Functional characterization of PI3K C2 domain mutations detected in breast cancer circulating tumor cells and metastatic cells.

BACKGROUND: In breast cancer, over one third of all patients harbor a somatic mutation in the PIK3CA gene, encoding the p110α catalytic subunit of the phosphatidylinositol 3-kinase (PI3K) in their tumor cells. Circulating tumor cells (CTCs) are cells shed from the primary tumor into the blood stream. Recently, the long-term stable breast cancer CTC-ITB-01 cell line with tumorigenic and metastatic capacity was established from liquid biopsy derived cells. The oncogenic hotspot PIK3CA mutation H1047R (kinase domain) was detected in the primary tumor, CTCs and metastasis of the same patient. Other PIK3CA mutations located within the C2 domain (E418K and E453K) were detected in the CTCs and the vaginal metastasis but not in the primary tumor. The goal of our study was to functionally characterize the impact of the rare E418K and E453K mutations within the C2 domain that were not detected in the primary tumor. METHODS: PIK3CA mutations E418K, E453K, H1047R were generated by site-directed mutagenesis and stably overexpressed in breast cancer cells by lentiviral transduction. Subsequent signaling pathway activation was examined by western blot analysis. The impact of PIK3CA mutations on biological processes was studied by live cell imaging using the Incucyte Zoom system. Structural modeling was conducted in Pymol. The membrane localization of the mutants was evaluated by separating the cytosolic and membrane fraction using ultracentrifugation. Drug susceptibility of CTC-ITB-01 cells was analyzed by live cell imaging. RESULTS: Western blot analysis of human MDA-MB-231, MCF-7 and T47D breast cancer cells stably overexpressing either the PIK3CA wildtype (WT) or one of the E418K, E453K or H1047R mutants revealed a significant increase in AKT phosphorylation in both C2 mutants (E418K and E453K) and the kinase domain mutant H1047R. Functional analysis showed a significantly increased proliferation of MDA-MB-231 cells overexpressing the E453K and H1047R mutants. Migration was increased in all cells overexpressing WT and each of the mutants. Interestingly, invasion and chemotaxis were only enhanced in the MDA-MB-231 cells overexpressing the C2 domain mutants, i.e. E418K and E453K. In addition, membrane localization of the two C2 domain mutants was increased. Structural modeling of the E453K mutation suggests a disruption of the interaction between the negative regulatory domain of the p85α subunit and the p110α catalytic subunit as a potential mechanism leading to the observed activation of PI3K/AKT/mTOR signaling. Dual targeting of AKT/mTOR pathway by MK2206 and RAD001 leads to very strong synergistic effects (IC50 MK2206: 148 nM, IC50 RAD001: 15 nM) with respect to proliferation in the CTC-ITB-01 line through apoptosis induction. CONCLUSIONS: Our results demonstrate that PIK3CA C2 domain mutations activate PI3K downstream AKT signaling and can increase proliferation, migration and invasion after stable lentiviral transduction. Although both investigated mutations - E418K and E453K - are located within the C2 domain, a different molecular mechanism can be proposed. The PIK3CA mutated CTC-ITB-01 shows a high susceptibility against dual inhibition of AKT/mTOR. Further studies are required to fully elucidate the oncogenic potential of rare PIK3CA mutations.

Comparison of ex vivo bioluminescence imaging, Alu-qPCR and histology for the quantification of spontaneous lung and bone metastases in subcutaneous xenograft mouse models.

