Development of clinically relevant in vivo metastasis models using human bone discs and breast cancer patient-derived xenografts.

BACKGROUND: Late-stage breast cancer preferentially metastasises to bone; despite advances in targeted therapies, this condition remains incurable. The lack of clinically relevant models for studying breast cancer metastasis to a human bone microenvironment has stunted the development of effective treatments for this condition. To address this problem, we have developed humanised mouse models in which breast cancer patient-derived xenografts (PDXs) metastasise to human bone implants with low variability and high frequency. METHODS: To model the human bone environment, bone discs from femoral heads of patients undergoing hip replacement surgery were implanted subcutaneously into NOD/SCID mice. For metastasis studies, 7 patient-derived xenograft tumours (PDX: BB3RC32, ER+ PR+ HER2-; BB2RC08, ER+ PR+ ER2-; BB6RC37, ER- PR- HER2- and BB6RC39, ER+ PR+ HER2+), MDA-MB-231-luc2, T47D-luc2 or MCF7-Luc2 cells were injected into the 4th mammary ducts and metastases monitored by luciferase imaging and confirmed on histological sections. Bone integrity, viability and vascularisation were assessed by uCT, calcein uptake and histomorphometry. Expression profiling of genes/proteins during different stages of metastasis were assessed by whole genome Affymetrix array, real-time PCR and immunohistochemistry. Importance of IL-1 was confirmed following anakinra treatment. RESULTS: Implantation of femoral bone provided a metabolically active, human-specific site for tumour cells to metastasise to. After 4 weeks, bone implants were re-vascularised and demonstrated active bone remodelling (as evidenced by the presence of osteoclasts, osteoblasts and calcein uptake). Restricting bone implants to the use of subchondral bone and introduction of cancer cells via intraductal injection maximised metastasis to human bone implants. MDA-MB-231 cells specifically metastasised to human bone (70% metastases) whereas T47D, MCF7, BB3RC32, BB2RC08, and BB6RC37 cells metastasised to both human bone and mouse bones. Importantly, human bone was the preferred metastatic site especially from ER+ PDX (100% metastasis human bone compared with 20-75% to mouse bone), whereas ER-ve PDX developed metastases in 20% of human and 20% of mouse bone. Breast cancer cells underwent a series of molecular changes as they progressed from primary tumours to bone metastasis including altered expression of IL-1B, IL-1R1, S100A4, CTSK, SPP1 and RANK. Inhibiting IL-1B signalling significantly reduced bone metastasis. CONCLUSIONS: Our reliable and clinically relevant humanised mouse models provide significant advancements in modelling of breast cancer bone metastasis.

Patient-derived Mammosphere and Xenograft Tumour Initiation Correlates with Progression to Metastasis.

Breast cancer specific mortality results from tumour cell dissemination and metastatic colonisation. Identification of the cells and processes responsible for metastasis will enable better prevention and control of metastatic disease, thus reducing relapse and mortality. To better understand these processes, we prospectively collected 307 patient-derived breast cancer samples (n = 195 early breast cancers (EBC) and n = 112 metastatic samples (MBC)). We assessed colony-forming activity in vitro by growing isolated cells in both primary (formation) and secondary (self-renewal) mammosphere culture, and tumour initiating activity in vivo through subcutaneous transplantation of fragments or cells into mice. Metastatic samples formed primary mammosphere colonies significantly more frequently than early breast cancers and had significantly higher primary mammosphere colony formation efficiency (0.9 % vs. 0.6 %; p < 0.0001). Tumour initiation in vivo was significantly higher in metastatic than early breast cancer samples (63 % vs. 38 %, p = 0.04). Of 144 breast cancer samples implanted in vivo, we established 20 stable patient-derived xenograft (PDX) models at passage 2 or greater. Lung metastases were detected in mice from 14 PDX models. Mammosphere colony formation in vitro significantly correlated with the ability of a tumour to metastasise to the lungs in vivo (p = 0.05), but not with subcutaneous tumour initiation. In summary, the breast cancer stem cell activities of colony formation and tumour initiation are increased in metastatic compared to early samples, and predict metastasis in vivo. These results suggest that breast stem cell activity will predict for poor outcome tumours, and therapy targeting this activity will improve outcomes for patients with metastatic disease.

