Amy-Jo Casbon

Invasive breast cancer reprograms early myeloid differentiation in the bone marrow to generate immunosuppressive neutrophils

Significance We show that tumor reprogramming of hematopoiesis in bone marrow occurs at the onset of malignant conversion and results in systemic expansion of circulating activated neutrophils that preferentially accumulate in lungs. Our data are, to our knowledge, the first to show that activation and not inhibition of myeloid differentiation is responsible for expansion and activity of T cell-suppressive myeloid cells; a tumor-derived factor targets the immature hematopoietic compartment to drive myeloid expansion; granulocyte-colony stimulating factor (G-CSF) is the only hematopoietic growth factor to increase in serum during early tumor development; prolonged G-CSF induces production of Rb1low neutrophils and not short-term mobilization; and G-CSF acts in a cell intrinsic manner to expand multipotent progenitors to increase production of tumor-derived Ly6G+ neutrophils. Expansion of myeloid cells associated with solid tumor development is a key contributor to neoplastic progression. Despite their clinical relevance, the mechanisms controlling myeloid cell production and activity in cancer remains poorly understood. Using a multistage mouse model of breast cancer, we show that production of atypical T cell-suppressive neutrophils occurs during early tumor progression, at the onset of malignant conversion, and that these cells preferentially accumulate in peripheral tissues but not in the primary tumor. Production of these cells results from activation of a myeloid differentiation program in bone marrow (BM) by a novel mechanism in which tumor-derived granulocyte-colony stimulating factor (G-CSF) directs expansion and differentiation of hematopoietic stem cells to skew hematopoiesis toward the myeloid lineage. Chronic skewing of myeloid production occurred in parallel to a decrease in erythropoiesis in BM in mice with progressive disease. Significantly, we reveal that prolonged G-CSF stimulation is both necessary and sufficient for the distinguishing characteristics of tumor-induced immunosuppressive neutrophils. These results demonstrate that prolonged G-CSF may be responsible for both the development and activity of immunosuppressive neutrophils in cancer.

Intravital imaging reveals distinct responses of depleting dynamic tumor-associated macrophage and dendritic cell subpopulations.

Significance Tumor-infiltrating myeloid cells fail to support antitumor immunity, and instead contribute to increased malignancy and poor prognosis in breast cancer. We used intravital microscopy in a model of breast cancer to provide unique insight into cellular composition and real-time dynamics of the stromal microenvironment. We characterized the effects of targeted therapy against CSF-1R, an important myeloid cell mitogen receptor. We demonstrate that by blocking accumulation and compromising survival, anti–CSF-1R treatment depletes a cell population sharing characteristics of tumor-associated macrophages and dendritic cells, which further comprises subgroups with different endocytic and matrix metalloproteinase activities. However, the resulting relatively modest delay in tumor growth and metastasis suggests that other cells, such as neutrophils or fibroblasts, may maintain the tumor trophic microenvironment. Tumor-infiltrating inflammatory cells comprise a major part of the stromal microenvironment and support cancer progression by multiple mechanisms. High numbers of tumor myeloid cells correlate with poor prognosis in breast cancer and are coupled with the angiogenic switch and malignant progression. However, the specific roles and regulation of heterogeneous tumor myeloid populations are incompletely understood. CSF-1 is a major myeloid cell mitogen, and signaling through its receptor CSF-1R is also linked to poor outcomes. To characterize myeloid cell function in tumors, we combined confocal intravital microscopy with depletion of CSF-1R–dependent cells using a neutralizing CSF-1R antibody in the mouse mammary tumor virus long-terminal region-driven polyoma middle T antigen breast cancer model. The depleted cells shared markers of tumor-associated macrophages and dendritic cells (M-DCs), matching the phenotype of tumor dendritic cells that take up antigens and interact with T cells. We defined functional subgroups within the M-DC population by imaging endocytic and matrix metalloproteinase activity. Anti–CSF-1R treatment altered stromal dynamics and impaired both survival of M-DCs and accumulation of new M-DCs, but did not deplete Gr-1+ neutrophils or block doxorubicin-induced myeloid cell recruitment, and had a minimal effect on lung myeloid cells. Nevertheless, prolonged treatment led to delayed tumor growth, reduced vascularity, and decreased lung metastasis. Because the myeloid infiltrate in metastatic lungs differed significantly from that in mammary tumors, the reduction in metastasis may result from the impact on primary tumors. The combination of functional analysis by intravital imaging with cellular characterization has refined our understanding of the effects of experimental targeted therapies on the tumor microenvironment.