Bioluminescence imaging (BLI) is a non-invasive state-of-the-art-method for longitudinal tracking of tumor cells in mice. The technique is commonly used to determine bone metastatic burden in vivo and also suitable ex vivo to detect even smallest bone micro-metastases in spontaneous metastasis xenograft models. However, it is unclear to which extent ex vivo BLI correlates with alternative methods for metastasis quantification. Here, we compared ex vivo BLI, human DNA-based Alu-qPCR, and histology for the quantification of bone vs. lung metastases, which are amongst the most common sites of metastasis in prostate cancer (PCa) patients and spontaneous PCa xenograft models. Data from 93 immunodeficient mice were considered, each of which were subcutaneously injected with luciferase/RGB-labeled human PCa PC-3 cells. The primary tumors were resected at ~ 0.75 cm³ and mice were sacrificed ~ 3 weeks after surgery and immediately examined by ex vivo BLI. Afterwards, the right lungs and hind limbs with the higher BLI signal (BLIHi bone) were processed for histology, whereas the left lung lobes and hind limbs with the lower BLI signal (BLILo bone) were prepared for Alu-qPCR. Our data demonstrate remarkable differences in the correlation coefficients of the different methods for lung metastasis detection (r ~ 0.8) vs. bone metastasis detection (r ~ 0.4). However, the BLI values of the BLIHi and BLILo bones correlated very strongly (r ~ 0.9), indicating that the method per se was reliable under identical limitations; the overall level of metastasis to contralateral bones was astonishingly similar. Instead, the level of lung metastasis only weakly to moderately correlated with the level of bone metastasis formation. Summarized, we observed a considerable discrepancy between ex vivo BLI and histology/Alu-qPCR in the quantification of bone metastases, which was not observed in the case of lung metastases. Future studies using ex vivo BLI for bone metastasis quantification should combine multiple methods to accurately determine metastatic load in bone samples.

ALDH1A1 drives prostate cancer metastases and radioresistance by interplay with AR- and RAR-dependent transcription.

Rationale: Current therapies for metastatic osseous disease frequently fail to provide a durable treatment response. To date, there are only limited therapeutic options for metastatic prostate cancer, the mechanisms that drive the survival of metastasis-initiating cells are poorly characterized, and reliable prognostic markers are missing. A high aldehyde dehydrogenase (ALDH) activity has been long considered a marker of cancer stem cells (CSC). Our study characterized a differential role of ALDH1A1 and ALDH1A3 genes as regulators of prostate cancer progression and metastatic growth. Methods: By genetic silencing of ALDH1A1 and ALDH1A3 in vitro, in xenografted zebrafish and murine models, and by comparative immunohistochemical analyses of benign, primary tumor, and metastatic specimens from patients with prostate cancer, we demonstrated that ALDH1A1 and ALDH1A3 maintain the CSC phenotype and radioresistance and regulate bone metastasis-initiating cells. We have validated ALDH1A1 and ALDH1A3 as potential biomarkers of clinical outcomes in the independent cohorts of patients with PCa. Furthermore, by RNAseq, chromatin immunoprecipitation (ChIP), and biostatistics analyses, we suggested the molecular mechanisms explaining the role of ALDH1A1 in PCa progression. Results: We found that aldehyde dehydrogenase protein ALDH1A1 positively regulates tumor cell survival in circulation, extravasation, and metastatic dissemination, whereas ALDH1A3 plays the opposite role. ALDH1A1 and ALDH1A3 are differentially expressed in metastatic tumors of patients with prostate cancer, and their expression levels oppositely correlate with clinical outcomes. Prostate cancer progression is associated with the increasing interplay of ALDH1A1 with androgen receptor (AR) and retinoid receptor (RAR) transcriptional programs. Polo-like kinase 3 (PLK3) was identified as a transcriptional target oppositely regulated by ALDH1A1 and ALDH1A3 genes in RAR and AR-dependent manner. PLK3 contributes to the control of prostate cancer cell proliferation, migration, DNA repair, and radioresistance. ALDH1A1 gain in prostate cancer bone metastases is associated with high PLK3 expression. Conclusion: This report provides the first evidence that ALDH1A1 and PLK3 could serve as biomarkers to predict metastatic dissemination and radiotherapy resistance in patients with prostate cancer and could be potential therapeutic targets to eliminate metastasis-initiating and radioresistant tumor cell populations.

Spontaneous metastasis xenograft models link CD44 isoform 4 to angiogenesis, hypoxia, EMT and mitochondria-related pathways in colorectal cancer.

