Irradiated Mammary Spheroids Elucidate Mechanisms of Macrophage-Mediated Breast Cancer Recurrence.

Introduction: While most patients with triple negative breast cancer receive radiation therapy to improve outcomes, a significant subset of patients continue to experience recurrence. Macrophage infiltration into radiation-damaged sites has been shown to promote breast cancer recurrence in pre-clinical models. However, the mechanisms that drive recurrence are unknown. Here, we developed a novel spheroid model to evaluate macrophage-mediated tumor cell recruitment. Methods: We characterized infiltrating macrophage phenotypes into irradiated mouse mammary tissue via flow cytometry. We then engineered a spheroid model of radiation damage with primary fibroblasts, macrophages, and 4T1 mouse mammary carcinoma cells using in vivo macrophage infiltration results to inform our model. We analyzed 4T1 infiltration into spheroids when co-cultured with biologically relevant ratios of pro-healing M2:pro-inflammatory M1 macrophages. Finally, we quantified interleukin 6 (IL-6) secretion associated with conditions favorable to tumor cell infiltration, and we directly evaluated the impact of IL-6 on tumor cell invasiveness in vitro and in vivo. Results: In our in vivo model, we observed a significant increase in M2 macrophages in mouse mammary glands 10 days post-irradiation. We determined that tumor cell motility toward irradiated spheroids was enhanced in the presence of a 2:1 ratio of M2:M1 macrophages. We also measured a significant increase in IL-6 secretion after irradiation both in vivo and in our model. This secretion increased tumor cell invasiveness, and tumor cell invasion and recruitment were mitigated by neutralizing IL-6. Conclusions: Our work suggests that interactions between infiltrating macrophages and damaged stromal cells facilitate breast cancer recurrence through IL-6 signaling. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-023-00775-x.

Ultrasound Imaging Enables Longitudinal Tracking of Vascular Changes that Correlate with Immune Cell Infiltration After Radiotherapy.

While immunotherapy shows great promise in patients with triple negative breast cancer, many will not respond to treatment, and predicting response is made difficult by significant tumor heterogeneity. Non-invasive imaging of the tumor vasculature enables the monitoring of treatment and has potential to aid in predicting therapeutic response. Here, we use ultrafast power doppler ultrasound (US) to track longitudinal changes in the vascular response to radiotherapy in two breast cancer models to correlate vascular and immune changes in the tumor microenvironment. Tumor volume and vascular index were calculated to evaluate the effects of radiation using US imaging. US tumor measurements and the quantified vascular response to radiation were confirmed with caliper measurements and immunohistochemistry observations, respectively, demonstrating a proof-of-principle method for non-invasive vascular monitoring. Additionally, we found significant infiltration of CD8+ T cells into irradiated tumors 10 days after radiation, which followed a sustained decline in vascular index that was first observed 1 day post-radiation. Taken together, our findings reveal the potential for ultrafast power doppler US to evaluate changes in tumor vasculature that may be indicative of the tumor-immune microenvironment and ultimately improve patient outcomes by predicting response to immunotherapy.