Balancing the innate immune system in tumor development

Cells of the innate immune system have a dual role in cancer development in both tumor initiation and progression. Innate immune cells can, on the one hand, aid malignant transformation and tumor outgrowth and, on the other hand, prevent tumor progression. The innate immune system has the ability to tune the inflammatory response and is a key player in cancer-related inflammation, which can precede the development of malignancy or be induced by oncogenic changes promoting a protumor inflammatory milieu. In this review, we discuss the emerging cellular and molecular mechanisms of the innate immune system and inflammation in tumor initiation and progression, and point to the outstanding questions that remain.

Adaptive Immune Regulation of Mammary Postnatal Organogenesis

Postnatal organogenesis occurs in an immune competent environment and is tightly controlled by interplay between positive and negative regulators. Innate immune cells have beneficial roles in postnatal tissue remodeling, but roles for the adaptive immune system are currently unexplored. Here we show that adaptive immune responses participate in the normal postnatal development of a non-lymphoid epithelial tissue. Since the mammary gland (MG) is the only organ developing predominantly after birth, we utilized it as a powerful system to study adaptive immune regulation of organogenesis. We found that antigen-mediated interactions between mammary antigen-presenting cells and interferon-γ (IFNγ)-producing CD4+ T helper 1 cells participate in MG postnatal organogenesis as negative regulators, locally orchestrating epithelial rearrangement. IFNγ then affects luminal lineage differentiation. This function of adaptive immune responses, regulating normal development, changes the paradigm for studying players of postnatal organogenesis and provides insights into immune surveillance and cancer transformation.

Protective effects of matrix metalloproteinase-12 following corneal injury

Summary Corneal scarring due to injury is a leading cause of blindness worldwide and results from dysregulated inflammation and angiogenesis during wound healing. Here we demonstrate that the extracellular matrix metalloproteinase MMP12 (macrophage metalloelastase) is an important regulator of these repair processes. Chemical injury resulted in higher expression of the fibrotic markers &agr;-smooth muscle actin and type I collagen, and increased levels of angiogenesis in corneas of Mmp12−/− mice compared with corneas of wild-type mice. In vivo, we observed altered immune cell dynamics in Mmp12−/− corneas by confocal imaging. We determined that the altered dynamics were the result of an altered inflammatory response, with delayed neutrophil infiltration during the first day and excessive macrophage infiltration 6 days later, mediated by altered expression levels of chemokines CXCL1 and CCL2, respectively. Corneal repair returned to normal upon inhibition of these chemokines. Taken together, these data show that MMP12 has a protective effect on corneal fibrosis during wound repair through regulation of immune cell infiltration and angiogenesis.

HIF signaling in osteoblast-lineage cells promotes systemic breast cancer growth and metastasis in mice

Significance Previous work showed that primary tumors instigate systemic macroenvironmental changes supporting cancer progression and metastasis. Here, we show that activation of HIF signaling in osteoblast-lineage cells also generates systemic changes promoting breast cancer growth and dissemination in bones and outside the skeleton. Our results indicate that loss of bone homeostasis through alterations of the bone anabolism could affect breast cancer progression and present the skeleton as an important organ of the tumor macroenvironment. They also suggest that targeting the bone microenvironment could limit systemic tumor growth and dissemination in breast cancer. Bone metastasis involves dynamic interplay between tumor cells and the local stromal environment. In bones, local hypoxia and activation of the hypoxia-inducible factor (HIF)-1α in osteoblasts are essential to maintain skeletal homeostasis. However, the role of osteoblast-specific HIF signaling in cancer metastasis is unknown. Here, we show that osteoprogenitor cells (OPCs) are located in hypoxic niches in the bone marrow and that activation of HIF signaling in these cells increases bone mass and favors breast cancer metastasis to bone locally. Remarkably, HIF signaling in osteoblast-lineage cells also promotes breast cancer growth and dissemination remotely, in the lungs and in other tissues distant from bones. Mechanistically, we found that activation of HIF signaling in OPCs increases blood levels of the chemokine C-X-C motif ligand 12 (CXCL12), which leads to a systemic increase of breast cancer cell proliferation and dissemination through direct activation of the CXCR4 receptor. Hence, our data reveal a previously unrecognized role of the hypoxic osteogenic niche in promoting tumorigenesis beyond the local bone microenvironment. They also support the concept that the skeleton is an important regulator of the systemic tumor environment.

