A long-standing unknown is how an immune synapse disassembles. In this study, Srpan et al. show that shedding of CD16 promotes the detachment of NK cells from target cells to aid serial engagement of multiple targets and to sustain NK cell viability.

Intercellular communication within the bone marrow niche significantly promotes leukemogenesis and provides protection of leukemic cells from therapy. Secreted factors, intercellular transfer of mitochondria and the receptor–ligand interactions have been shown as mediators of this protection. Here we report that tunneling nanotubes (TNTs)—long, thin membranous structures, which have been identified as a novel mode of intercellular cross-talk—are formed in the presence of stroma and mediate transfer of cellular vesicles from stroma to leukemic cells. Importantly, transmission of vesicles via TNTs from stromal cells increases resistance of leukemic cells to the tyrosine kinase inhibitor, imatinib. Using correlative light-electron microscopy and electron tomography we show that stromal TNTs contain vesicles, provide membrane continuity with the cell bodies and can be open-ended. Moreover, trans-SILAC studies to reveal the non-autonomous proteome showed that specific sets of proteins are transferred together with cellular vesicles from stromal to leukemic cells, with a potential role in survival and adaptation. Altogether, our findings provide evidence for the biological role of the TNT-mediated vesicle exchange between stromal and leukemic cells, implicating the direct vesicle and protein transfer in the stroma-provided protection of leukemic cells.

No abstract

Intercellular communication within the bone marrow niche significantly influences leukemogenesis and the sensitivity of leukemic cells to therapy. Tunneling nanotubes (TNTs) are a novel mode of intercellular cross-talk. They are long, thin membranous protrusions that enable the direct transfer of various cargo between cells. Here we show that TNTs are formed between leukemic and bone marrow stromal cells. Fluorescence confocal microscopy with 3D reconstructions, correlative light-electron microscopy and electron tomography provided evidence that TNTs transfer cellular vesicles between cells. The quantitative analysis demonstrated that the stromal cells stimulate TNT-mediated vesicle transfer towards leukemic cells. Transfer of vesicular cargo from stromal cells correlated with increased resistance to anti-leukemic treatment. Moreover, specific sets of proteins with a potential role in survival and the drug response were transferred within these vesicles. Altogether, we found that TNTs are involved in the leukemia-stroma cross-talk and the stroma-mediated cytoprotection of leukemic cells. Our findings implicate TNT connections as a possible target for therapeutic interventions within the leukemia microenvironment to attenuate stroma-conferred protection.

Human CMV (HCMV) is a ubiquitous pathogen that indelibly shapes the NK cell repertoire. Using transcriptomic, epigenomic, and proteomic approaches to evaluate peripheral blood NK cells from healthy human volunteers, we find that prior HCMV infection promotes NK cells with a T celllike gene profile, including the canonical markers CD3e, CD5, and CD8b, as well as the T cell lineagecommitment transcription factor Bcl11b. Although Bcl11b expression is upregulated during NK maturation from CD56 bright to CD56 dim , we find a Bcl11b-mediated signature at the protein level for FceRIg, PLZF, IL-2Rb, CD3g, CD3d, and CD3e in later-stage, HCMV-induced NK cells. BCL11B is targeted by Notch signaling in T cell development, and culture of NK cells with Notch ligand increases cytoplasmic CD3e expression. The Bcl11b-mediated gain of CD3e, physically associated with CD16 signaling molecules Lck and CD247 in NK cells is correlated with increased Ab-dependent effector function, including against HCMV-infected cells, identifying a potential mechanism for their prevalence in HCMV-infected individuals and their prospective clinical use in Ab-based therapies.

33 34 Immune cells identify cancer cells by recognizing characteristic biochemical 35 features indicative of oncogenic transformation. Cancer cells have characteristic 36 mechanical features, as well, but whether these biophysical properties also contribute to 37 destruction by the immune system is not known. In the present study, we found that 38 enhanced expression of myocardin related transcription factors (MRTFs), which promote 39 migration and metastatic invasion, paradoxically compromised lung colonization by 40 melanoma and breast carcinoma cells in an immune-mediated manner. Cancer cells with 41 increased MRTF signaling were also more sensitive to immune checkpoint blockade 42 therapy in mice and humans. The basis for this vulnerability was not biochemical, but 43 biophysical. MRTF expression strengthened the actin cytoskeleton, increasing the 44 rigidity of cancer cells and thereby making them more vulnerable to cytotoxic T 45 lymphocytes and natural killer cells. These results reveal a mechanical dimension of 46 immunosurveillance, which we call mechanosurveillance, that is particularly relevant to 47 109 cytoskeletal components. Morphoregulation by MRTF is absolutely required for metastatic 110 invasion and subsequent proliferative expansion (Er et al., 2018). However, because analogous 111 shape changes have been shown to increase cell stifffness (Kasza et al., 2009), it is tempting to 112 speculate that MRTF signaling might also mechanically sensitize cancer cells to cytotoxic 113 lymphocytes. 114In the present study, we explored this hypothesis by analyzing the effects of MRTF on 115 the mechanical properties of cancer cells, their immune sensitivity, and their capacity to colonize 116 tissues in vivo. Our results reveal a novel, mechanical form of immunosurveillance that enables 117 cytotoxic lymphocytes to target the specific biophysical features of metastatic cancer cells. 118 119 120 5 RESULTS 121 122 MRTF overexpression sensitizes metastatic cells to the immune system 123 Given the importance of MRTF-mediated mechanotransduction for migration and 124 metastasis, and the unique position of MRTF in F-actin regulation, we reasoned that 125 manipulating MRTFA and MRTFB levels might reveal novel vulnerabilities associated with 126 cancer cell architecture (Fig. 1A). To explore this idea, we employed an established model of 127 metastatic colonization in which malignant cancer cells expressing luciferase are injected 128 intravenously into congenic mice and their subsequent growth in the lung monitored by 129 bioluminescent imaging (Fig. 1B). shRNA-mediated suppression of MRTFA and MRTFB130 reduced lung colonization by both B16F10 melanoma and E0771 breast cancer cells in this 131 model (Fig. 1C-D and Fig. S1A), consistent with previous data showing that MRTF is required 132 for metastatic seeding (Er et al., 2018; Kim et al., 2017; Medjkane et al., 2009). However, 133 B16F10 and E0771 cell lines overexpressing MRTFB (B16F10-MRTFB and E0771-MRTFB) 134 exhibited dramatically reduced lung colonization ...

