Metastasis-Entrained Eosinophils Enhance Lymphocyte-Mediated Antitumor Immunity.

The recognition of the immune system as a key component of the tumor microenvironment (TME) led to promising therapeutics. Because such therapies benefit only subsets of patients, understanding the activities of immune cells in the TME is required. Eosinophils are an integral part of the TME especially in mucosal tumors. Nonetheless, their role in the TME and the environmental cues that direct their activities are largely unknown. We report that breast cancer lung metastases are characterized by resident and recruited eosinophils. Eosinophil recruitment to the metastatic sites in the lung was regulated by G protein-coupled receptor signaling but independent of CCR3. Functionally, eosinophils promoted lymphocyte-mediated antitumor immunity. Transcriptome and proteomic analyses identified the TME rather than intrinsic differences between eosinophil subsets as a key instructing factor directing antitumorigenic eosinophil activities. Specifically, TNFα/IFNγ-activated eosinophils facilitated CD4+ and CD8+ T-cell infiltration and promoted antitumor immunity. Collectively, we identify a mechanism by which the TME trains eosinophils to adopt antitumorigenic properties, which may lead to the development of eosinophil-targeted therapeutics. SIGNIFICANCE: These findings demonstrate antitumor activities of eosinophils in the metastatic tumor microenvironment, suggesting that harnessing eosinophil activity may be a viable clinical strategy in patients with cancer.

Evolution of fibroblasts in the lung metastatic microenvironment is driven by stage-specific transcriptional plasticity.

Mortality from breast cancer is almost exclusively a result of tumor metastasis, and lungs are one of the main metastatic sites. Cancer-associated fibroblasts are prominent players in the microenvironment of breast cancer. However, their role in the metastatic niche is largely unknown. In this study, we profiled the transcriptional co-evolution of lung fibroblasts isolated from transgenic mice at defined stage-specific time points of metastases formation. Employing multiple knowledge-based platforms of data analysis provided powerful insights on functional and temporal regulation of the transcriptome of fibroblasts. We demonstrate that fibroblasts in lung metastases are transcriptionally dynamic and plastic, and reveal stage-specific gene signatures that imply functional tasks, including extracellular matrix remodeling, stress response, and shaping the inflammatory microenvironment. Furthermore, we identified Myc as a central regulator of fibroblast rewiring and found that stromal upregulation of Myc transcriptional networks is associated with disease progression in human breast cancer.

Fibroblast-Derived IL33 Facilitates Breast Cancer Metastasis by Modifying the Immune Microenvironment and Driving Type 2 Immunity.

Lungs are one of the main sites of breast cancer metastasis. The metastatic microenvironment is essential to facilitate growth of disseminated tumor cells. Cancer-associated fibroblasts (CAF) are prominent players in the microenvironment of breast cancer. However, their role in the formation of a permissive metastatic niche is unresolved. Here we show that IL33 is upregulated in metastases-associated fibroblasts in mouse models of spontaneous breast cancer metastasis and in patients with breast cancer with lung metastasis. Upregulation of IL33 instigated type 2 inflammation in the metastatic microenvironment and mediated recruitment of eosinophils, neutrophils, and inflammatory monocytes to lung metastases. Importantly, targeting of IL33 in vivo resulted in inhibition of lung metastasis and significant attenuation of immune cell recruitment and type 2 immunity. These findings demonstrate a key function of IL33 in facilitating lung metastatic relapse by modulating the immune microenvironment. Our study shows a novel interaction axis between CAF and immune cells and reveals the central role of CAF in establishing a hospitable inflammatory niche in lung metastasis. SIGNIFICANCE: This study elucidates a novel role for fibroblast-derived IL33 in facilitating breast cancer lung metastasis by modifying the immune microenvironment at the metastatic niche toward type 2 inflammation.

Bone metastasis is associated with acquisition of mesenchymal phenotype and immune suppression in a model of spontaneous breast cancer metastasis.

