Malignant melanoma is the deadliest of skin cancers. Melanoma frequently metastasizes to the brain, resulting in dismal survival. Nevertheless, mechanisms that govern early metastatic growth and the interactions of disseminated metastatic cells with the brain microenvironment are largely unknown. To study the hallmarks of brain metastatic niche formation, we established a transplantable model of spontaneous melanoma brain metastasis in immunocompetent mice and developed molecular tools for quantitative detection of brain micrometastases. Here we demonstrate that micrometastases are associated with instigation of astrogliosis, neuroinflammation, and hyperpermeability of the blood-brain barrier. Furthermore, we show a functional role for astrocytes in facilitating initial growth of melanoma cells. Our findings suggest that astrogliosis, physiologically instigated as a brain tissue damage response, is hijacked by tumor cells to support metastatic growth. Studying spontaneous melanoma brain metastasis in a clinically relevant setting is the key to developing therapeutic approaches that may prevent brain metastatic relapse.

Highlights d CXCL10 is upregulated in metastases-associated astrocytes in vivo d Astrocyte-derived CXCL10 enhances melanoma cell migration toward astrocytes d CXCR3, the receptor for CXCL10, is upregulated in braintropic melanoma cells d Targeting CXCR3 expression attenuates the formation of melanoma brain metastases

The major cause of melanoma mortality is metastasis to distant organs, including lungs and brain. Reciprocal interactions of metastasizing tumor cells with stromal cells in secondary sites play a critical role in all stages of tumorigenesis and metastasis. Changes in the metastatic microenvironment were shown to precede clinically relevant metastases, and may occur prior to the arrival of disseminated tumor cells to the distant organ, thus creating a hospitable “premetastatic niche.” Exosomes secreted by tumor cells were demonstrated to play an important role in the preparation of a hospitable metastatic niche. However, the functional role of melanoma‐derived exosomes on metastatic niche formation, and the downstream pathways activated in stromal cells at the metastatic niche are largely unresolved. Here we show that extracellular vesicles (EVs) secreted by metastatic melanoma cells that spontaneously metastasize to lungs and to brain, activate proinflammatory signaling in lung fibroblasts and in astrocytes. Interestingly, unlike paracrine signaling by melanoma cells, EVs secreted by metastatic melanoma cells instigated a proinflammatory gene signature in lung fibroblasts but did not activate wound‐healing functions, suggesting that tumor cell‐secreted EVs activate distinct CAF characteristics and tumor‐promoting functions. Moreover, melanoma‐secreted EVs also activated proinflammatory signaling in astrocytes, indicating that EV‐mediated reprogramming of stromal cells is a general mechanism of modulating the metastatic niche in multiple distant organs. Thus, our study demonstrates that melanoma‐derived EVs reprogram tumor‐promoting functions in stromal cells in a distinct manner, implicating a central role for tumor‐derived EV signaling in promoting the formation of an inflammatory metastatic niche.

Brain metastases are prevalent in various types of cancer and are often terminal, given the low efficacy of available therapies. Therefore, preventing them is of utmost clinical relevance, and prophylactic treatments are perhaps the most efficient strategy. Here, we show that systemic prophylactic administration of a toll-like receptor (TLR) 9 agonist, CpG-C, is effective against brain metastases. Acute and chronic systemic administration of CpG-C reduced tumor cell seeding and growth in the brain in three tumor models in mice, including metastasis of human and mouse lung cancer, and spontaneous melanoma-derived brain metastasis. Studying mechanisms underlying the therapeutic effects of CpG-C, we found that in the brain, unlike in the periphery, natural killer (NK) cells and monocytes are not involved in controlling metastasis. Next, we demonstrated that the systemically administered CpG-C is taken up by endothelial cells, astrocytes, and microglia, without affecting blood-brain barrier (BBB) integrity and tumor brain extravasation. In vitro assays pointed to microglia, but not astrocytes, as mediators of CpG- C effects through increased tumor killing and phagocytosis, mediated by direct microglia-tumor contact. In vivo, CpG-C–activated microglia displayed elevated mRNA expression levels of apoptosis-inducing and phagocytosis-related genes. Intravital imaging showed that CpG-C–activated microglia cells contact, kill, and phagocytize tumor cells in the early stages of tumor brain invasion more than nonactivated microglia. Blocking in vivo activation of microglia with minocycline, and depletion of microglia with a colony-stimulating factor 1 inhibitor, indicated that microglia mediate the antitumor effects of CpG-C. Overall, the results suggest prophylactic CpG-C treatment as a new intervention against brain metastasis, through an essential activation of microglia.