Metastatic dissemination of cancer cells to vital secondary organs pose a lifelong lethal threat unless they can successfully be eliminated by the immune system. Interestingly, despite their ability to evade immunity at primary organs, the vast majority of disseminated cancer cells are killed at secondary organs, suggesting that the process of dissemination renders cancer cells sensitive to immunity. Understanding how dissemination renders cancer cells sensitive to immunity would therefore provide exciting new opportunities to target metastases, but the relationship between dissemination and the immune response is not clear. In our effort to study this relationship, we manipulated cancer cells' expression of myocardin related transcription factors A and B (MRTFA/B), which are central in regulation of actin cytoskeleton and are indispensable for metastatic invasion, dissemination and colonization. Surprisingly, despite MRTFA/B's established roles in promoting metastasis, we found that MRTFA/B expression in cancer cells downregulated metastatic colonization in fully immuno-competent mouse models. MRTFA/B mediated decrease in colonization was completely reversed upon immune cell depletion suggesting that the immune cells exploit MRTFA/B expression in cancer cells for cytotoxicity. Among various cancer cell parameters involved in immune cell activation, T- and NK cell activation most prominently correlated with MRTFA/B driven mechanical compliance of cancer cells. Mechanical compliance, also known as stiffness, of antigen presenting cells and synthetic substrates are well-known to activate T- and NK cells through biophysical and mechano-chemical signal transduction. As such, perturbing cancer cells' stiffness downregulated the immune response and cytotoxicity. Thus, we termed this process mechanosurveillance. Our work on mechanosurveillance will help us understand how disseminated cancer cells are targeted by immunity and how they evade it at secondary organs. Importantly the concept of mechanosurveillance will help guide the use of existing therapeutics that alter tissue stiffness and immune boosting therapies to combat metastases. Citation Format: Maria Tello-Lafoz, Katja Srpan, Jing Hu, Yevgeniy Romin, Annalisa Calo, Katharine C. Hsu, Joan Massagué, Morgan Huse, Ekrem Emrah Er. Mechanosurveillance by immunity targets mechanical compliance of metastatic cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2825.

Metastatic dissemination of cancer cells to vital secondary organs pose a lifelong lethal threat unless they can successfully be eliminated by the immune system. Interestingly, despite their ability to evade immunity at primary organs, the vast majority of disseminated cancer cells are killed at secondary organs, suggesting that the process of dissemination renders cancer cells sensitive to immunity. Understanding how dissemination renders cancer cells sensitive to immunity would therefore provide exciting new opportunities to target metastases, but the relationship between dissemination and the immune response is not clear. In our effort to study this relationship, we manipulated cancer cells’ expression of myocardin related transcription factors A and B (MRTFA/B), which are central in regulation of actin cytoskeleton and are indispensable for metastatic invasion, dissemination and colonization. Surprisingly, despite MRTFA/B’s established roles in promoting metastasis, we found that MRTFA/B expression in cancer cells downregulated metastatic colonization in fully immuno-competent mouse models. MRTFA/B mediated decrease in colonization was completely reversed upon immune cell depletion suggesting that the immune cells exploit MRTFA/B expression in cancer cells for cytotoxicity. Among various cancer cell parameters involved in immune cell activation, T- and NK cell activation most prominently correlated with MRTFA/B driven mechanical compliance of cancer cells. Mechanical compliance, also known as stiffness, of antigen presenting cells and synthetic substrates are well-known to activate T- and NK cells through biophysical and mechano-chemical signal transduction. As such, perturbing cancer cells’ stiffness downregulated the immune response and cytotoxicity. Thus, we termed this process mechanosurveillance. Our work on mechanosurveillance will help us understand how disseminated cancer cells are targeted by immunity and how they evade it at secondary organs. Importantly the concept of mechanosurveillance will help guide the use of existing therapeutics that alter tissue stiffness and immune boosting therapies to combat metastases. Citation Format: Maria Tello-Lafoz, Katja Srpan, Jing Hu, Yevgeniy Romin, Annalisa Calò, Katharine C. Hsu, Joan Massagué, Morgan Huse, Ekrem Emrah Er. Mechanosurveillance eliminates disseminated cancer cells by sensing their mechanical compliance [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr LT016.