The most common site of breast cancer metastasis is the bone, occurring in approximately 70% of patients with advanced disease. Bone metastasis is associated with severe morbidities and high mortality. Therefore, deeper understanding of the mechanisms that enable bone-metastatic relapse are urgently needed. We report the establishment and characterization of a bone-seeking variant of breast cancer cells that spontaneously forms aggressive bone metastases following surgical resection of primary tumor. We characterized the modifications in the immune milieu during early and late stages of metastatic relapse and found that the formation of bone metastases is associated with systemic changes, as well as modifications of the bone microenvironment towards an immune suppressive milieu. Furthermore, we characterized the intrinsic changes in breast cancer cells that facilitate bone-tropism and found that they acquire mesenchymal and osteomimetic features. This model provides a clinically relevant platform to study the functional interactions between breast cancer cells and the bone microenvironment, in an effort to identify novel targets for intervention.

NLRP3 inflammasome in fibroblasts links tissue damage with inflammation in breast cancer progression and metastasis.

Cancer-Associated Fibroblasts (CAFs) were shown to orchestrate tumour-promoting inflammation in multiple malignancies, including breast cancer. However, the molecular pathways that govern the inflammatory role of CAFs are poorly characterised. In this study we found that fibroblasts sense damage-associated molecular patterns (DAMPs), and in response activate the NLRP3 inflammasome pathway, resulting in instigation of pro-inflammatory signalling and secretion of IL-1ß. This upregulation was evident in CAFs in mouse and in human breast carcinomas. Moreover, CAF-derived inflammasome signalling facilitated tumour growth and metastasis, which was attenuated when NLRP3 or IL-1ß were specifically ablated. Functionally, CAF-derived inflammasome promoted tumour progression and metastasis by modulating the tumour microenvironment towards an immune suppressive milieu and by upregulating the expression of adhesion molecules on endothelial cells. Our findings elucidate a mechanism by which CAFs promote breast cancer progression and metastasis, by linking the physiological tissue damage response of fibroblasts with tumour-promoting inflammation.

Inflammatory Activation of Astrocytes Facilitates Melanoma Brain Tropism via the CXCL10-CXCR3 Signaling Axis.

Melanoma is the deadliest skin cancer due to its high rate of metastasis, frequently to the brain. Brain metastases are incurable; therefore, understanding melanoma brain metastasis is of great clinical importance. We used a mouse model of spontaneous melanoma brain metastasis to study the interactions of melanomas with the brain microenvironment. We find that CXCL10 is upregulated in metastasis-associated astrocytes in mice and humans and is functionally important for the chemoattraction of melanoma cells. Moreover, CXCR3, the receptor for CXCL10, is upregulated in brain-tropic melanoma cells. Targeting melanoma expression of CXCR3 by nanoparticle-mediated siRNA delivery or by shRNA transduction inhibits melanoma cell migration and attenuates brain metastasis in vivo. These findings suggest that the instigation of pro-inflammatory signaling in astrocytes is hijacked by brain-metastasizing tumor cells to promote their metastatic capacity and that the CXCL10-CXCR3 axis may be a potential therapeutic target for the prevention of melanoma brain metastasis.

Melanoma-derived extracellular vesicles instigate proinflammatory signaling in the metastatic microenvironment.

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.

Bone marrow-derived fibroblasts are a functionally distinct stromal cell population in breast cancer.

Cancer-associated fibroblasts (CAFs) are highly prominent in breast tumors, but their functional heterogeneity and origin are still largely unresolved. We report that bone marrow (BM)-derived mesenchymal stromal cells (MSCs) are recruited to primary breast tumors and to lung metastases and differentiate to a distinct subpopulation of CAFs. We show that BM-derived CAFs are functionally important for tumor growth and enhance angiogenesis via up-regulation of Clusterin. Using newly generated transgenic mice and adoptive BM transplantations, we demonstrate that BM-derived fibroblasts are a substantial source of CAFs in the tumor microenvironment. Unlike resident CAFs, BM-derived CAFs do not express PDGFRα, and their recruitment resulted in a decrease in the percentage of PDGFRα-expressing CAFs. Strikingly, decrease in PDGFRα in breast cancer patients was associated with worse prognosis, suggesting that BM-derived CAFs may have deleterious effects on survival. Therefore, PDGFRα expression distinguishes two functionally unique CAF populations in breast tumors and metastases and may have important implications for patient stratification and precision therapeutics.