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Type I interferon-mediated activation of immune cells can facilitate the generation of productive tumor antigen-specific T cell responses in solid tumors. The cGAS/STING DNA sensing pathway is a critical upstream mediator of type I interferon production and is an important regulator of anti-tumor immunity. Numerous STING pathway agonists are now being tested in clinical trials, but the effectiveness of this approach is not yet clear and a better understanding of the relative importance of this pathway in various tumor settings is needed. We have evaluated syngeneic tumor models with different baseline inflammatory states to determine the contributions of STING activity in both tumor and non-tumor cellular compartments to anti-tumor immune responses. We find that productive anti-tumor immune responses in the poorly immunogenic B16F10 model show a strong dependence on STING expression in non-tumor cells. In the immunogenic MC38 model, constitutive STING activation in tumor cells can partially bypass the requirement for STING-dependent activity from immune cells. Our findings reveal multiple, context-dependent roles for STING activity in the regulation of anti-tumor immunity and the response to immunotherapy. In preclinical models where STING is basally active, checkpoint inhibition is more likely to have a therapeutic effect and removal of STING signaling from either the tumor or the non-tumor compartment has a minimal effect. Removal of STING signaling in both, however, diminishes the efficacy derived from checkpoint therapy. Further work is needed to understand the heterogeneity of STING signaling in patients, both in tumor cells and the tumor microenvironment, and the best means of harnessing this pathway to generate anti-tumor immunity and improve therapeutic outcomes.

T cell exhaustion resulting from chronic antigen stimulation and immunosuppression in the tumor microenvironment (TME) limits CAR T efficacy in the solid tumor setting. We have previously shown that engineering therapeutic T cell products using CRISPR-based gene insertion of a dual shRNA cassette targeting Fas and PTPN2 significantly increased antitumor efficacy of Integrated Circuit T cells (ICTs) in ovarian cancer models. We sought to build on this finding to induce additional gene perturbations that improve the efficacy of ICTs. Transforming growth factor (TGF)-b is an immunosuppressive cytokine that potently inhibits T cell responses and is present at high levels in numerous solid tumors, including renal cell carcinoma (RCC). In order to render ICT cells less susceptible to TGF-b-mediated suppression, we developed a quadruple shRNA cassette that simultaneously targets Fas, PTPN2, and TGFBR. Candidate TGFBR-targeting shRNAs were selected for their ability to reduce surface TGFBR receptor expression and impair proximal (pSMAD) or distal (CD103, PD-1) signaling through TGFBR. While single shRNAs against TGFBR did not rescue ICT cell activity in the presence of TGF-b, likely due to partial knockdown of TGFBR signaling, a cassette encoding two shRNAs against TGFBR restored ICT function to similar levels observed in the absence of TGF-b. The quadruple shRNA cassettes targeting Fas/PTPN2/TGFBR significantly enhanced antitumor activity of ICT cells in multiple xenograft tumor models relative to Fas/PTPN2 cassettes. These results demonstrate the utility of multiplexed shRNA strategies to render therapeutic T cells resistant to orthogonal suppressive pathways in solid tumors. Citation Format: Thomas J. Gardner, Adam Litterman, Brenal K. Singh, Luisa Silva, Mukund Hari, Stanley Zhou, Colin Tang, Sahil Joshi, John Gagnon, Oliver Takacsi-Nagy, Jason Hall, Hans Pope, James Zhang, Alma Gomez, Jeremy Chen, Suchismita Mohanty, Vince Thomas, Nicholas Quant, Beatriz Millare, Amy-Jo Casbon, Natalie Bezman, Levi Gray-Rupp, Angela C. Boroughs, W. Nicholas Haining. Multiplexed shRNA cassettes targeting orthogonal pathways (FAS/PTPN2/TGFBR) enhance the potency of integrated circuit T cells (ICTs) in multiple solid tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1768.