Hematogenous metastasis limits the survival of colorectal cancer (CRC) patients. Here, we illuminated the roles of CD44 isoforms in this process. Isoforms 3 and 4 were predominantly expressed in CRC patients. CD44 isoform 4 indicated poor outcome and correlated with epithelial-mesenchymal transition (EMT) and decreased oxidative phosphorylation (OxPhos) in patients; opposite associations were found for isoform 3. Pan-CD44 knockdown (kd) independently impaired primary tumor formation and abrogated distant metastasis in CRC xenografts. The xenograft tumors mainly expressed the clinically relevant CD44 isoforms 3 and 4. Both isoforms were enhanced in the paranecrotic, hypoxic tumor regions but were generally absent in lung metastases. Upon CD44 kd, tumor angiogenesis was increased in the paranecrotic areas, accompanied by reduced hypoxia-inducible factor-1α and CEACAM5 but increased E-cadherin expression. Mitochondrial genes and proteins were induced upon pan-CD44 kd, as were OxPhos genes. Hypoxia increased VEGF release from tumor spheres, particularly upon CD44 kd. Genes affected upon CD44 kd in xenografts specifically overlapped concordantly with genes correlating with CD44 isoform 4 (but not isoform 3) in patients, validating the clinical relevance of the used model and highlighting the metastasis-promoting role of CD44 isoform 4.

Identification of potential classes of glycoligands mediating dynamic endothelial adhesion of human tumor cells.

One critical step of metastasis formation is the extravasation of circulating tumor cells from the bloodstream. This process requires the dynamic interaction of cell adhesion molecules like E-selectin on endothelial cells with carbohydrate ligands on tumor cells. To characterize these glycans in a comprehensible approach, the rolling, tethering, and firm adhesion of nine human tumor cell lines on human umbilical vein endothelial cells was analyzed using laminar flow adhesion assays. The tumor cell lines were grouped into three subsets by their canonical E-selectin ligand status (sialyl-Lewis A and X +/+, -/+, -/-) and their adhesiveness was compared after enzymatic, pharmacologic, chemical treatment or antibody blockade of the tumor cells or endothelial cells, respectively. Tumor cells were also screened regarding their glycosyltransferase expression profile. We found that although E-selectin and terminal α2,3-sialic acid largely determined firm adhesion, adhesive events did not exclusively depend on the presence of sialyl-Lewis A and/or sialyl-Lewis X. Nevertheless, two of the three sialyl-Lewis A/X-/- tumor cells additionally or fully depended on vascular cell adhesion molecule-1 for firm adhesion. The significance of O-GalNAc- and N-glycans for adhesion varied remarkably among the tumor cells. The sialyl-Lewis A/X+/+ subset showed glycoprotein-independent adhesion, suggesting a role of glycolipids as well. All sialyl-Lewis A/X-/- tumor cells lacked FUT3 and FUT7 expression as opposed to sialyl-Lewis A/X+/+ or -/+ cell lines. In summary, the glycans on tumor cells mediating endothelial adhesion are not as much restricted to sialyl-Lewis A /X as previously assumed. The present study specifically suggests α2,3-linked sialic acid, O-GalNAc glycans, glycosphingolipids, and FUT3/FUT7 products as promising targets for future studies.

Efficacy of zoledronic acid for the elimination of disseminated tumor cells in a clinically relevant, spontaneously metastatic prostate cancer xenograft model.

