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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.

Resistance to checkpoint-blockade treatments is a challenge in the clinic. We found that although treatment with combined anti-CTLA-4 and anti-PD-1 improved control of established tumors, this combination compromised anti-tumor immunity in the low tumor burden (LTB) state in pre-clinical models as well as in melanoma patients. Activated tumor-specific Tcells expressed higher amounts of interferon-γ (IFN-γ) receptor and were more susceptible to apoptosis than naive Tcells. Combination treatment induced deletion of tumor-specific Tcells and altered the Tcell repertoire landscape, skewing the distribution of Tcells toward lower-frequency clonotypes. Additionally, combination therapy induced higher IFN-γ production in the LTB state than in the high tumor burden (HTB) state on a per-cell basis, reflecting a less exhausted immune status in the LTB state. Thus, elevated IFN-γ secretion in the LTB state contributes to the development of an immune-intrinsic mechanism of resistance to combination checkpoint blockade, highlighting the importance of achieving the optimal magnitude of immune stimulation for successful combination immunotherapy strategies.

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.

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.

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.

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 α-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.

IFNγ regulates trafficking and synthesis of flavocytochrome b558, suggesting a role to control superoxide production in macrophages. Flavocytochrome b558, the catalytic core of the phagocyte NADPH oxidase (NOX2), mediates electron transfer from NADPH to molecular oxygen to generate superoxide, the precursor of highly ROS for host defense. Flavocytochrome b558 is an integral membrane heterodimer consisting of a large glycosylated subunit, gp91phox, and a smaller subunit, p22phox. We recently showed in murine macrophages that flavocytochrome b558 localizes to the PM and Rab11-positive recycling endosomes, whereas in primary hMDMs, gp91phox and p22phox reside in the PM and the ER. The antimicrobial activity of macrophages, including ROS production, is greatly enhanced by IFN-γ, but how this is achieved is incompletely understood. To further define the mechanisms by which IFN-γ enhances macrophage NADPH oxidase activity, we evaluated changes in flavocytochrome b558 expression and localization, along with NADPH oxidase activity, in IFN-γ stimulated RAW 264.7 cells and primary murine BMDMs and hMDMs. We found that enhanced capacity for ROS production is, in part, a result of increased protein expression of gp91phox and p22phox but also demonstrate that IFN-γ induced a shift in the predominant localization of gp91phox and p22phox from intracellular membrane compartments to the PM. Our results are the first to show that a cytokine can change the distribution of macrophage flavocytochrome b558 and provide a potential, new mechanism by which IFN-γ modulates macrophage antimicrobial activity. Altogether, our data suggest that the mechanisms by which IFN-γ regulates antimicrobial activity of macrophages are more complex than previously appreciated.

Flavocytochrome b558, the catalytic core of the phagocytic NADPH oxidase, mediates the transfer of electrons from NADPH to molecular oxygen to generate superoxide for host defense. Flavocytochrome b is a membrane heterodimer consisting of a large subunit gp91phox (NOX2) and a smaller subunit, p22phox. Although in neutrophils flavocytochrome b has been shown to localize to the plasma membrane and specific granules, little is known about its distribution in macrophages. Using immunofluorescent staining and live cell imaging of fluorescently tagged gp91phox and p22phox, we demonstrate in a Chinese hamster ovary cell model system and in RAW 264.7 and primary murine bone marrow-derived macrophages that flavocytochrome b is found in the Rab11-positive recycling endocytic compartment, as well as in Rab5-positive early endosomes and plasma membrane. Additionally, we show that unassembled p22phox and gp91phox subunits localize to the endoplasmic reticulum, which redistribute to the cell surface and endosomal compartments following heterodimer formation. These studies show for the first time that flavocytochrome b localizes to intracellular compartments in macrophages that recycle to the plasma membrane, which may act as a reservoir to deliver flavocytochrome b to the cell surface and phagosome membranes.

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

In 1863, Virchow hypothesized that the cancer originated at sites of chronic tissue injury and that the ensuing inflammation they cause enhances cell proliferation. While it is now clear that proliferation of tumor cells alone does not cause cancer, sustained cell proliferation in an environment rich in inflammatory cells, growth factors, activated stroma certainly potentiates and/or promotes neoplastic risk and leads to metastasis. In fact, sometimes the trigger for neoplastic progression may come from signals within the stromal microenvironment. The stromal microenvironment consists of fibroblasts, macrophages, neutrophils, vascular components, nerves and inflammatory cells of the innate and acquired immune response, as well as the extracellular matrix that they elaborate along with the molecules that are concentrated and immobilized on it. All of these components communicate with each other and with the neoplastic cells to contribute to the aberrant tumor organ. Factors elaborated by tumors cause dramatic increases in inflammatory myeloid cells in the microenvironment of the primary tumor as well as in distant sites upon the malignant conversion of the tumor cells. These myeloid cells modulate the tumor ecology and regulate metastatic take, altering vascular permeability and responses to chemotherapy. The dynamic interplay of tumor cells and host cells responding to the tumor in the microenvironment contribute to tumor evolution, metastasis and evasion of therapeutic responses. Supported by grants from the National Cancer Institute. 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. [2013 Jan 27, PMCID: PMC3660859. Chou, J., P. Shahi & Z. Werb (2013). MicroRNA-mediated regulation of the tumor microenvironment. Cell Cycle. In press. Egeblad, M., A. J. Ewald, H. A. Askautrud, B. E. Welm, M. 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GATA-3 links tumor differentiation and dissemination in a luminal breast cancer model. Cancer Cell. 13:141-152. PMCID: PMC2262951. Littlepage, L. E., M. D. Sternlicht, N. Rougier, J. Phillips, E. Gallo, Y. Yu, K. Williams, A. Brenot, J. I. Gordon & Z. Werb (2010). Matrix metalloproteinases contribute distinct roles in neuroendocrine prostate carcinogenesis, metastasis, and angiogenesis progression. Cancer Res. 70:2224-2234. PMCID: PMC2840052. Littlepage, L.E., A. S. Adler, H. Kouros-Mehr, G. Huang, J. Chou, S. R. Krig, O. L. Griffith, J. E. Korkola, K. Qu, D. A. Lawson, Q. Xue, M. D. Sternlicht, G. J. P. Dijkgraaf, P. Yaswen, H. S. Rugo, C. A. Sweeney, C. C. Collins, J. W. Gray, H. Y. Chang & Z. Werb (2012). The transcription factor ZNF217 is a prognostic biomarker and therapeutic target during breast cancer progression. Cancer Discov. 2 : 638-651. PMCID : PMC3546490. Lu, P., V. M. Weaver & Z. Werb (2012). Extracellular matrix: a dynamic niche component during cancer progression. J. 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