Delineating CSF-1-dependent regulation of myeloid cell diversity in tumors

Myeloid cells contribute to increased malignancy and poor prognosis in breast cancer. We demonstrate that anti-CSF-1R therapy depletes a cell population sharing characteristics of tumor-associated macrophages (TAMs) and dendritic cells (DCs). Intravital imaging combined with cellular characterization has refined our understanding of anti-CSF-1R therapy on the tumor microenvironment.

NLRP3 Inflammasome Mediates Dormant Neutrophil Recruitment following Sterile Lung Injury and Protects against Subsequent Bacterial Pneumonia in Mice

Sterile lung injury is an important clinical problem that complicates the course of severely ill patients. Interruption of blood flow, namely ischemia–reperfusion (IR), initiates a sterile inflammatory response in the lung that is believed to be maladaptive. The rationale for this study was to elucidate the molecular basis for lung IR inflammation and whether it is maladaptive or beneficial. Using a mouse model of lung IR, we demonstrate that sequential blocking of inflammasomes [specifically, NOD-, LRR-, and pyrin domain-containing 3 (NLRP3)], inflammatory caspases, and interleukin (IL)-1β, all resulted in an attenuated inflammatory response. IL-1β production appeared to predominantly originate in conjunction with alveolar type 2 epithelial cells. Lung IR injury recruited unactivated or dormant neutrophils producing less reactive oxygen species thereby challenging the notion that recruited neutrophils are terminally activated. However, lung IR inflammation was able to limit or reduce the bacterial burden from subsequent experimentally induced pneumonia. Notably, inflammasome-deficient mice were unable to alter this bacterial burden following IR. Thus, we conclude that the NLRP3 inflammasome, through IL-1β production, regulates lung IR inflammation, which includes recruitment of dormant neutrophils. The sterile IR inflammatory response appears to serve an important function in inducing resistance to subsequent bacterial pneumonia and may constitute a critical part of early host responses to infection in trauma.

Abstract IA09: Building the metastatic microenvironment

The major cause of cancer-associated mortality is metastasis, but our understanding of this process is far from complete. Tumor cells invade the surrounding tissue of the primary tumor, intravasate into blood and lymphatic vessels, survive and translocate to distant tissues, extravasate, adapt to the new microenvironment and eventually seed, proliferate and colonize to form metastases. Nearly 125 years ago, Paget enunciated the seed and soil hypothesis of cancer. More recently, emerging data suggested that the cellular and extracellular matrix (ECM) microenvironments—both in the primary tumor and in metastatic sites—are crucial at multiple stages of metastasis. Significantly, targeting the tumor microenvironment in metastasis might hold promise for therapy as stromal cells are not mutated and the effects may be widespread as the ECM interacts with multiple tumor cells. The ECM, which is a rich reservoir of pro- and anti-angiogenic cues that regulate neovascularization of the tumor, a crucial process in tumor cell dissemination. The developing metastatic lesions result from a complex crosstalk between disseminating tumor cells and the different players in the microenvironment of the metastatic lesion. The specific recruitment of distinct populations of leukocytes and stromal cells with overlapping functions in metastasis, may open new avenues to the development of metastasis-targeted therapies. Finally, the function of the tumor microenvironment in modulating sensitivity to chemotherapy is of clinical importance. But the aspects of the microenvironment contribute to loss of drug efficacy are still poorly understood in the metastatic setting. The microenvironment is an important source of anticancer drug resistance and also a therapeutic target, as drugs might not need to be completely penetrant to be effective because altering some immune cells, ECM components or the vasculature may have profound effects as seen with regulation of microRNAs. The microenvironment faced by cells at metastatic sites also determines whether these cells die, proliferate or become dormant. How does the microenvironment evolve from the pre-metastatic stage to established metastases? Determining whether the metastatic niche arises from changes in the ECM, decreased immune surveillance or changes in specific pro-inflammatory molecules poses a challenge for the future. We need a more complete understanding of the role of the metastatic microenvironment to uncover how these processes promote metastasis. Chou, J., J.H. Lin, A. Brenot, J.-w. Kim, S. Provot & Z. Werb (2013). GATA3 suppresses metastasis and modulates the tumor microenvironment by regulating miR-29 expression. Nat. Cell Biol. 15: 201-213. PMCID: PMC3660859. Egeblad, M., A. J. Ewald, H. A. Askautrud, B. E. Welm, M. Truitt, E. Bainbridge, G. Peeters, M. Krummell & Z. Werb (2008). Visualizing stromal cell dynamics in different tumor microenvironments by spinning disk confocal microscopy. Dis. Model. Mech. 1:155-167. PMCID: PMC2562195. Kessenbrock, K., G.J.P. Dijkgraaf, D. A. Lawson, L. E. Littlepage, P. Shahi, U. Pieper & Z. Werb (2013). A role for matrix metalloproteinases in regulating mammary stem cell function via the Wnt signaling pathway. Cell Stem Cell. 13:300-313. PMCID: PMC3769456. Lu, P., V. M. Weaver & Z. Werb (2012). Extracellular matrix: a dynamic niche component during cancer progression. J. Cell Biol. 196:396-406. PMCID: PMC3283993. Nakasone, E., H. A.Askautrud, T. Kees, V. Plaks, A. J. Ewald, M. G. Rasch, Y. X. Tan, J. Qin, M. Fein, J. Park, P. Sinha, M. J. Bissell, E. Frengen, Z. Werb & M. Egeblad (2012). Imaging tumor-stroma interactions during chemotherapy reveals microenvironmental contributions to chemoresistance. Cancer Cell. 21:488-503. PMCID: PMC3332002. Citation Format: Amy-Jo Casbon, Vicki Plaks, Zena Werb. Building the metastatic microenvironment. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr IA09. doi:10.1158/1538-7445.CHTME14-IA09

