Expanding the use of monoclonal antibody therapy of cancer by using ionising radiation to upregulate antibody targets

Abstract

Background: Monoclonal antibody (mAb) therapy for the treatment of solid and haematologic malignancies has shown poor response rates as a monotherapy. Furthermore, its use is limited to tumours expressing certain molecular targets. It has been shown that single-dose radiation can induce immunogenic modulation that is characterised by cell-surface phenotypic changes leading to augmented tumour cell/cytotoxic T-cell interaction.

Methods: We examined radiation's ability to upregulate mAb therapy targets. We also used radiation to sensitise tumour cells to antibody-dependent cell-mediated cytotoxicity (ADCC).

Results: Radiation significantly increased cell-surface and total protein expression of mAb targets HER2, EGFR, and CD20. Focusing on HER2, targeted by trastuzumab, we observed significant upregulation of HER2 following radiation of 3 out of 3 breast cancer cell lines, one of which was triple negative, as well as in residential stem-cell populations. HER2 upregulation was sustained up to 96 h following radiation exposure and was largely dependent on intracellular reactive oxygen species. Improved ADCC and sensitisation to the antiproliferative effects of trastuzumab demonstrated the functional significance of radiation-induced HER2 upregulation.

Conclusions: We show that single-dose radiation enhances mAb therapy. These findings highlight a mechanism for combining radiation with immunotherapy and expand the patient population that can be treated with targeted therapy.

Figures

Figure 1

Radiation increases total and cell-surface expression of target molecules. Tumour-cell lines were mock-irradiated (open histograms) or treated with radiation (shaded histograms). After 48 h of culture, expression of target molecules was examined by flow cytometry and western blot analysis. (A) Expression of HER2 in MCF-7 breast carcinoma cells. (B) Expression of EGFR in HN4 HNSCC cells. (C) Expression of CD20 in Ramos NHL cells. Insets: numbers indicate % positive cells and MFI (parentheses). Cells used for analysis were viable as determined by 7AAD staining, and staining was corrected for isotype control binding. This experiment was repeated two times with similar results. Comparisons of protein expression HER2 (A), EGFR (B), or CD20 (C) were determined by using band densitometry analysis using ImageJ software normalising β-actin relative to control. ‘*' denotes statistical significance relative to untreated cells (P<0.05).

Figure 2

Radiation-induced upregulation of HER2 cell-surface expression visualised by immunofluorescence. (A) ZR75-1 breast carcinoma cells were mock irradiated (open histograms) or irradiated with 10 Gy (shaded histograms), and HER2 expression was examined by flow cytometry and western blot 48 h post exposure. Insets: numbers indicate % positive cells and MFI (parentheses). Cells used for analysis were viable as determined by 7AAD staining, and staining was corrected for isotype control binding. ‘*' denotes statistical significance relative to untreated cells (P<0.05). (B) ZR75-1 cells were mock irradiated or irradiated with 10 Gy, and HER2 expression was examined by immunofluorescence 48 h post exposure. Nuclei were stained with DAPI; images represent × 20 magnification. Insets: average HER2 expression per cell corrected by total cell fluorescence±s.d. The images presented are representative of three independent experiments with similar results.

Figure 3

Radiation increased HER2 expression in TNBC cells. (A) The TNBC cell line MDA-MB-231 was mock irradiated (open histograms) or irradiated with 10 Gy (shaded histograms), and HER2 expression was examined by flow cytometry 48 h post exposure. Inset: Numbers indicate % positive cells and MFI (parentheses). (B, C) Stem-cell populations of (B) mock-irradiated and (C) irradiated (10 Gy) MDA-MB-231 cells were examined by flow cytometry. Insets: numbers indicate % positive cells. (D) HER2 expression of gated MDA-MB-231 stem-cell populations, as shown in B, C respectively, that were mock-irradiated (open histograms) or irradiated with 10 Gy (shaded histograms). Inset: numbers indicate % positive cells and MFI (parentheses). ‘*' denotes statistical significance relative to untreated cells (P<0.05).

Figure 4

Radiation-induced HER2 upregulation is time- and dose dependent. (A) MCF-7 cells were mock-irradiated (open circles) or irradiated with 10 Gy (closed circles). After treatment, cells were harvested daily up to 96 h and examined for HER2 expression by flow cytometry. (B) MCF-7 cells were mock-irradiated (open circles) or irradiated with 5 Gy (closed diamonds). After treatment, cells were harvested daily up to 96 h and examined for HER2 expression by flow cytometry. Relative fluorescence was calculated as treated sample MFI÷control sample MFI at each time point. ‘*' denotes statistical significance relative to mock-irradiated cells at each 24-h time point (P<0.05). This experiment was repeated three additional times with similar results.

Figure 5

Radiation-induced HER2 upregulation is mediated by ROS and associated with phosphorylation of NF-κB. (A) Intracellular ROS and cell-surface HER2 were measured by flow cytometry. MCF-7 cells were mock irradiated (open histograms), irradiated with 10 Gy (shaded histograms), treated with ROS inhibitor (N-acetyl-L-cysteine) (shaded histograms), or irradiated with 10 Gy in the presence of ROS inhibitor (shaded histograms). (B) MFI of ROS measurements taken from histograms in A. (C) MFI of HER2 cell-surface expression taken from histograms in A. (D) MCF-7 cells were mock irradiated, irradiated with 10 Gy, treated with ROS inhibitor, or irradiated with 10 Gy in the presence of ROS inhibitor. Expression of HER2, phospho-NF-κB-p65, and β-actin was examined by western blot analysis 24 h post treatment. ‘*' denotes statistical significance (P<0.05). The data presented is representative of three independent experiments with similar results.

Figure 6

Radiation exposure augments tumour-cell killing in an in vitro ADCC assay and sensitises tumour cells to the antiproliferative effects of trastuzumab. (A) Peripheral blood mononuclear cells isolated from a normal donor were used as effector cells. Number indicates percent NK cells in sample. MCF-7 cells were mock irradiated or irradiated with 10 Gy 48 h before the ADCC assay. Triplicate wells of untreated cells (open symbols) and irradiated cells (closed symbols) were incubated with trastuzumab (triangles), isotype control antibody (rituximab) (circles), or left untreated (squares). (B) Natural killer cells were purified from the normal-donor PBMCs used in A and used as effector cells. Number indicates NK cell purity. MCF-7 cells were mock-irradiated or irradiated with 10 Gy 48 h before the ADCC assay. Three replicate wells of untreated cells (open symbols) and irradiated cells (closed symbols) were incubated with trastuzumab (triangles), incubated with isotype control antibody (rituximab) (circles), or left untreated (squares). ‘*' denotes statistical significance relative to untreated cells incubated with trastuzumab (P<0.05). (C) MCF-7 cells were irradiated (mock or 10 Gy) and cultured in triplicate wells of a 96-well plate for 4 days. Trastuzumab or isotype control antibody (rituximab) was added for the final 48 h of culture. Proliferation was then measured by incorporation of 3H-thymidine, which was added for the final 24 h of culture. The data presented is representative of three independent experiments with similar results. Data were analysed using a non-paired Student's t-test. ‘*' denotes statistical significance (P<0.05).