Insights into immuno-oncology drug development landscape with focus on bone metastasis

Bone is among the main sites of metastasis in breast, prostate and other major cancers. Bone metastases remain incurable causing high mortality, severe skeletal-related effects and decreased quality of life. Despite the success of immunotherapies in oncology, no immunotherapies are approved for bone metastasis and no clear benefit has been observed with approved immunotherapies in treatment of bone metastatic disease. Therefore, it is crucial to consider unique features of tumor microenvironment in bone metastasis when developing novel therapies. The vicious cycle of bone metastasis, referring to crosstalk between tumor and bone cells that enables the tumor cells to grow in the bone microenvironment, is a well-established concept. Very recently, a novel osteoimmuno-oncology (OIO) concept was introduced to the scientific community. OIO emphasizes the significance of interactions between tumor, immune and bone cells in promoting tumor growth in bone metastasis, and it can be used to reveal the most promising targets for bone metastasis. In order to provide an insight into the current immuno-oncology drug development landscape, we used 1stOncology database, a cancer drug development resource to identify novel immunotherapies in preclinical or clinical development for breast and prostate cancer bone metastasis. Based on the database search, 24 immunotherapies were identified in preclinical or clinical development that included evaluation of effects on bone metastasis. This review provides an insight to novel immuno-oncology drug development in the context of bone metastasis. Bone metastases can be approached using different modalities, and tumor microenvironment in bone provides many potential targets for bone metastasis. Noting current increasing interest in the field of OIO, more therapeutic opportunities that primarily target bone metastasis are expected in the future

Dovitinib dilactic acid reduces tumor growth and tumor-induced bone changes in an experimental breast cancer bone growth model

Advanced breast cancer has a high incidence of bone metastases. In bone, breast cancer cells induce osteolytic or mixed bone lesions by inducing an imbalance in bone formation and resorption. Activated fibroblast growth factor receptors (FGFRs) are important in regulation of tumor growth and bone remodeling. In this study we used FGFR1 and FGFR2 gene amplifications containing human MFM223 breast cancer cells in an experimental xenograft model of breast cancer bone growth using intratibial inoculation technique. This model mimics bone metastases in breast cancer patients. The effects of an FGFR inhibitor, dovitinib dilactic acid (TKI258) on tumor growth and tumor-induced bone changes were evaluated. Cancer-induced bone lesions were smaller in dovitinib-treated mice as evaluated by X-ray imaging. Peripheral quantitative computed tomography imaging showed higher total and cortical bone mineral content and cortical bone mineral density in dovitinib-treated mice, suggesting better preserved bone mass. CatWalk gait analysis indicated that dovitinib-treated mice experienced less cancer-induced bone pain in the tumor-bearing leg. A trend towards decreased tumor growth and metabolic activity was observed in dovitinib-treated mice quantified by positron emission tomography imaging with 2-[ 18 F]fluoro-2-deoxy-D-glucose at the endpoint. We conclude that dovitinib treatment decreased tumor burden, cancer-induced changes in bone, and bone pain. The results suggest that targeting FGFRs could be beneficial in breast cancer patients with bone metastases.</p

Immunotherapies and Metastatic Cancers: Understanding Utility and Predictivity of Human Immune Cell Engrafted Mice in Preclinical Drug Development

Metastases cause high mortality in several cancers and immunotherapies are expected to be effective in the prevention and treatment of metastatic disease. However, only a minority of patients benefit from immunotherapies. This creates a need for novel therapies that are efficacious regardless of the cancer types and metastatic environments they are growing in. Preclinical immuno-oncology models for studying metastases have long been limited to syngeneic or carcinogenesis-inducible models that have murine cancer and immune cells. However, the translational power of these models has been questioned. Interactions between tumor and immune cells are often species-specific and regulated by different cytokines in mice and humans. For increased translational power, mice engrafted with functional parts of human immune system have been developed. These humanized mice are utilized to advance understanding the role of immune cells in the metastatic process, but increasingly also to study the efficacy and safety of novel immunotherapies. From these aspects, this review will discuss the role of immune cells in the metastatic process and the utility of humanized mouse models in immuno-oncology research for metastatic cancers, covering several models from the perspective of efficacy and safety of immunotherapies