Bone metastases develop in >90 % of patients with castration-resistant prostate cancer (PCa) through complex interactions between the bone microenvironment and tumor cells. Previous androgen-deprivation therapy (ADT), which is known to cause bone loss, as well as anti-resorptive agents such as zoledronic acid (ZA), used to prevent skeletal complications, may influence these interactions and thereby the growth of disseminated tumor cells (DTC) in the bone marrow (BM). Here, a spontaneously metastatic xenograft tumor model of human PCa was further optimized to mimic the common clinical situation of ADT (castration) combined with primary tumor resection in vivo. The effects of these interventions, alone or in combination with ZA treatment, on tumor cell dissemination to the BM and other distant sites were analyzed. Metastatic burden was quantified by human-specific Alu-qPCR, bioluminescence imaging (BLI), and immunohistochemistry. Further, bone remodeling was assessed by static histomorphometry and serum parameters. Initial comparative analysis between NSG and SCID mice showed that spontaneous systemic dissemination of subcutaneous PC-3 xenograft tumors was considerably enhanced in NSG mice. Primary tumor resection and thereby prolonged observational periods resulted in a higher overall metastatic cell load at necropsy and tumor growth alone caused significant bone loss, which was further augmented by surgical castration. In addition, castrated mice showed a strong trend towards higher bone metastasis loads. Weekly treatment of mice with ZA completely prevented castration- and tumor-induced bone loss but had no effect on bone metastasis burden. Conversely, the total lung metastasis load as determined by BLI was significantly decreased upon ZA treatment. These findings provide a basis for future research on the role of ZA not only in preventing skeletal complications but also in reducing metastasis to other organs.

MicroRNAs: Emerging Regulators of Metastatic Bone Disease in Breast Cancer.

Bone metastasis is a frequent complication in patients with advanced breast cancer. Once in the bone, cancer cells disrupt the tightly regulated cellular balance within the bone microenvironment, leading to excessive bone destruction and further tumor growth. Physiological and pathological interactions in the bone marrow are mediated by cell-cell contacts and secreted molecules that include soluble proteins as well as RNA molecules. MicroRNAs (miRNAs) are short non-coding RNAs that post-transcriptionally interfere with their target messenger RNA (mRNA) and subsequently reduce protein abundance. Since their discovery, miRNAs have been identified as critical regulators of physiological and pathological processes, including breast cancer and associated metastatic bone disease. Depending on their targets, miRNAs can exhibit pro-tumorigenic or anti-tumorigenic functions and serve as diagnostic and prognostic biomarkers. These properties have encouraged pre-clinical and clinical development programs to investigate miRNAs as biomarkers and therapeutic targets in various diseases, including metastatic cancers. In this review, we discuss the role of miRNAs in metastatic bone disease with a focus on breast cancer and the bone microenvironment and elaborate on their potential use for diagnostic and therapeutic purposes in metastatic bone disease and beyond.

Interleukins as Mediators of the Tumor Cell-Bone Cell Crosstalk during the Initiation of Breast Cancer Bone Metastasis.

Patients with advanced breast cancer are at high risk of developing bone metastasis. Despite treatment advances for primary breast cancer, metastatic bone disease remains incurable with a low relative survival. Hence, new therapeutic approaches are required to improve survival and treatment outcome for these patients. Bone is among the most frequent sites of metastasis in breast cancer. Once in the bone, disseminated tumor cells can acquire a dormant state and remain quiescent until they resume growth, resulting in overt metastasis. At this stage the disease is characterized by excessive, osteoclast-mediated osteolysis. Cells of the bone microenvironment including osteoclasts, osteoblasts and endothelial cells contribute to the initiation and progression of breast cancer bone metastasis. Direct cell-to-cell contact as well as soluble factors regulate the crosstalk between disseminated breast cancer cells and bone cells. In this complex signaling network interleukins (ILs) have been identified as key regulators since both, cancer cells and bone cells secrete ILs and express corresponding receptors. ILs regulate differentiation and function of bone cells, with several ILs being reported to act pro-osteoclastogenic. Consistently, the expression level of ILs (e.g., in serum) has been associated with poor prognosis in breast cancer. In this review we discuss the role of the most extensively investigated ILs during the establishment of breast cancer bone metastasis and highlight their potential as therapeutic targets in preventing metastatic outgrowth in bone.

High Sensitivity of Circulating Tumor Cells Derived from a Colorectal Cancer Patient for Dual Inhibition with AKT and mTOR Inhibitors.

