Mouse Models of Melanoma Bone Metastasis.

Melanomas are aggressive cancers of the skin with high metastatic capacity. Mouse models are necessary to delineate the mechanisms of cancer metastasis and xenograft models can also allow examining the role of the host using different genetically-modified mouse models. In this chapter, I provide a detailed protocol for the preparation and inoculation of tumor cells intra-cardially and intra-tibially to achieve bone metastasis.

Fra-2 Expression in Osteoblasts Regulates Systemic Inflammation and Lung Injury through Osteopontin.

Inflammatory responses require mobilization of innate immune cells from the bone marrow. The functionality of this process depends on the state of the bone marrow microenvironment. We therefore hypothesized that molecular changes in osteoblasts, which are essential stromal cells of the bone marrow microenvironment, influence the inflammatory response. Here, we show that osteoblast-specific expression of the AP-1 transcription factor Fra-2 (Fra-2Ob-tet) induced a systemic inflammatory state with infiltration of neutrophils and proinflammatory macrophages into the spleen and liver as well as increased levels of proinflammatory cytokines, such as interleukin-1ß (IL-1ß), IL-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF). By in vivo inhibition of osteopontin (OPN) in Fra-2Ob-tet mice, we demonstrated that this process was dependent on OPN expression, which mediates alterations of the bone marrow niche. OPN expression was transcriptionally enhanced by Fra-2 and stimulated mesenchymal stem cell (MSC) expansion. Furthermore, in a murine lung injury model, Fra-2Ob-tet mice showed increased inflammatory responses and more severe disease features via an enhanced and sustained inflammatory response to lipopolysaccharide (LPS). Our findings demonstrate for the first time that molecular changes in osteoblasts influence the susceptibility to inflammation by altering evasion of innate immune cells from the bone marrow space.

Somatic genome editing with the RCAS-TVA-CRISPR-Cas9 system for precision tumor modeling.

To accurately recapitulate the heterogeneity of human diseases, animal models require to recreate multiple complex genetic alterations. Here, we combine the RCAS-TVA system with the CRISPR-Cas9 genome editing tools for precise modeling of human tumors. We show that somatic deletion in neural stem cells of a variety of known tumor suppressor genes (Trp53, Cdkn2a, and Pten) leads to high-grade glioma formation. Moreover, by simultaneous delivery of pairs of guide RNAs we generate different gene fusions with oncogenic potential, either by chromosomal deletion (Bcan-Ntrk1) or by chromosomal translocation (Myb-Qk). Lastly, using homology-directed-repair, we also produce tumors carrying the homologous mutation to human BRAF V600E, frequently identified in a variety of tumors, including different types of gliomas. In summary, we have developed an extremely versatile mouse model for in vivo somatic genome editing, that will elicit the generation of more accurate cancer models particularly appropriate for pre-clinical testing.

Chronic systemic inflammation originating from epithelial tissues.

Chronic systemic inflammation (CSI) has recently been identified as a major contributor to common diseases ranging from cancer to metabolic disorders and neurologic alterations. In the last decade, we and others have generated genetically engineered mouse models for inflammatory diseases, which enable studying the molecular mechanisms of CSI. Recently, organ cross-talk induced by CSI under homeostatic and pathological conditions has begun to be appreciated. In this review, we will revisit whole organism physiology in relation to CSI originating from epithelial tissues, such as the skin and gut. Furthermore, we will discuss the current knowledge regarding the mechanisms, the specific immune cells and molecules responsible for inducing the most common comorbidities, such as cardiovascular, metabolic, and neurological complications, as well as bone loss, in heterogeneous diseases like psoriasis, atopic dermatitis, and inflammatory bowel disease. As it would be impossible to discuss all comorbidities of these diseases as well as all epithelial tissues, we present an overview with a special emphasis on our recent findings linking skin inflammation to bone loss.

Chronic skin inflammation leads to bone loss by IL-17-mediated inhibition of Wnt signaling in osteoblasts.

