Dual blockade of CD47 and HER2 eliminates radioresistant breast cancer cells

Radiotherapy (RT) is increasingly used to treat breast cancer (BC) due to its relatively less systemic side effects compared to chemotherapy. Meta-analyses have shown significant benefits in reducing recurrence and mortality. However, tumor radioresistance remains a challenge, leading to recurrent and metastatic disease. Combining RT with targeted immunotherapy (TI) offers potential improvements. Radiation induces changes in tumor cells and the surrounding stroma, influencing immune responses. Beyond well-known immune checkpoints like PD-1 and PD-L1, several other regulators are involved. Understanding the communication between RT-surviving tumor cells and immune cells is crucial for enhancing RT efficacy with TI. CD47, a myeloid-specific immune checkpoint protein, is expressed in many cancers. It binds to SIRPα on macrophages, inhibiting phagocytosis. Humanized CD47 antibodies are under development to enhance macrophage-mediated tumor cell clearance and show anti-tumor efficacy with tolerable toxicity. Besides promoting phagocytosis, CD47 blockade improves antigen presentation and inhibits breast cancer stem cell (BCSC) aggressiveness. The interplay between CD47 and growth factors, especially in radioresistant cells, requires further investigation. This study focuses on the coordinated regulation of CD47 and HER2 in radioresistant BC cells to elucidate the mechanisms of radioresistance and explore a potential therapeutic strategy.

The existing literature supports the efficacy of radiotherapy in breast cancer treatment, although radioresistance remains a significant hurdle. Studies have highlighted the involvement of various microRNAs and interferon-stimulated genes in mediating radioresistance. The combination of radiotherapy and immunotherapy is emerging as a promising therapeutic approach, with ongoing clinical trials investigating the synergistic benefits. Research has established the role of CD47 as an immune checkpoint, where its blockade enhances macrophage-mediated tumor cell clearance. Furthermore, evidence suggests that CD47 blockade can also modulate other aspects of the anti-tumor response, including antigen presentation and the suppression of cancer stem cell characteristics. However, the precise mechanisms through which CD47 contributes to radioresistance and its interplay with other growth factors remain incompletely understood. This lack of understanding necessitates further research to optimize treatment strategies.

This study employed a multi-faceted approach involving in silico analysis, in vitro experiments, and in vivo mouse models. The analysis of the TCGA database using the GEPIA web tool was utilized to investigate the expression of CD47 and HER2 across various cancers, including breast cancer. In vitro studies utilized radioresistant breast cancer cell lines (MCF7/C6 and MDA-MB-231/C5) and normal breast epithelial cells (MCF10A) to assess the expression and regulation of CD47 and HER2. Techniques including immunoblotting, flow cytometry, and immunohistochemistry (IHC) were used to analyze protein expression levels. The role of HER2 in regulating CD47 expression was investigated using Lapatinib, a HER2 inhibitor, and CRISPR-mediated gene knockouts. Functional assays, such as mammosphere formation, transwell invasion, and gap-filling assays, were used to assess the impact of CD47 and HER2 on cell aggressiveness. Macrophage-mediated phagocytosis assays were conducted to evaluate the effect of CD47 blockade on immune cell activity. In vivo studies involved the creation of syngeneic mouse orthotopic breast tumors using radioresistant 4T1 cells. The impact of radiotherapy, either alone or in combination with anti-CD47 and/or anti-HER2 antibodies, on tumor growth was assessed. Gene editing techniques such as CRISPR-Cas9 were used to generate cell lines with knockouts of CD47 and HER2. Immunofluorescence was used to analyze the infiltration of macrophages and phagocytosis in tumor tissues. Various statistical tests, including the Student's t-test, ANOVA, and log-rank test, were used for data analysis.

The study revealed a strong correlation between CD47 and HER2 expression in radioresistant breast cancer cells. Both receptors were upregulated in irradiated cells, RT-treated mouse tumors, and recurrent tumors from patients with poor prognosis. Blocking either CD47 or HER2 resulted in reduced expression of both receptors, decreased clonogenicity, and increased phagocytosis. CRISPR-mediated dual knockout of CD47 and HER2 significantly inhibited clonogenicity and enhanced macrophage-mediated attack. The combination of dual antibody blockade of CD47 and HER2 synergized with RT in controlling syngeneic mouse breast tumors. Mechanistically, the study demonstrated that CD47 transcription is regulated via the HER2-NF-κB pathway. Inhibition of HER2 with Lapatinib reduced CD47 expression, while CRISPR-mediated HER2 knockout abolished radiation-induced CD47 expression. Dual blockade of CD47 and HER2 was most effective in enhancing macrophage phagocytosis and radiosensitization, suggesting a synergistic interaction between the two receptors in promoting radioresistance. In vivo studies using radioresistant 4T1/C2 mouse breast tumor models showed that dual CD47/HER2 deficiency enhanced radiosensitivity, leading to improved tumor control. The combination of RT with dual antibody blockade further synergized tumor inhibition compared to either antibody alone.

The findings of this study highlight the crucial role of the coordinated upregulation of CD47 and HER2 in mediating radioresistance in breast cancer. The synergistic effect of dual blockade of these receptors, in combination with radiotherapy, offers a promising therapeutic strategy. The mechanistic link between HER2 and CD47 expression via the NF-κB pathway reveals a novel mechanism of radioresistance. The study's findings support the development of targeted therapies that concurrently target both CD47 and HER2 to overcome radioresistance and improve clinical outcomes in breast cancer patients. The observed synergistic effect of dual blockade underscores the importance of considering multiple therapeutic targets in combating cancer.

This research demonstrates a novel mechanism of radioresistance in breast cancer driven by the coordinated action of CD47 and HER2. The findings highlight the potential of a dual blockade strategy targeting these two receptors, in combination with radiotherapy, to overcome radioresistance. Future studies could focus on exploring the clinical translation of this dual blockade approach and investigating other factors contributing to radioresistance in breast cancer. The identification of additional radiation-responsive immunomodulatory factors could further enhance therapeutic efficacy.

The study primarily used in vitro and syngeneic mouse models, which may not fully recapitulate the complexity of human breast cancer. The clinical translatability of the findings needs to be confirmed in larger clinical trials. While the mechanistic studies provide insights into the HER2-NF-κB-CD47 pathway, other signaling pathways could also be involved. Furthermore, the study focused on the combination of radiotherapy and dual blockade; other combinations of therapies could be investigated.