Circulating tumor cells (CTCs) are cells shed from the primary tumor into the bloodstream. While many studies on solid tumor cells exist, data on CTCs are scarce. The mortality of cancer is mostly associated with metastasis and recent research identified CTCs as initiators of metastasis. The PI3K/AKT/mTOR signaling pathway is an intracellular pathway that regulates essential functions including protein biosynthesis, cell growth, cell cycle control, survival and migration. Importantly, activating oncogenic mutations and amplifications in this pathway are frequently observed in a wide variety of cancer entities, underlining the significance of this signaling pathway. In this study, we analyzed the functional role of the PI3K/AKT/mTOR signaling pathway in the CTC-MCC-41 line, derived from a patient with metastatic colorectal cancer. One striking finding in our study was the strong sensitivity of this CTC line against AKT inhibition using MK2206 and mTOR inhibition using RAD001 within the nanomolar range. This suggests that therapies targeting AKT and mTOR could have been beneficial for the patient from which the CTC line was isolated. Additionally, a dual targeting approach of AKT/mTOR inside the PI3K/AKT/mTOR signaling pathway in the colorectal CTCs showed synergistic effects in vitro. Depending on the phenotypical behavior of CTC-MCC-41 in cell culture (adherent vs. suspension), we identified altered phosphorylation levels inside the PI3K/AKT/mTOR pathway. We observed a downregulation of the PI3K/AKT/mTOR signaling pathway, but not of the RAS/RAF/MAPK pathway, in CTCs growing in suspension in comparison to adherent CTCs. Our results highlight distinct functions of AKT isoforms in CTC-MCC-41 cells with respect to cell proliferation. Knockdown of AKT1 and AKT2 leads to significantly impaired proliferation of CTC-MCC-41 cells in vitro. Therefore, our data demonstrate that the PI3K/AKT/mTOR signaling pathway plays a key role in the proliferation of CTC-MCC-41.

The Endosteal Niche in Breast Cancer Bone Metastasis.

The establishment of bone metastasis remains one of the most frequent complications of patients suffering from advanced breast cancer. Patients with bone metastases experience high morbidity and mortality caused by excessive, tumor-induced and osteoclast-mediated bone resorption. Anti-resorptive treatments, such as bisphosphonates, are available to ease skeletal related events including pain, increased fracture risk, and hypercalcemia. However, the disease remains incurable and 5-year survival rates for these patients are below 25%. Within the bone, disseminated breast cancer cells localize in "metastatic niches," special microenvironments that are thought to regulate cancer cell colonization and dormancy as well as tumor progression and subsequent development into overt metastases. Precise location and composition of this "metastatic niche" remain poorly defined. However, it is thought to include an "endosteal niche" that is composed of key bone cells that are derived from both, hematopoietic stem cells (osteoclasts), and mesenchymal stromal cells (osteoblasts, fibroblasts, adipocytes). Our knowledge of how osteoclasts drive the late stage of the disease is well-established. In contrast, much less is known about the interaction between osteogenic cells and disseminated tumor cells prior to the initiation of the osteolytic phase. Recent studies suggest that mesenchymal-derived cells, including osteoblasts and fibroblasts, play a key role during the early stages of breast cancer bone metastasis such as tumor cell homing, bone marrow colonization, and tumor cell dormancy. Hence, elucidating the interactions between breast cancer cells and mesenchymal-derived cells that drive metastasis progression could provide novel therapeutic approaches and targets to treat breast cancer bone metastasis. In this review we discuss evidences reporting the interaction between tumor cells and endosteal niche cells during the early stages of breast cancer bone metastasis, with a particular focus on mesenchymal-derived osteoblasts and fibroblasts.

Breast cancer bone metastases are attenuated in a Tgif1-deficient bone microenvironment.