Osteoimmuno-Oncology: Therapeutic Opportunities for Targeting Immune Cells in Bone Metastasis

Immunotherapies provide a potential treatment option for currently incurable bone metastases. Bone marrow is an important secondary lymphoid organ with a unique immune contexture. Even at non-disease state immune cells and bone cells interact with each other, bone cells supporting the development of immune cells and immune cells regulating bone turnover. In cancer, tumor cells interfere with this homeostatic process starting from formation of pre-metastatic niche and later supporting growth of bone metastases. In this review, we introduce a novel concept osteoimmuno-oncology (OIO), which refers to interactions between bone, immune and tumor cells in bone metastatic microenvironment. We also discuss therapeutic opportunities of targeting immune cells in bone metastases, and associated efficacy and safety concerns

Human Immune System Increases Breast Cancer-Induced Osteoblastic Bone Growth in a Humanized Mouse Model without Affecting Normal Bone

Bone metastases are prevalent in many common cancers such as breast, prostate, and lung cancers, and novel therapies for treating bone metastases are needed. Human immune system-engrafted models are used in immuno-oncology (IO) studies for subcutaneous cancer cell or patient-derived xenograft implantations that mimic primary tumor growth. Novel efficacy models for IO compounds on bone metastases need to be established. The study was performed using CIEA NOG (NOG) mice engrafted with human CD34+ hematopoietic stem cells (huNOG) and age-matched immunodeficient NOG mice. Bone phenotyping was performed to evaluate baseline differences. BT-474 human breast cancer cells were inoculated into the tibia bone marrow, and cancer-induced bone changes were monitored by X-ray imaging. Bone content and volume were analyzed by dual X-ray absorptiometry and microcomputed tomography. Tumor-infiltrating lymphocytes (TILs) and the expression of immune checkpoint markers were analyzed by immunohistochemistry. Bone phenotyping showed no differences in bone architecture or volume of the healthy bones in huNOG and NOG mice, but the bone marrow fat was absent in huNOG mice. Fibrotic areas were observed in the bone marrow of some huNOG mice. BT-474 tumors induced osteoblastic bone growth. Bone lesions appeared earlier and were larger, and bone mineral density was higher in huNOG mice. huNOG mice had a high number of human CD3-, CD4-, and CD8-positive T cells and CD20-positive B cells in immune-related organs. A low number of TILs and PD-1-positive cells and low PD-L1 expression were observed in the BT-474 tumors at the endpoint. This study reports characterization of the first breast cancer bone growth model in huNOG mice. BT-474 tumors represent a “cold” tumor with a low number of TILs. This model can be used for evaluating the efficacy of combination treatments of IO therapies with immune-stimulatory compounds or therapeutic approaches on bone metastatic breast cancer

Regulation of FGFR1 mRNA expression in sh cells and MCF-7 cells.

<p>Expression of total FGFR1 or FGFR1IgIIIc was quantified by qRT-PCR in the cells cultured as follows: A) The cells grown in standard growth medium were treated with PD173074 for 24 h. B) The cells were grown without testosterone (Te) for 5 days and without serum for 48 h. C) The FGF-8b-overexpressing S115 cell lines (FGF8b1 and FGF8b14) and the transfection control cell lines (Mock1 and Mock3) were cultured in 4% DC-FBS in the absence of Te. D) shLacZ cells were grown without Te for 2 days and without serum for 24 h followed by treatment with FGF-8b (25 ng/ml) for 24 h. E) MCF-7 cells were grown in standard growth medium and treated similarly with FGF-8. The experiment was performed as triplicates and repeated twice with similar results. The statistical difference between the vehicle- and FGF-8b-treated cells was determined by independent sample t-test, * <i>P</i><0.05.</p

Growth of sh cells in nude mouse tumors.

