title: The in vitro immunogenic potential of caspase-3 proficient breast cancer cells with basal low immunogenicity is increased by hypofractionated irradiation.
abstract: BACKGROUND
  Radiotherapy is an integral part of breast cancer treatment. Immune activating properties of especially hypofractionated irradiation are in the spotlight of clinicians, besides the well-known effects of radiotherapy on cell cycle and the reduction of the clonogenic potential of tumor cells. Especially combination of radiotherapy with further immune stimulation induces immune-mediated anti-tumor responses. We therefore examined whether hypofractionated irradiation alone or in combination with hyperthermia as immune stimulants is capable of inducing breast cancer cells with immunogenic potential.
  
  
  METHODS
  Clonogenic assay, AnnexinA5-FITC/Propidium iodide assay and ELISA analyses of heat shock protein 70 and high mobility group box 1 protein were applied to characterize colony forming capability, cell death induction, cell death forms and release of danger signals by breast cancer cells in response to hypofractionated radiation (4x4Gy, 6x3Gy) alone and in combination with hyperthermia (41.5 °C for 1 h). Caspase-3 deficient, hormone receptor positive, p53 wild type MCF-7 and caspase-3 intact, hormone receptor negative, p53 mutated MDA-MB231 breast cancer cells, the latter in absence or presence of the pan-caspase inhibitor zVAD-fmk, were used. Supernatants of the treated tumor cells were analyzed for their potential to alter the surface expression of activation markers on human-monocyte-derived dendritic cells.
  
  
  RESULTS
  Irradiation reduced the clonogenicity of caspase deficient MCF-7 cells more than of MDA-B231 cells. In contrast, higher amounts of apoptotic and necrotic cells were induced in MDA-B231 cells after single irradiation with 4Gy, 10Gy, or 20Gy or after hypofractionated irradiation with 4x4Gy or 6x3Gy. MDA-B231 cells consecutively released higher amounts of Hsp70 and HMGB1 after hypofractionated irradiation. However, only the release of Hsp70 was further increased by hyperthermia. Both, apoptosis induction and release of the danger signals, was dependent on caspase-3. Only supernatants of MDA-B231 cells after hypofractionated irradiation resulted in slight changes of activation markers on dendritic cells; especially that of CD86 was upregulated and HT did not further impact on it.
  
  
  CONCLUSIONS
  Hypofractionated irradiation is the main stimulus for cell death induction and consecutive dendritic cell activation in caspase proficient breast cancer cells. For the assessment of radiosensitivity and immunological effects of radio- and immunotherapies the readout system is crucial.
authors.affiliation: Department of Radiation Oncology, University Hospital Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany. bernhard.koetter@gmail.com.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Kötter
authors.affiliation: Department of Radiation Oncology, University Hospital Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany. benjamin.frey@uk-erlangen.de.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Frey
authors.affiliation: Department of Radiation Oncology, University Hospital Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Winderl
authors.affiliation: Department of Radiation Oncology, University Hospital Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany. yvonne.rubner@uk-erlangen.de.
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Rubner
authors.affiliation: Department of Radiotherapy and Radiation Oncology, Ludwig Maximilian University Munich, D-81377, Munich, Germany. Heike.Scheithauer@med.uni-muenchen.de.
authors.forename: Heike
authors.initials: H
authors.lastname: Scheithauer
authors.affiliation: Department of Radiation Oncology, University Hospital Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany. renate.sieber@uk-erlangen.de.
authors.forename: Renate
authors.initials: R
authors.lastname: Sieber
authors.affiliation: Department of Radiation Oncology, University Hospital Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany. rainer.fietkau@uk-erlangen.de.
authors.forename: Rainer
authors.initials: R
authors.lastname: Fietkau
authors.affiliation: Department of Radiation Oncology, University Hospital Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany. udo.gaipl@uk-erlangen.de.
authors.forename: Udo S
authors.initials: US
authors.lastname: Gaipl
mesh_terms: D001943:Breast Neoplasms / Q000276:immunology* / Q000378:metabolism / Q000532:radiotherapy*; D053148:Caspase 3 / Q000378:metabolism*; D016923:Cell Death / Q000528:radiation effects; D045744:Cell Line, Tumor; D003713:Dendritic Cells / Q000276:immunology / Q000528:radiation effects; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D006979:Hyperthermia, Induced / Q000379:methods; D066298:In Vitro Techniques; D000069473:Radiation Dose Hypofractionation; D011836:Radiation Tolerance / Q000502:physiology; D011878:Radiotherapy / Q000379:methods*
chemical_list: C505407:CASP3 protein, human; D053148:Caspase 3