BACKGROUND: Osteoclast activation is a hallmark of breast cancer-induced bone disease while little is known about the role of osteoblasts in this process. Recently, we identified the homeodomain protein TG-interacting factor-1 (Tgif1) as a crucial regulator of osteoblast function. In this study, we demonstrate that lack of Tgif1 also restricts the progression of breast cancer bone metastases. METHODS: Transwell migration assays were used to investigate the osteoblast-breast cancer cell interaction in vitro. Molecular analyses included RNA sequencing, immunoblotting, and qRT-PCR. To determine the role of Tgif1 in metastatic bone disease, 4T1 breast cancer cells were injected intracardially into mice with a germ line deletion of Tgif1 (Tgif1-/-) or control littermates (Tgif1+/+). Progression of bone metastases and alterations in the bone microenvironment were assessed using bioluminescence imaging, immunofluorescence staining, confocal microscopy, and histomorphometry. RESULTS: Medium conditioned by osteoblasts stimulated breast cancer cell migration, indicating a potential role of osteoblasts during bone metastasis progression. Tgif1 expression was strongly increased in osteoblasts upon stimulation by breast cancer cells, demonstrating the implication of Tgif1 in the osteoblast-breast cancer cell interaction. Indeed, conditioned medium from osteoblasts of Tgif1-/- mice failed to induce breast cancer cell migration compared to control, suggesting that Tgif1 in osteoblasts augments cancer cell motility. Semaphorin 3E (Sema3E), which is abundantly secreted by Tgif1-/- osteoblasts, dose-dependently reduced breast cancer cell migration while silencing of Sema3E expression in Tgif1-/- osteoblasts partially restored the impaired migration. In vivo, we observed a decreased number of breast cancer bone metastases in Tgif1-/- mice compared to control littermates. Consistently, the presence of single breast cancer cells or micro-metastases in the tibiae was reduced in Tgif1-/- mice. Breast cancer cells localized in close proximity to Endomucin-positive vascular cells as well as to osteoblasts. Although Tgif1 deficiency did not affect the bone marrow vasculature, the number and activity of osteoblasts were reduced compared to control. This suggests that the protective effect on bone metastases might be mediated by osteoblasts rather than by the bone marrow vasculature. CONCLUSION: We propose that the lack of Tgif1 in osteoblasts increases Sema3E expression and attenuates breast cancer cell migration as well as metastases formation.

Pathological Crosstalk between Metastatic Breast Cancer Cells and the Bone Microenvironment.

Bone is the most common metastatic site in breast cancer. Upon arrival to the bone, disseminated tumor cells can undergo a period of dormancy but often eventually grow and hijack the bone microenvironment. The bone marrow microenvironment consists of multiple cell types including the bone cells, adipocytes, endothelial cells, and nerve cells that all have crucial functions in the maintenance of bone homeostasis. Tumor cells severely disturb the tightly controlled cellular and molecular interactions in the bone marrow fueling their own survival and growth. While the role of bone resorbing osteoclasts in breast cancer bone metastases is well established, the function of other bone cells, as well as adipocytes, endothelial cells, and nerve cells is less understood. In this review, we discuss the composition of the physiological bone microenvironment and how the presence of tumor cells influences the microenvironment, creating a pathological crosstalk between the cells. A better understanding of the cellular and molecular events that occur in the metastatic bone microenvironment could facilitate the identification of novel cellular targets to treat this devastating disease.

Modulating Bone Marrow Hematopoietic Lineage Potential to Prevent Bone Metastasis in Breast Cancer.

The presence of disseminated tumor cells in breast cancer patient bone marrow aspirates predicts decreased recurrence-free survival. Although it is appreciated that physiologic, pathologic, and therapeutic conditions impact hematopoiesis, it remains unclear whether targeting hematopoiesis presents opportunities for limiting bone metastasis. Using preclinical breast cancer models, we discovered that marrow from mice treated with the bisphosphonate zoledronic acid (ZA) are metastasis-suppressive. Specifically, ZA modulated hematopoietic myeloid/osteoclast progenitor cell (M/OCP) lineage potential to activate metastasis-suppressive activity. Granulocyte-colony stimulating factor (G-CSF) promoted ZA resistance by redirecting M/OCP differentiation. We identified M/OCP and bone marrow transcriptional programs associated with metastasis suppression and ZA resistance. Analysis of patient blood samples taken at randomization revealed that women with high-plasma G-CSF experienced significantly worse outcome with adjuvant ZA than those with lower G-CSF levels. Our findings support discovery of therapeutic strategies to direct M/OCP lineage potential and biomarkers that stratify responses in patients at risk of recurrence.Significance: Bone marrow myeloid/osteoclast progenitor cell lineage potential has a profound impact on breast cancer bone metastasis and can be modulated by G-CSF and bone-targeting agents. Cancer Res; 78(18); 5300-14. ©2018 AACR.