<p>1*10⁶ cells were inoculated subcutaneously (s.c.) into male nude mice (n = 5–6). A) Tumor growth was monitored every 3–4 days with a caliper until the end of the experiment. Representative growth curves from one of the two experiments with similar results are shown. Mean ± SE from each group is shown. Statistical significance of the growth rate differences were tested by Repeated Measures ANOVA, * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001. B) FGFR inhibitor PD173074 was administered to mice bearing shR2 cell tumors, starting from day 13 (indicated by an arrow), at a dose of 25 mg/kg 5 times/week until the end of the experiment. Mean ± SE from each group is shown. C) FGFR mRNA levels were quantified by qRT-PCR analysis from shLacZ, shR1 and shR2 tumors from a separate experiment, in which the tumors were grown for 8 weeks in nude mice. Statistical difference between mRNA levels were tested by independent sample t-test, * <i>P</i><0.05. D) P-HisH3 immunohistochemical staining of shLacZ, shR1, shR2 and shR3 tumor sections (upper panel). Scale bar = 100 µm. Lower panel shows the number of P-HisH3-positive cells per field. P-HisH3-positive cells were counted in 5–15 fields per tumor section and each group contained six tumor sections. Statistical differences between groups were tested by ANOVA followed by Bonferroni's multiple comparison test, * <i>P</i><0.05.</p

FGF-induced ERK1/2 activation in sh cells.

<p>Cells were pre-cultured in DC-FBS for 48 h and then treated with A) 25 ng/ml FGF-8b, B) 10 ng/ml FGF-2 or 100 ng/ml FGF-7 or PBS vehicle for indicated time periods. Whole-cell lysates were generated from cells and protein was subjected to SDS-PAGE and immunoblotted with antibodies against p-ERK1/2 and ERK1/2. The intensity of the bands was determined by scanning densitometry and is presented in columns as the p-ERK1/2 expression relative to ERK expression. The experiment was repeated twice with similar results.</p

Proliferation and cyclin expression of sh cells <i>in vitro</i>.

<p>A) shLacZ, shR1, shR2 and shR3 cells were grown in full growth medium and cell proliferation was measured at 24 h intervals. Columns represent [³H]-thymidine incorporation, and data are expressed as mean ± SD cpm/well (<i>n</i> = 8). The experiment was repeated once, with similar results. B) Effect of the FGFR inhibitor PD173074 on FGF-8b-induced proliferation in sh cells analyzed by [³H]-thymidine incorporation. sh cells were pre-cultured in DC-FBS for 48 h and then treated with FGF-8b (25 ng/ml) and/or PD173074 or PBS vehicle for 48 h. The inhibitor was added 30 minutes before addition of FGF-8b. Data are expressed as mean ± SD cpm/well (<i>n</i> = 8). The statistical differences between groups were analyzed using one-way ANOVA followed by Bonferroni's multiple comparison test. * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001. C) Cyclin D1 and cyclin B protein expression in untreated sh cells. Whole-cell lysates were generated from sh cells grown in full growth medium. Protein was subjected to SDS-PAGE and immunoblotted with antibodies against cyclin D1, cyclin B, 1 and β-actin. The experiment was repeated once, with similar results.</p

Morphology and vascularization of the sh cell tumors.

<p>A) Morphology of shLacZ, shR1, shR2 and shR3 tumors visualized by H-E staining with 100× magnification, scale bar = 100 µm and B) 40× magnification, scale bar = 500 µm. Necrotic areas are indicated by arrows. C) Vascularization in tumors was demonstrated by Pecam-1 immunoreactivity. The upper panel shows representative photomicrographs of immunohistochemical Pecam-1 staining in shLacZ, shR1, shR2 and shR3 tumors, scale bar = 100 µm. The density of Pecam-1-positive capillaries was counted in a blinded manner from 3 fields per tumor, 4–6 tumors per group and is presented as graphs (lower panel). The difference in the number of positive capillaries between the shLacZ tumors and the other tumors was tested by ANOVA followed by Bonferroni's multiple comparison test, * <i>P</i><0.05.</p