Inflammation has important roles in tissue regeneration, autoimmunity, and cancer. Different inflammatory stimuli can lead to bone loss by mechanisms that are not well understood. We show that skin inflammation induces bone loss in mice and humans. In psoriasis, one of the prototypic IL-17A-mediated inflammatory human skin diseases, low bone formation and bone loss correlated with increased serum IL-17A levels. Similarly, in two mouse models with chronic IL-17A-mediated skin inflammation,K14-IL17A(ind)andJunB(Δep), strong inhibition of bone formation was observed, different from classical inflammatory bone loss where osteoclast activation leads to bone degradation. We show that under inflammatory conditions, skin-resident cells such as keratinocytes, γδ T cells, and innate lymphoid cells were able to express IL-17A, which acted systemically to inhibit osteoblast and osteocyte function by a mechanism involving Wnt signaling. IL-17A led to decreased Wnt signaling in vitro, and importantly, pharmacological blockade of IL-17A rescued Wnt target gene expression and bone formation in vivo. These data provide a mechanism where IL-17A affects bone formation by regulating Wnt signaling in osteoblasts and osteocytes. This study suggests that using IL-17A blocking agents in psoriasis could be beneficial against bone loss in these patients.

Signalling in inflammatory skin disease by AP-1 (Fos/Jun).

Skin inflammation is a physiological reaction to tissue injury, pathogen invasion and irritants. During this process, innate and/or adaptive immune cells are activated and recruited to the site of inflammation to either promote or suppress inflammation. The sequential recruitment and activation of immune cells is modulated by a combination of cytokines and chemokines, which are regulated by transcription factors, such as AP-1 (Fos/Jun), NF-κB, NFATs, and STATs. Here we review the present evidence and the underlying mechanisms of how Jun/AP-1 proteins control skin inflammation. Genetically engineered mouse models (GEMMs) in which AP-1 proteins are deleted in the epidermis have revealed that these proteins control cytokine expression at multiple levels. Constitutive epidermal deletion of JunB in mice leads to a multi-organ disease characterised by increased levels of pro-inflammatory cytokines. These JunB-deficient mutant mice display several phenotypes from skin inflammation to a G-CSF-dependent myeloproliferative disease, as well as kidney atrophy and bone loss, reminiscent of psoriasis and systemic lupus erythematosus. Importantly, epidermal deletion of both JunB and c-Jun in an inducible manner in adult mice leads to a psoriasis-like disease, in which the epidermal proteome expression profile is comparable to the one from psoriasis patient samples. In this GEMM and in psoriasis patient-derived material, S100A8/A9-dependent C3/CFB complement activation, as well as a miR-21-dependent TIMP-3/TACE pathway leading to TNF-α shedding, plays causal roles in disease development. The newly identified therapeutic targets from GEMMs together with investigations in human patient samples open up new avenues for therapeutic interventions for psoriasis and related inflammatory skin diseases.

Preclinical mouse models of osteosarcoma.

Osteosarcoma is the most common form of primary bone tumors with high prevalence in children. Survival rates of osteosarcoma are low, especially in the case of metastases. Mouse models of this disease have been very valuable in investigation of mechanisms of tumorigenesis, metastasis, as well as testing possible therapeutic options. In this chapter, we summarize currently available mouse models for osteosarcoma and provide detailed methodology for the isolation of cell lines from genetically engineered mouse models (GEMMs), gene modification and tumor cell injection methods, as well as imaging techniques.

Characterization of mouse model-derived osteosarcoma (OS) cells in vitro and in vivo.

Osteosarcoma (OS) is the most common primary tumor of bone with a high incidence in children. Treatment options for OS are limited, and once metastasized, the prognosis is very poor. Genetically engineered mouse models (GEMMs) are valuable tools to understand the mechanisms of tumorigenesis and to test possible therapies. In this chapter, we summarize the methods related to the isolation, characterization, and transplantation of OS cells obtained from GEMMs.

Fos-dependent induction of Chk1 protects osteoblasts from replication stress.