Targeting the Metastatic Bone Microenvironment by MicroRNAs.

Bone metastases are a common and devastating feature of late-stage breast cancer. Metastatic bone disease is a consequence of disturbed bone remodeling due to pathological interactions between cancer cells and the bone microenvironment (BME). In the BME, breast cancer cells severely alter the balanced bone formation and bone resorption driven by osteoblasts and osteoclasts. The complex cellular cross talk in the BME is governed by secreted molecules, signaling pathways and epigenetic cues including non-coding RNAs. MicroRNAs (miRNAs) are small non-coding RNAs that reduce protein abundance and regulate several biological processes, including bone remodeling. Under pathological conditions, abnormal miRNA signaling contributes to the progression of diseases, such as bone metastasis. Recently miRNAs have been demonstrated to regulate several key drivers of bone metastasis. Furthermore, miRNAs are implicated as important regulators of cellular interactions within the metastatic BME. As a consequence, targeting the BME by miRNA delivery or antagonism has been reported to limit disease progression in experimental and preclinical conditions positioning miRNAs as emerging novel therapeutic tools in metastatic bone disease. This review will summarize our current understanding on the composition and function of the metastatic BME and discuss the recent advances how miRNAs can modulate pathological interactions in the bone environment.

Zoledronic acid alters hematopoiesis and generates breast tumor-suppressive bone marrow cells.

BACKGROUND: The bone-targeting agent zoledronic acid (ZOL) increases breast cancer survival in subsets of patients, but the underlying reasons for this protective effect are unknown. ZOL modulates the activity of osteoclasts and osteoblasts, which form hematopoietic stem cell niches, and therefore may affect hematopoietic cells that play a role in breast cancer progression. METHOD: Immunocompetent and immunocompromised strains of mice commonly used for breast cancer research were injected with a single, clinically relevant dose of ZOL (100 µg/kg) or vehicle control. The effects of ZOL on the bone marrow microenvironment (bone volume, bone cell number/activity, extracellular matrix composition) were established at various time points following treatment, using micro-computed tomography (µCT) analysis, histomorphometry, ELISA and immunofluorescence. The effects on peripheral blood and bone marrow hematopoietic progenitor populations were assessed using a HEMAVET® hematology analyzer and multicolor flow cytometry, respectively. Tumor support function of bone marrow cells was determined using an in vivo functional assay developed in our laboratory. RESULTS: Using multiple mouse strains, we observed transient changes in numbers of hematopoietic stem cells, myeloid-biased progenitor cells, and lymphoid-biased cells concurrent with changes to hematopoietic stem cell niches following ZOL administration. Importantly, bone marrow cells from mice treated with a single, clinically relevant dose of ZOL inhibited breast tumor outgrowth in vivo. The ZOL-induced tumor suppressive function of the bone marrow persisted beyond the time point at which numbers of hematopoietic progenitor cells had returned to baseline. CONCLUSIONS: These findings provide novel evidence that alterations to the bone marrow play a role in the anti-tumor activity of ZOL and suggest possibilities for capitalizing on the beneficial effects of ZOL in reducing breast cancer development and progression.

Rapid modification of the bone microenvironment following short-term treatment with Cabozantinib in vivo.