22Brain metastases are prevalent in various types of cancer, and are often terminal given low 23 efficacy of available therapies. Therefore, preventing them is of outmost clinical relevance 24 and prophylactic treatments are perhaps the most efficient strategy. Here, we show that 25 systemic prophylactic administration of a TLR9 agonist, CpG-C, is effective against brain 26 metastases. Acute and chronic systemic administration of CpG-C reduced tumor cell 27 seeding and growth in the brain in three tumor models in mice, including metastasis of 28 human and mouse lung cancer, and spontaneous melanoma-derived brain metastasis. 29Studying mechanisms underlying the therapeutic effects of CpG-C, we found that in the 30 brain, unlike in the periphery, NK cells and monocytes are not involved in controlling 31 metastasis. Next, we demonstrated that the systemically administered CpG-C is taken up by 32 endothelial cells, astrocytes, and microglia, without affecting blood-brain barrier integrity and 33 tumor brain extravasation. In vitro assays pointed to microglia, but not astrocytes, as 34 mediators of CpG-C effects through increased tumor killing and phagocytosis, mediated by 35 direct microglia-tumor contact. In vivo, CpG-C-activated microglia displayed elevated mRNA 36 expression levels of apoptosis-inducing and phagocytosis-related genes. Intravital imaging 37 showed that CpG-C-activated microglia cells contact, kill, and phagocytize tumor cells in the 38 early stages of tumor brain invasion more than non-activated microglia. Blocking in vivo 39 activation of microglia with minocycline, and depletion of microglia with a colony-stimulating 40 factor 1 inhibitor, indicated that microglia mediate the anti-tumor effects of CpG-C. Overall, 41 the results suggest prophylactic CpG-C treatment as a new intervention against brain 42 metastasis, through an essential activation of microglia. 43 44 Summary 45 Brain metastases are prevalent and often terminal. Thus, reducing their occurrence could 46 markedly improve cancer outcome. We show that systemic prophylactic and perioperative 47 administration of a TLR9 agonist, CpG-C, reduced metastatic growth in experimental and 48 spontaneous brain metastasis models, employing mouse and human tumors. CpG-C was 49 taken up in the brain, without affecting blood-brain barrier integrity and tumor extravasation. 50In vitro assays, imaging flow cytometry, and intravital imaging pointed to microglia as 51 mediators of CpG-C effects through contact-dependent tumor killing and phagocytosis; 52 corresponding with in vivo mRNA profile. In vivo depletion studies proved that microglia, but 53 not NK cells or monocytes, mediated the beneficial effects of CpG-C; Also hindered by 54 blocking microglial activation. In-toto, perioperative treatment with CpG-C should be 55 considered clinically relevant. 57Significance 58 Preventing brain metastases is paramount, as they are considered incurable and their 59 incidence is on the rise due to prolonged survival of cancer patients. Here, we demonstrate 60 th...

In cancer, two unique and seemingly contradictory behaviors are evident: on the one hand, tumors are typically stiffer than the tissues in which they grow, and this high stiffness promotes their malignant progression; on the other hand, cancer cells are anchorage-independent—namely, they can survive and grow in soft environments that do not support cell attachment. How can these two features be consolidated? Recent findings on the mechanisms by which cells test the mechanical properties of their environment provide insight into the role of aberrant mechanosensing in cancer progression. In this review article, we focus on the role of high stiffness on cancer progression, with particular emphasis on tumor growth; we discuss the mechanisms of mechanosensing and mechanotransduction, and their dysregulation in cancerous cells; and we propose that a ‘yin and yang’ type phenomenon exists in the mechanobiology of cancer, whereby a switch in the type of interaction with the extracellular matrix dictates the outcome of the cancer cells.

Both cell–cell and cell–matrix adhesions are regulated by mechanical signals, but the mechanobiological processes that mediate the cross talk between these structures are poorly understood. Here we show that α-catenin, a mechanosensitive protein that is classically linked with cadherin-based adhesions, associates with and regulates integrin adhesions. α-Catenin is recruited to the edges of mesenchymal cells, where it interacts with F-actin. This is followed by mutual retrograde flow of α-catenin and F-actin from the cell edge, during which α-catenin interacts with vinculin within integrin adhesions. This interaction affects adhesion maturation, stress-fiber assembly, and force transmission to the matrix. In epithelial cells, α-catenin is present in cell–cell adhesions and absent from cell–matrix adhesions. However, when these cells undergo epithelial-to-mesenchymal transition, α-catenin transitions to the cell edge, where it facilitates proper mechanosensing. This is highlighted by the ability of α-catenin–depleted cells to grow on soft matrices. These results suggest a dual role of α-catenin in mechanosensing, through both cell–cell and cell–matrix adhesions.

<p>Incidence of spontaneous micrometastases</p>