Stable Fos expression in the osteoblast lineage results in the development of osteosarcomas (OS) in mice, yet the underlying mechanisms are poorly understood. Using a genetic system in which Fos expression can be induced in osteoblasts in a doxycycline-dependent manner and through subsequent RNA sequencing and gene set enrichment analysis, we were able to identify novel transcriptional targets of Fos in osteoblasts. These included a distinct activation of cellular response toward replication stress (RS), exemplified by a Fos-dependent induction of the RS-suppressing Chk1 kinase. Importantly, Fos expression protects osteoblasts from RS and DNA damage likely through upregulation of Chk1 and facilitates transformation by Ras/E1A oncogenes. These data reveal a novel function of Fos in safeguarding genome stability during replication, which is particularly relevant in conditions of oncogene-induced S-phase entry.

The ADP receptor P2RY12 regulates osteoclast function and pathologic bone remodeling.

The adenosine diphosphate (ADP) receptor P2RY12 (purinergic receptor P2Y, G protein coupled, 12) plays a critical role in platelet aggregation, and P2RY12 inhibitors are used clinically to prevent cardiac and cerebral thrombotic events. Extracellular ADP has also been shown to increase osteoclast (OC) activity, but the role of P2RY12 in OC biology is unknown. Here, we examined the role of mouse P2RY12 in OC function. Mice lacking P2ry12 had decreased OC activity and were partially protected from age-associated bone loss. P2ry12-/- OCs exhibited intact differentiation markers, but diminished resorptive function. Extracellular ADP enhanced OC adhesion and resorptive activity of WT, but not P2ry12-/-, OCs. In platelets, ADP stimulation of P2RY12 resulted in GTPase Ras-related protein (RAP1) activation and subsequent αIIbß3 integrin activation. Likewise, we found that ADP stimulation induced RAP1 activation in WT and integrin ß3 gene knockout (Itgb3-/-) OCs, but its effects were substantially blunted in P2ry12-/- OCs. In vivo, P2ry12-/- mice were partially protected from pathologic bone loss associated with serum transfer arthritis, tumor growth in bone, and ovariectomy-induced osteoporosis: all conditions associated with increased extracellular ADP. Finally, mice treated with the clinical inhibitor of P2RY12, clopidogrel, were protected from pathologic osteolysis. These results demonstrate that P2RY12 is the primary ADP receptor in OCs and suggest that P2RY12 inhibition is a potential therapeutic target for pathologic bone loss.

Dissection of platelet and myeloid cell defects by conditional targeting of the beta3-integrin subunit.

The purpose of this work was to determine platelet and myeloid cell-specific requirements for beta3-containing integrins in hemostasis, bone resorption, and tumor growth. LoxP-flanked mice were generated to study the conditional deletion of beta3-integrin in platelets [knockout in platelets (KOP)] and myeloid cells [knockout in myeloid (KOM)]. Using the beta3KOP and beta3KOM strains of mice, we studied the role of beta3-integrin in hemostasis, bone resorption, and subcutaneous tumor growth. Tissue-specific deletion of platelet beta3-integrins in beta3KOP mice did not affect bone mass but resulted in a severe bleeding phenotype. No growth difference of tumor xenografts or in neoangiogenesis were found in beta3KOP mice, in contrast to the defects observed in germline beta3(-/-) mice. Conditional deletion of myeloid beta3-integrins in beta3KOM mice resulted in osteopetrosis but had no effect on hemostasis or mortality. Tumor growth in beta3KOM mice was increased and accompanied by decreased macrophage infiltration, without increase in blood vessel number. Platelet beta3-integrin deficiency was sufficient to disrupt hemostasis but had no effect on bone mass or tumor growth. Myeloid-specific beta3-integrin deletion was sufficient to perturb bone mass and enhance tumor growth due to reduced macrophage infiltration in the tumors. These results suggest that beta3-integrins have cell-specific roles in complex biological processes.-Morgan, E. A., Schneider, J. G., Baroni, T. E., Uluçkan, O., Heller, E., Hurchla, M. A., Deng, H., Floyd, D., Berdy, A., Prior, J. L., Piwnica-Worms, D., Teitelbaum, S. L., Ross, F. P., Weilbaecher, K. N. Dissection of platelet and myeloid cell defects by conditional targeting of the beta3-integrin subunit.