INTRODUCTION: Bone metastasis remains incurable with treatment restricted to palliative care. Cabozantinib (CBZ) is targeted against multiple receptor tyrosine kinases involved in tumour pathobiology, including hepatocyte growth factor receptor (MET) and vascular endothelial growth factor receptor 2 (VEGFR-2). CBZ has demonstrated clinical activity in advanced prostate cancer with resolution of lesions visible on bone scans, implicating a potential role of the bone microenvironment as a mediator of CBZ effects. We characterised the effects of short-term administration of CBZ on bone in a range of in vivo models to determine how CBZ affects bone in the absence of tumour. METHODS: Studies were performed in a variety of in vivo models including male and female BALB/c nude mice (age 6-17-weeks). Animals received CBZ (30 mg/kg, 5× weekly) or sterile H2O control for 5 or 10 days. Effects on bone integrity (µCT), bone cell activity (PINP, TRAP ELISA), osteoblast and osteoclast number/mm trabecular bone surface, area of epiphyseal growth plate cartilage, megakaryocyte numbers and bone marrow composition were assessed. Effects of longer-term treatment (15-day & 6-week administration) were assessed in male NOD/SCID and beige SCID mice. RESULTS: CBZ treatment had significant effects on the bone microenvironment, including reduced osteoclast and increased osteoblast numbers compared to control. Trabecular bone structure was altered after 8 administrations. A significant elongation of the epiphyseal growth plate, in particular the hypertrophic chondrocyte zone, was observed in all CBZ treated animals irrespective of administration schedule. Both male and female BALB/c nude mice had increased megakaryocyte numbers/mm(2) tissue after 10-day CBZ treatment, in addition to vascular ectasia, reduced bone marrow cellularity and extravasation of red blood cells into the extra-vascular bone marrow. All CBZ-induced effects were transient and rapidly lost following cessation of treatment. CONCLUSION: Short-term administration of CBZ induces rapid, reversible effects on the bone microenvironment in vivo highlighting a potential role in mediating treatment responses.

Modifying the osteoblastic niche with zoledronic acid in vivo-potential implications for breast cancer bone metastasis.

INTRODUCTION: Bone metastasis is the most common complication of advanced breast cancer. The associated cancer-induced bone disease is treated with bone-sparing agents like zoledronic acid. Clinical trials have shown that zoledronic acid also reduces breast cancer recurrence in bone; potentially by modifying the bone microenvironment surrounding disseminated tumour cells. We have characterised the early effects of zoledronic acid on key cell types of the metastatic niche in vivo, and investigated how these modify the location of breast tumour cells homing to bone. METHODS: Female mice were treated with a single, clinically achievable dose of zoledronic acid (100µg/kg) or PBS. Bone integrity, osteoclast and osteoblast activity and number/mm trabecular bone on 1, 3, 5 and 10days after treatment were assessed using µCT, ELISA (TRAP, PINP) and bone histomorphometry, respectively. The effect of zoledronic acid on osteoblasts was validated in genetically engineered mice with GFP-positive osteoblastic cells. The effects on growth plate cartilage were visualised by toluidine blue staining. For tumour studies, mice were injected i.c. with DID-labelled MDA-MB-231-NW1-luc2 breast cancer cells 5days after zoledronic acid treatment, followed by assessment of tumour cell homing to bone and soft tissues by multiphoton microscopy, flow cytometry and ex vivo cultures. RESULTS: As early as 3days after treatment, animals receiving zoledronic acid had significantly increased trabecular bone volume vs. control. This rapid bone effect was reflected in a significant reduction in osteoclast and osteoblast number/mm trabecular bone and reduced bone marker serum levels (day 3-5). These results were confirmed in mice expressing GFP in osteoblastic linage cells. Pre-treatment with zoledronic acid caused accumulation of an extra-cellular matrix in the growth plate associated with a trend towards preferential [1] homing of tumour cells to osteoblast-rich areas of bone, but without affecting the total number of tumour cells. The number of circulating tumour cells was reduced in ZOL treated animals. CONCLUSION: A single dose of zoledronic acid caused significant changes in the bone area suggested to contain the metastatic niche. Tumour cells arriving in this modified bone microenvironment appeared to preferentially locate to osteoblast-rich areas, supporting that osteoblasts may be key components of the bone metastasis niche and therefore a potential therapeutic target in breast cancer.