra Tietz-Dalfuß 1, Sylvia Trinh1,2, Dorothee Albrecht1, Ramon Lopez Perez1,2, Peter Huber1,3
1German Cancer Research Center (DKFZ), Molecular and Radiation Oncology, Heidelberg, Germany; 2University Hospital Heidelberg, Radiation Oncology, Heidelberg, Germany; 3 University Hospital Heidelberg, Radiation Oncology, Heidelberg, Germany
Radiotherapy for cancer of organs in the thorax including lung cancer, breast cancer or Hodgkin’s disease can lead to unwanted irradiation of the heart. Because the heart in general is considered relatively radioresistant, the symptoms of radiotherapy-induced heart disease (RIHD) such as fibrosis may require decades to manifest. However RIHD is becoming more important due to the increasing cancer patient survival, and RIHD may substantially contribute to the causes of death in these cancer survivors. Despite the many clinical data, preclinical and cell mechanistic investigations are sparse. Therefore, here we hypothesized that the interplay of immune cells with cardiac fibroblasts play a role in radiation induced cardiac fibrogenesis, and analyzed in vitro the TGFß, PDGF, and CTGF related signaling program including senescence potentially playing a role in this process.
To this end we established a radiation induced primary human cardiac fibroblast (HCF) - human macrophage (from PBMC monocytes) activation model, measured proliferation, senescence (ß-gal), clonogenic survival, analyzed protein expression by Western blot and ELISA, and transcriptomic changes in the cells alone and in coculture with and without specific small molecule inhibitors of PDGF and TGFß signaling (Sunitinib/Galunisertib analogues).
We found that radiation upregulated profibrotic genes including αSMA, Col 1a1, Cola 3a1, Col 4a1, Col 5a1, TGFß1, CTGF, chemokines such as IL6 and senescence markers in HCF on the mRNA or the protein level. The upregulation of profibrotic signaling was markedly enhanced after coculturing the HCF with macrophages, after direct and indirect cell contact, and additionally enhanced after radiation. This profibrotic macrophage effect was associated with predominantly M2 marker polarization after HCF coculture which was in turn enhanced after radiation, and accompanied by IL10 and IL1b induction. Collectively, inhibition of PDGF and TGF-ß signaling reduced the profibrotic effects in the HCF/macrophage system.
In conclusion our results suggest that macrophage - human cardiac fibroblast interactions prompted by radiation play an important role in the initiation of cardiac fibrosis, that TGF-β, CTGF and PDGF have a role in this process, and that inhibitors of these pathways may be able to attenuate RIHD.