Nanocharacterization plays a vital role in understanding the complex nanoscale organization of cells and organelles. Understanding cellular function requires high-resolution information about how the cellular structures evolve over time. A number of techniques exist to resolve static nanoscale structure of cells in great detail (super-resolution optical microscopy, EM, AFM). However, time-resolved imaging techniques tend to either have a lower resolution, are limited to small areas, or cause damage to the cells, thereby preventing long-term time-lapse studies. Scanning probe microscopy methods such as atomic force microscopy (AFM) combine high-resolution imaging with the ability to image living cells in physiological conditions. The mechanical contact between the tip and the sample, however, deforms the cell surface, disturbs the native state, and prohibits long-term time-lapse imaging. Here, we develop a scanning ion conductance microscope (SICM) for high-speed and long-term nanoscale imaging of eukaryotic cells. By utilizing advances in nanopositioning, nanopore fabrication, microelectronics, and controls engineering, we developed a microscopy method that can resolve spatiotemporally diverse three-dimensional (3D) processes on the cell membrane at sub-5-nm axial resolution. We tracked dynamic changes in live cell morphology with nanometer details and temporal ranges of subsecond to days, imaging diverse processes ranging from endocytosis, micropinocytosis, and mitosis to bacterial infection and cell differentiation in cancer cells. This technique enables a detailed look at membrane events and may offer insights into cell–cell interactions for infection, immunology, and cancer research.

BackgroundActivin-A, a transforming growth factor β family member, is secreted by many cancer types and is often associated with poor disease prognosis. Previous studies have shown that Activin-A expression can promote cancer progression and reduce the intratumoral frequency of cytotoxic T cells. However, the underlying mechanisms and the significance of Activin-A expression for cancer therapies are unclear.MethodsWe analyzed the expression of the Activin-A encoding gene INHBA in melanoma patients and the influence of its gain- or loss-of-function on the immune infiltration and growth of BRAF-driven YUMM3.3 and iBIP2 mouse melanoma grafts and in B16 models. Using antibody depletion strategies, we investigated the dependence of Activin-A tumor-promoting effect on different immune cells. Immune-regulatory effects of Activin-A were further characterized in vitro and by an adoptive transfer of T cells. Finally, we assessed INHBA expression in melanoma patients who received immune checkpoint therapy and tested whether it impairs the response in preclinical models.ResultsWe show that Activin-A secretion by melanoma cells inhibits adaptive antitumor immunity irrespective of BRAF status by inhibiting CD8+ T cell infiltration indirectly and even independently of CD4 T cells, at least in part by attenuating the production of CXCL9/10 by myeloid cells. In addition, we show that Activin-A/INHBA expression correlates with anti-PD1 therapy resistance in melanoma patients and impairs the response to dual anti-cytotoxic T-Lymphocyte associated protein 4/anti-PD1 treatment in preclinical models.ConclusionsOur findings suggest that strategies interfering with Activin-A induced immune-regulation offer new therapeutic opportunities to overcome CD8 T cell exclusion and immunotherapy resistance.

Understanding cellular function requires high-resolution information about cellular structures as well as their evolution over time. The major challenge is to obtain three-dimensional (3D) information at nanometer resolution without affecting the viability of the cells and avoiding interference with the process. Here, we develop a scanning ion conductance microscope (SICM) for high-speed and long term imaging that can resolve spatiotemporally diverse processes on the cell membrane. We tracked dynamic changes in live cell morphology with nanometer details and temporal ranges of sub-second to days, imagining diverse processes ranging from endocytosis, micropinocytosis, and mitosis, to bacterial infection and cell differentiation in cancer cells. This technique enables a detailed look at membrane events and may offer insights into cell-cell interactions for infection, immunology, and cancer research.