Abstract IA2: Microenvironmental control of bone metastasis

Breast cancer is often associated with a poor prognosis largely due to its tendency to metastasize. Breast cancer preferentially metastasizes to the skeleton. Since Paget9s seed and soil hypothesis from more than a century ago, it is known that bones constitute a very hospitable soil that attracts and allows breast cancer cells to thrive. Surprisingly, however, only a few studies have addressed the role of the bone microenvironment in bone metastasis, and most of them emphasized the role of osteoclasts (bone-destructing cells). Osteoclasts are known to stimulate bone metastasis because of their bone resorbing activity, which releases into the bone marrow space growth factors normally embedded in the bone matrix. On the other hand, the role of osteoblasts (bone-making cells) in this process has not been addressed directly. There is growing evidence that osteoblasts9 function is not limited to making bone. In particular, these cells regulate various aspects of hematopoiesis, insulin secretion in the pancreas, or even male fertility in mice. We hypothesized that osteoblasts play an important role in breast cancer metastasis to bone. We tested this possibility by inoculating a breast cancer cell line (PyMT-FVB-Luc+ cells) into either the mammary fat pad, or directly into the blood stream of syngeneic recipient mice, which presented either an increased or a decreased osteoblast number. Hypoxia and the hypoxia inducible factor 1alpha (Hif-1alpha) play a fundamental role in bone by stimulating the differentiation of the osteoblasts in hypoxic niches where metastatic cells coming from distant tumors are often found. However, the role of the hypoxic microenvironment in bone metastasis is unknown. We used a conditional knockout approach to suppress either Hif-1alpha or von Hippel Lindau (VHL, an E3 ubiquitin-ligase that targets Hif-1alpha for degradation) specifically in osteoblasts (ΔHif-1alphaOB and ΔVHLOB mice). VHL suppression leads to ectopic expression of Hif-1alpha, and thus functions as a gain-of-function model for Hif-1alpha. Tissue specific ablation of Hif-1alpha and VHL in osteoblasts was performed by mating floxed Hif-1alpha or VHL mice, with mice expressing the Cre recombinase under the control of the osterix promoter, which is solely activated in immature osteoblasts. ΔHif-1alphaOB mice presented a decreased number of osteoblasts and reduced bone mass. Conversely, ΔVHLOB mice exhibited a dramatic increase in osteoblast number and bone mass. Intracardiac injection of PyMT-FVB-Luc+ cells into these loss- or gain-of-function models revealed that osteoblasts directly or indirectly stimulate breast cancer metastasis to bone. Strikingly, our results also show that osteoblasts produce systemic changes, controlling breast cancer metastasis to the lungs, as well as the growth of primary mammary tumors. These results were obtained by either transplanting PyMT-FVB-Luc+ cells into the mammary gland, or by tail vein (intravenous) injections. Thus, osteoblasts can affect distant tissues, well beyond the bone microenvironment. By providing the first evidence that the skeleton exerts a systemic control of breast cancer growth and dissemination, our study expands the biological importance of this organ. Citation Format: Sylvain Provot, Audrey Brenot, Amy-Jo Casbon, Ying Yu, Zena Werb. Microenvironmental control of bone metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr IA2.