CD47 regulates bone mass and tumor metastasis to bone.

CD47, also called integrin-associated protein, plays a critical role in the innate immune response and is an atypical member of the immunoglobulin superfamily that interacts with and activates beta3 integrins. beta3 integrin(-/-) mice have defective platelet and osteoclast function and are protected from bone metastasis. The role of CD47 in skeletal homeostasis and bone metastasis has not been described. CD47(-/-) mice had increased bone mass and defective osteoclast function in vivo. Although the number of functional osteoclasts formed by differentiating CD47(-/-) bone marrow macrophages was decreased, high doses of RANKL rescued differentiation and function of CD47(-/-) osteoclasts ex vivo and rescued the osteoclast defect in CD47(-/-) mice. Inhibition of nitric oxide (NO) synthase, which is expressed at higher levels in CD47(-/-) osteoclasts, also rescued the osteoclast defect in CD47(-/-) cells. We then examined the consequences of this osteoclast defect in bone metastasis. In a model of tumor metastasis to bone, bone tumor burden was decreased in the CD47(-/-) mice compared with wild-type (WT) controls, with no decrease in s.c. tumor growth in CD47(-/-) mice. There was decreased tumor-associated bone destruction in the CD47(-/-) mice compared with WT controls, consistent with a defect in osteoclast function that was not rescued by the presence of tumor. Our data show that CD47 regulates osteoclastogenesis, in part, via regulation of NO production, and its disruption leads to a decrease in tumor bone metastasis. CD47 is a novel therapeutic target to strengthen bone mass and diminish metastatic tumor growth in bone.

Interferon-gamma targets cancer cells and osteoclasts to prevent tumor-associated bone loss and bone metastases.

Interferon-gamma (IFN-gamma) has been shown to enhance anti-tumor immunity and inhibit the formation of bone-resorbing osteoclasts. We evaluated the role of IFN-gamma in bone metastases, tumor-associated bone destruction, and hypercalcemia in human T cell lymphotrophic virus type 1-Tax transgenic mice. Compared with Tax(+)IFN-gamma(+/+) mice, Tax(+)IFN-gamma(-/-) mice developed increased osteolytic bone lesions and soft tissue tumors, as well as increased osteoclast formation and activity. In vivo administration of IFN-gamma to tumor-bearing Tax(+)IFN-gamma(-/-) mice prevented new tumor development and resulted in decreased bromodeoxyuridine uptake by established tumors. In vitro, IFN-gamma directly decreased the viability of Tax(+) tumor cells through inhibition of proliferation, suppression of ERK phosphorylation, and induction of apoptosis and caspase 3 cleavage. IFN-gamma also inhibited macrophage colonystimulating factor-mediated proliferation and survival of osteoclast progenitors in vitro. Administration of IFN-gamma to C57BL/6 mice decreased Tax(+) tumor growth and prevented tumor-associated bone loss and hypercalcemia. In contrast, IFN-gamma treatment failed to protect IFN-gammaR1(-/-) mice from Tax(+) tumor-induced skeletal complications, despite decreasing tumor growth. These data demonstrate that IFN-gamma suppressed tumor-induced bone loss and hypercalcemia in Tax(+) mice by inhibiting both Tax(+) tumor cell growth and host-induced osteolysis. These data suggest a protective role for IFN-gamma in patients with bone metastases and hypercalcemia of malignancy.

APT102, a novel adpase, cooperates with aspirin to disrupt bone metastasis in mice.

