Rheology of tumor-associated macrophages using photothermal actuation AFM

Introduction. AFM-based micro-rheology is a powerful approach for quantifying viscoelastic properties of soft biological samples. Here, we used AFM micro-rheology with photothermal excitation to extend the frequency range accessible with conventional piezoelectric actuation (<500 Hz) to the kHz range, enabling a more complete understanding of cell viscoelastic behavior.1,2 We applied this approach to perform a nanomechanical characterization of different phenotypes of tumor-associated macrophages (TAMs),3,4 as a first step in the determination of a multi-scale and multi-technique characterization procedure of immune cell mechanics in the tumor microenvironment. Methods. THP-1-derived macrophages were polarised into anti-tumor (M1) and pro-tumor (M2) phenotypes and cultured on glass substrates.5 A Nanosurf Drive AFM mounted on an inverted optical microscope (Zeiss Axio Observer) was used to perform single-cell micro-rheology with piezoelectric and photothermal actuation and extract the storage modulus (G′), loss modulus (G″), and loss tangent in the frequency ranges of 1-300 Hz and 100-1500 Hz, respectively. Results. Anti-tumor and pro-tumor macrophages exhibited a complex modulus well described by a double-power-law, with a dominant elastic response at low frequencies and an almost purely viscous behavior at high frequencies. Anti-tumor macrophages exhibited a softer and more viscous profile compared to pro-tumor macrophages as indicated by i) lower storage modulus and higher loss tangent in the entire range of frequencies, and ii) higher values of both low-frequency and high-frequency exponents. Notably, low- and high-frequency curves, acquired using the piezoelectric and photothermal actuation, showed good overlap in the shared frequency range, validating the multi-frequency approach. Conclusion. These findings highlight the potential of multi-frequency AFM micro-rheology as a tool for probing immune cell mechanics in tumor contexts. This approach, coupled with other techniques (e.g., FluidFM and immunostaining) and mechanobiology tools, will enable us to investigate the mechanobiology of macrophages in response to physical and chemical stimuli in the tumor microenvironment. References. 1. Gunstheimer, H., Fläschner, G., Adams, J. D., Hölscher, H. & Hoogenboom, B. W. High‐Speed Quantitative Nanomechanical Mapping by Photothermal Off‐Resonance Atomic Force Microscopy. Small 21, (2025). 2. Garcia, R. & Tejedor, J. R. Advances in nanomechanical property mapping by atomic force microscopy. Nanoscale Adv. 7, 6286–6307 (2025). 3. Choi, Y. et al. Role of Tumor-Associated Macrophages in Cervical Cancer: Integrating Classical Perspectives with Recent Technological Advances. 14, 443 (2024). 4. Xiong, J. et al. Matrix stiffness affects tumor-associated macrophage functional polarization and its potential in tumor therapy. J. Transl. Med. 22, 85 (2024). 5. Pedraza-Brindis, E. J. et al. Culture supernatants of cervical cancer cells induce an M2 phenotypic profile in THP-1 macrophages. Cell. Immunol. 310, 42–52 (2016).