Clinically effective adoptive T cell therapy for the treatment of solid tumors will require robust T cell expansion, persistence, and potency. The Janus-kinase signal transducer and activator of transcription (JAK-STAT) pathway governs T cell activation and differentiation, and can thereby serve as a critical regulator of these properties. To take advantage of the benefits of STAT signaling in programming an antitumor T-cell response, we used synthetic biology to create a library of proteins, termed Synthetic Pathway Activators (SPAs) which constitutively drive STAT signaling without the need for external cytokine input. SPAs can be designed to engage activity of multiple STAT family transcription factors at variable levels through rational design. We have developed several classes of SPAs, including but not limited to Class I SPAs, which primarily increase pSTAT3 activity, and Class II SPAs, which increase pSTAT5 activity. When constitutively expressed in ArsenalBio Integrated Circuit T (ICT) cells, SPAs result in significant enhancements in T-cell potency and expansion. Repetitive stimulation assays, wherein T cells are challenged with tumor cells every 2 days, reveal that Class I SPAs result in 6-log or higher improved tumor cell clearance over a 2-week assay period. Across various mouse xenograft models, SPA-expressing ICTs reach at least 6-fold improved tumor growth inhibition. RNAseq and ATACseq analysis indicate dramatic changes to gene expression profiles in T cells expressing Class I SPAs, with maintenance of T cell stem-like phenotypes, and restricted accessibility of various exhaustion marker genes. Importantly, despite significantly increased levels of expansion, ICTs equipped with SPAs are not immortalized, showing no signs of cytokine-independent outgrowth. In addition, SPA-expressing ICT cells rapidly contract following tumor clearance in-vivo. The SPA platform represents a novel, tunable, and T cell intrinsic approach for engineering cell fates that result in potent anti-tumor properties. Citation Format: Thomas J. Gardner, Beatriz Millare, Anzhi Yao, Ashley Cass, Suchismita Mohanty, Jeremy Chen, Alma Gomez, David DeTomaso, Manching Ku, Lionel Berthoin, Meng Lim, Azalea Ong, Vince Thomas, Nicholas Quant, Brian Hsu, Amy-Jo Casbon, Natalie Bezman, Aaron Cooper, Levi Gray-Rupp, Angela C. Boroughs, W. Nicholas Haining. Tunable STAT activation by synthetic pathway activators (SPAs) increases engineered T-cell potency and persistence. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4073.

While traumatic brain injury (TBI) is the leading cause of death and disability in children, we have yet to identify those pathogenic events that determine the extent of recovery. Neutrophils are best known as “first responders” to sites of infection and trauma where they become fully activated, killing pathogens via proteases that are released during degranulation. However, this activational state may generate substantial toxicity in the young brain after TBI that is partially due to developmentally regulated inadequate antioxidant reserves. Neutrophil degranulation is triggered via a downstream signaling pathway that is dependent on spleen tyrosine kinase (Syk). To test the hypothesis that the activational state of neutrophils is a determinant of early pathogenesis and long-term recovery, we compared young, brain-injured conditional knockouts of Syk (sykf/fMRP8-cre+) to congenic littermates (sykf/f). Based upon flow cytometry, there was an extended recruitment of distinct leukocyte subsets, including Ly6G+/Ly6C− and Ly6G+/Ly6Cint, over the first several weeks post-injury which was similar between genotypes. Subsequent assessment of the acutely injured brain revealed a reduction in blood-brain barrier disruption to both high and low molecular weight dextrans and reactive oxygen species in sykf/fMRP8-cre+ mice compared to congenic littermates, and this was associated with greater preservation of claudin 5 and neuronal integrity, as determined by Western blot analyses. At adulthood, motor learning was less affected in brain-injured sykf/fMRP8-cre+ mice as compared to sykf/f mice. Performance in the Morris Water Maze revealed a robust improvement in hippocampal-dependent acquisition and short and long-term spatial memory retention in sykf/fMRP8-cre+ mice. Subsequent analyses of swim path lengths during hidden platform training and probe trials showed greater thigmotaxis in brain-injured sykf/f mice than sham sykf/f mice and injured sykf/fMRP8-cre+ mice. Our results establish the first mechanistic link between the activation state of neutrophils and long-term functional recovery after traumatic injury to the developing brain. These results also highlight Syk kinase as a novel therapeutic target that could be further developed for the brain-injured child.

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.

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.

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.

