Tumor development and metastasis are dependent on tumor infiltrating immune cells which form a characteristic tumor microenvironment (TME). Activated monocytes secrete the protein heterodimer S100A8/A9 promoting TME formation. Monocyte-dependent proteases facilitate local tumor cell invasion by degradation of the extracellular matrix. We aimed for target specific in vivo imaging of S100A8 and proteases to provide differentiating biomarkers for local tumor growth and metastatic potential.
Murine breast cancer cells of the 4T1 model with graduated metastatic potential (4T1 and 4T07: both hematogenous metastasis > 168FAR: lymph-node metastasis > 67NR: no metastasis) were orthotopically implanted into female BALB/c mice. At 4 mm size, tumors were investigated by injecting the protease-specific probe ProSense 750EX (PerkinElmer, 4T1 n = 7, 4T07 n = 10, 168FAR n = 16, 67NR n = 15) and anti-S100A8-Cy5.5 (n = 6 each) and performing fluorescence reflectance imaging at 0 and 24 h after injection. In vivo imaging was validated with immunohistochemistry.
At 24 h, S100A8-specific signals in 4T1 and 4T07 were significantly higher (1714.05/1683.45 AU) as compared to 168FAR and 67NR (174.85/167.95 AU, p = 0.0012/p = 0.0003), reflecting the capability of hematogenous spread. Protease-specific signals were significantly higher in 4T1 and 4T07 (348.01/409.93 AU) as compared to 168FAR (214.91 AU) and 67NR (129.78 AU p < 0.0001 each), reflecting local vessel invasion and tumor cell shedding. Immunohistology supported the in vivo imaging results.
Non-invasive in vivo imaging of S100A8 and monocytic proteases allows for differentiation of the tumors’ local invasive and systemic metastatic potential in reflecting the TME formation. While proteases augment local tumor cell invasion, solid metastases seem to be dependent on a pro-tumoral microenvironment.
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We thank Heike Berheide, Klaudia Niepagenkemper, Claudia Terwesten-Solé, and Ina Winkler for excellent technical support.
This study was funded in part by the Interdisciplinary Center for Clinical Research of the University of Muenster (Vo2/014/09, PIX) and the German Research Foundation (DFG) CRC 1009 B8, B9, and EI 878 1-1.
All animal experiments in this study have been approved by the responsible authorities (Protocol No. 8.87-50.10.36.08.191).
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Helfen, A., Große Hokamp, N., Geyer, C. et al. Target-Specific Imaging of Cathepsin and S100A8/A9 Reflects Specific Features of Malignancy and Enables Estimation of Tumor Malignancy. Mol Imaging Biol 22, 66–72 (2020). https://doi.org/10.1007/s11307-019-01370-1
- Tumor microenvironment
- Tumor escape
- Breast neoplasms
- Molecular imaging