Platelets contribute to the development of metastasis, the most common cause of mortality in cancer patients, but the precise role that anti-platelet drugs play in cancer treatment is not defined. Metastatic tumor cells can produce platelet alphaIIb beta3 activators, such as ADP and thromboxane A(2) (TXA(2)). Inhibitors of platelet beta3 integrins decrease bone metastases in mice but are associated with significant bleeding. We examined the role of a novel soluble apyrase/ADPase, APT102, and an inhibitor of TXA(2) synthesis, acetylsalicylic acid (aspirin or ASA), in mouse models of experimental bone metastases. We found that treatment with ASA and APT102 in combination (ASA + APT102), but not either drug alone, significantly decreased breast cancer and melanoma bone metastases in mice with fewer bleeding complications than observed with alphaIIb beta3 inhibition. ASA + APT102 diminished tumor cell induced platelet aggregation but did not directly alter tumor cell viability. Notably, APT102 + ASA treatment did not affect initial tumor cell distribution and similar results were observed in beta3-/- mice. These results show that treatment with ASA + APT102 decreases bone metastases without significant bleeding complications. Anti-platelet drugs such as ASA + APT102 could be valuable experimental tools for studying the role of platelet activation in metastasis as well as a therapeutic option for the prevention of bone metastases.

Disruption of CXCR4 enhances osteoclastogenesis and tumor growth in bone.

CXCR4 regulates hematopoietic and tumor cell homing to bone, but its role during osteoclast (OC) development is unknown. We investigated the role of CXCR4 in osteoclastogenesis and in a model of bone metastasis. Compared with controls, mice reconstituted with CXCR4 null hematopoietic cells exhibited elevated markers of bone resorption, increased OC perimeter along bone, and increased bone loss. CXCR4-/- OCs demonstrated accelerated differentiation and enhanced bone resorption in vitro. Furthermore, tumor growth specifically in bone was significantly increased in mice reconstituted with CXCR4-/- hematopoietic cells. Finally, enhancement of bone tumor growth in the absence of CXCR4 was abrogated with the OC inhibitor, zoledronic acid. These data demonstrate that disruption of CXCR4 enhances osteoclastogenesis and suggest that inhibition of CXCR4 may enhance established skeletal tumor burden by increasing OC activity.

Granulocyte colony-stimulating factor enhances bone tumor growth in mice in an osteoclast-dependent manner.

Inhibition of osteoclast (OC) activity has been associated with decreased tumor growth in bone in animal models. Increased recognition of factors that promote osteoclastic bone resorption in cancer patients led us to investigate whether increased OC activation could enhance tumor growth in bone. Granulocyte colony-stimulating factor (G-CSF) is used to treat chemotherapy-induced neutropenia, but is also associated with increased markers of OC activity and decreased bone mineral density (BMD). We used G-CSF as a tool to investigate the impact of increased OC activity on tumor growth in 2 murine osteolytic tumor models. An 8-day course of G-CSF alone (without chemotherapy) significantly decreased BMD and increased OC perimeter along bone in mice. Mice administered G-CSF alone demonstrated significantly increased tumor growth in bone as quantitated by in vivo bioluminescence imaging and histologic bone marrow tumor analysis. Short-term administration of AMD3100, a CXCR4 inhibitor that mobilizes neutrophils with little effect on bone resorption, did not lead to increased tumor burden. However, OC-defective osteoprotegerin transgenic (OPG(Tg)) mice and bisphosphonate-treated mice were resistant to the effects of G-CSF administration upon bone tumor growth. These data demonstrate a G-CSF-induced stimulation of tumor growth in bone that is OC dependent.

Skeletal complications of breast cancer therapies.

Nonsurgical treatment options, such as hormonal therapy, chemotherapy, radiation, and bisphosphonate therapy, are undoubtedly improving outcomes for women with breast cancer; however, these therapies also carry significant skeletal side effects. For example, adjuvant hormonal treatments, such as aromatase inhibitors that disrupt the estrogen-skeleton axis, have the potential to cause decreased bone mineral density. Similarly, chemotherapy often induces primary ovarian failure in premenopausal women, resulting in decreased levels of circulating estrogen and subsequent osteopenia. In both cases, women receiving these therapies are at an increased risk for the development of osteoporosis and skeletal fracture. Furthermore, women undergoing radiation therapy to the upper body may have an increased incidence of rib fracture, and those receiving bisphosphonates may be vulnerable to the development of osteonecrosis of the jaw. Therefore, women with breast cancer who are undergoing any of these therapies should be closely monitored for bone mineral loss and advised of skeletal health maintenance strategies.