A myriad of combination immunotherapies is being developed as cancer treatments with the goal of enhancing antitumor immunity. We found that depending on the disease setting, combined immune checkpoint blockade may not always lead to synergistic antitumor effects. While combined anti-CTLA-4 and anti-PD-1 improves control of established tumors, this combination can paradoxically compromise antitumor immunity in the low tumor burden state in preclinical models as well as in melanoma patients. This paradoxical outcome results from treatment-induced apoptosis of tumor-specific T cells. These changes further alter the overall T-cell receptor repertoire. Activated tumor-specific T cells express higher levels of IFN-γ receptor and are more susceptible to apoptosis. Deficiency of IFN-γ receptor on immune cells rescues this phenotype and restores antitumor activity. Additionally, tumor-specific T cells lacking the IFN-γ receptor demonstrate a significant survival advantage compared to their wild-type counterparts in tumor-bearing mice receiving combination therapy. Finally, combination therapy induces significantly higher levels of IFN-γ in the low versus high tumor burden state on a per cell basis, reflecting their less exhausted immune status. Thus, the optimal immunotherapy strategy may depend on disease context, and may not always favor more potent combination immunotherapies. Citation Format: Chien-Chun Steven Pai, Xiaoqing Lu, Donald Simons, Michel DuPage, Kole T. Roybal, Mingyi Chen, Serena Kwek, Amy-Jo Casbon, Gillian A. Kinsbury, Lawrence Fong. Clonal deletion of tumor-specific T cells by combination checkpoint blockade compromises antitumor efficacy in low tumor burden states [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2981.

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.

© 2015, Taylor & Francis Group, LLC. 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.

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

The heterodimeric flavocytochrome b558, comprised of the two integral membrane proteins p22phox and gp91phox, mediates the transfer of electrons from NADPH to molecular oxygen in the phagocyte NADPH oxidase to generate the superoxide precursor of microbicidal oxidants. This study uses deletion mutagenesis to identify regions of p22phox required for maturation of gp91phox and for NADPH oxidase activity. N-terminal, C-terminal, or internal deletions of human p22phox were generated and expressed in Chinese hamster ovary cells with transgenes for gp91phox and two other NADPH oxidase subunits, p47phox, and p67phox. The results demonstrate that p22phox-dependent maturation of gp91phox carbohydrate, cell surface expression of gp91phox, and the enzymatic function of flavocytochrome b558 are closely correlated. Whereas the 5 N-terminal and 25 C-terminal amino acids are dispensable for these functions, the N-terminal 11 amino acids of p22phox are required, as is a hydrophilic region between amino acids 65 and 90. Upon deletion of 54 residues at the C terminus of p22phox (amino acids 142-195), maturation and cell surface expression of gp91phox was still preserved, although NADPH oxidase activity was absent, as expected, due to removal of a proline-rich domain between amino acids 151-160 that is required for recruitment of p47phox. Antibody binding studies indicate that the extreme N terminus of p22phox is inaccessible in the absence of cell permeabilization, supporting a model in which both the N- and C-terminal domains of p22phox extend into the cytoplasm, anchored by two membrane-embedded regions.

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. 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. Egeblad, M., N. Nakasone & Z. Werb (2010). Tumors as organs: complex tissues that interface with the entire organism. Dev. Cell. 18:884-901. PMCID: PMC2905377. Kessenbrock, K., V. Plaks & Z. Werb (2010). Matrix metalloproteinases: Regulators of the tumor microenvironment. Cell. 141: 52-67. PMCID: PMC286205. 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. [Epub ahead of print July 18]. PMID: 23871604. Kouros-Mehr, H., S. K. Bechis, E.M. Slorach, L. E. Littlepage, M. Egeblad, A. J. Ewald, S.-Y. Pai, I-C. Ho & Z. Werb (2008). 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. 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. Nguyen-Ngoc, K.-V., K. J. Cheung, A. Brenot, E. R. Shamir, R. S. Gray, W. C. Hines, P. Yaswen, Z. Werb* & A. J. Ewald* (2012). The ECM microenvironment regulates collective migration and local dissemination in normal and malignant mammary epithelium. Proc. Natl. Acad. Sci. USA. 109:E2595-E2604. PMCID: PMC3465416. Sounni, N. E., K. Dehne, L. van Kempen, M. Egeblad, N.I. Affara, I. Cuevas, J. Wiesen, J. Junankar, L. Korets, L. Lee, J. Shen, C. J. Morrison, C. M. Overall, S. M. Krane, Z. Werb, N. Boudreau & L. M. Coussens (2010). Stromal regulation of vessel stability by MMP9 and TGFβ. Dis. Model. Mech. 3: 317-332. PMCID: PMC20223936. Citation Format: Amy Jo Casbon, Marja Lohela, Vicki Plaks, Zena Werb. How tumors regulate their microenvironment in primary sites and metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr IA24.

Breast cancer is often associated with a poor prognosis largely due to its tendency to metastasize. Breast cancer preferentially metastasizes to the skeleton. Since Paget's 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 osteoblasts' 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.