Neutrophils seeking new neighbors: radiotherapy affects the cellular framework and the spatial organization in a murine breast cancer model

Abstract

Neutrophils are known to contribute in many aspects of tumor progression and metastasis. The presence of neutrophils or neutrophil-derived mediators in the tumor microenvironment has been associated with poor prognosis in several types of solid tumors. However, the effects of classical cancer treatments such as radiation therapy on neutrophils are poorly understood. Furthermore, the cellular composition and distribution of immune cells in the tumor is of increasing interest in cancer research and new imaging technologies allow to perform more complex spatial analyses within tumor tissues. Therefore, we aim to offer novel insight into intra-tumoral formation of cellular neighborhoods and communities in murine breast cancer. To address this question, we performed image mass cytometry on tumors of the TS/A breast cancer tumor model, performed spatial neighborhood analyses of the tumor microenvironment and quantified neutrophil-extracellular trap degradation products in serum of the mice. We show that irradiation with 2 × 8 Gy significantly alters the cellular composition and spatial organization in the tumor, especially regarding neutrophils and other cells of the myeloid lineage. Locally applied radiotherapy further affects neutrophils in a systemic manner by decreasing the serum neutrophil extracellular trap concentrations which correlates positively with survival. In addition, the intercellular cohesion is maintained due to radiotherapy as shown by E-Cadherin expression. Radiotherapy, therefore, might affect the epithelial-mesenchymal plasticity in tumors and thus prevent metastasis. Our findings underscore the growing importance of the spatial organization of the tumor microenvironment, particularly with respect to radiotherapy, and provide insight into potential mechanisms by which radiotherapy affects epithelial-mesenchymal plasticity and tumor metastasis.

Keywords: Cancer; Cellular neighborhood; Image mass cytometry; Neutrophil; Radiotherapy.

Fig. 1

Experimental setup and analysis strategy. A IMC method. Tissue samples are stained with metal-conjugated antibodies and acquired by the Hyperion device. Therefore, a UV laser ablates the region of interest pixel by pixel and analyzes the material regarding the masses to distinguish the antibodies. As a result, a two-dimensional pseudo picture of each marker in its spatial arrangement in the tissue is created. Created with BioRender. B Experimental workflow. Created with BioRender. C IMC staining of DNA (red) in a breast carcinoma sample with cell mask (white). Close-up to cell mask with cellular DNA signal in red and membrane/background signal in black (white arrow)

Fig. 2

Neutrophils dominate the TME of untreated breast carcinomas. A Expression dependent differentiation of five cell types in breast carcinoma samples. B Normalized cell populations of breast carcinomas after irradiation therapy (IT) or without therapy (NT). The frequencies of all cell types were normalized based on the total cell count of the sample. C Ratio of neutrophils to lymphocytes in the analyzed tissue samples shown as tissue-NLR (tNLR). D Treatment dependent survival of tumor bearing mice. e Correlation of NE-DNA complexes and survival in serum of tumor bearing mice. Statistical differences between cell populations were calculated using a 2-way ANOVA. Statistical differences between tNLRs were calculated using an unpaired t-test. Correlation between two variables were calculated using Pearson correlation coefficient. Statistical significances in the survival were calculated using the Kaplan–Meier method. Significance values: *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001. nNT = 3, nIT = 3

Fig. 3

Neutrophil dominated cellular neighborhoods (CNs) are significantly decreased in breast carcinoma due to radiotherapy. A Heat map of the cellular composition of the eight defined CNs in treated and untreated tumors. B Number of cells dedicated to the CNs three and eight in IT and NT samples. C Normalized cellular and immune cellular composition of the CNs three and eight. The frequencies of all CNs were normalized based on the total cell count. D CN in IT and NT samples. E Close-up to intercellular interactions in tumor parts dedicated to the cellular neighborhoods three and eight. Cell dots are colored according to their CN, interaction lines are colored according to the cell type. F Interaction analyses between neutrophils and other neutrophils, tumor cells and cytotoxic T-lymphocytes (CTL). Statistical differences between CNs were calculated using a 2-way ANOVA. Statistical differences between two groups were calculated using an unpaired t-test. Significance values: *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001. nNT = 3, nIT = 3

Fig. 4

Radiotherapy prevents E-Cadherin loss in murine breast carcinoma. A Origin of signals from the cell and membrane/background area from breast carcinoma sections. B The mean signal intensity (MSI) of E-Cadherin is significantly decreased in the cell area and in the membrane area of NT. C MSI of ecDNA in the cell area and the membrane area. D Molecular pathway of epithelial to mesenchymal transition (EMT). Created with BioRender. E MSI of TGF-β, SMAD4 and Slug in the cell area. Statistical differences between two groups were calculated using an unpaired t-test. Significance values: *: p ≤ 0. 05, **: p ≤ 0.01, ***: p ≤ 0.001. nNT = 3, nIT = 3