Reversing paclitaxel resistance in ovarian cancer cells via inhibition of the ABCB1 expressing side population.

The majority of deaths in ovarian cancer are caused by recurrent metastatic disease which is usually multidrug resistant. This progression has been hypothesised to be due in part to the presence of cancer stem cells, a subset of cells which are capable of self-renewal and are able to survive chemotherapy and migrate to distant sites. Side population (SP) cells, identified by the efflux of the DNA-binding dye Hoechst 33342 through ATP-binding cassette (ABC) transporters, are a known adult stem cell group and have been suggested as a cancer stem cell in various cancers. Despite the identification of SP cells in cancer cell lines and patient samples, little attention has been paid to the identification of specific ABC transporters within this cell fraction which efflux Hoechst dye and thus may facilitate drug resistance. In this study, we demonstrate that SP cells can be detected in both ovarian cancer cell lines and ascitic fluid samples, and these SP cells possess stem cell and drug resistance properties. We show that ABCB1 is the functioning ABC transporter in ovarian cancer cell lines, and expression of ABCB1 is associated with a paclitaxel-resistant phenotype. Moreover, silencing of ABCB1 using a specific morpholino oligonucleotide results in an inhibition of the SP phenotype and a sensitising of ovarian cancer cell lines to paclitaxel. ABCB1 should therefore be considered as a therapeutic target in ovarian cancer.

Reactive arthritis incidence in a community cohort following a large waterborne campylobacteriosis outbreak in Havelock North, New Zealand.

OBJECTIVES: In August 2016, Campylobacter spp contaminated an untreated reticulated water supply resulting in a large-scale gastroenteritis outbreak affecting an estimated 8320 people. We aimed to determine the incidence of probable reactive arthritis (ReA) cases in individuals with culture-confirmed campylobacteriosis (CC), self-reported probable campylobacteriosis (PC) and those reporting no diarrhoea (ND). DESIGN: We conducted a retrospective cohort study to identify incidence of probable ReA cases. We identified cases with new ReA symptoms using an adapted acute ReA (AReA) telephone questionnaire. Those reporting ≥1 symptom underwent a telephone interview with the study rheumatologist. Probable ReA was defined as spontaneous onset of pain suggestive of inflammatory arthritis in ≥1 previously asymptomatic joint for ≥3 days occurring ≤12 weeks after outbreak onset. SETTING: Population-based epidemiological study in Havelock North, New Zealand. PARTICIPANTS: We enrolled notified CC cases with gastroenteritis symptom onsets 5 August 2016-6 September 2016 and conducted a telephone survey of households supplied by the contaminated water source to enrol PC and ND cases. RESULTS: One hundred and six (47.3%) CC, 47 (32.6%) PC and 113 (34.3%) ND cases completed the AReA telephone questionnaire. Of those reporting ≥1 new ReA symptom, 45 (75.0%) CC, 13 (68.4%) PC and 14 (82.4%) ND cases completed the rheumatologist telephone interview. Nineteen CC, 4 PC and 2 ND cases developed probable ReA, resulting in minimum incidences of 8.5%, 2.8% and 0.6% and maximum incidences of 23.9%, 12.4% and 2.15%. DISCUSSION: We describe high probable ReA incidences among gastroenteritis case types during a very large Campylobacter gastroenteritis outbreak using a resource-efficient method that is feasible to employ in future outbreaks.

The epidemiology of bone cancer in 0 - 39 year olds in northern England, 1981 - 2002.

BACKGROUND: There is a paucity of recent epidemiological data on bone cancers. The aim of this study was to describe incidence and survival patterns for bone cancers diagnosed during 1981 - 2002. METHODS: Cases aged 0 - 39 years (236 osteosarcomas, 166 Ewing sarcomas and 73 chondrosarcomas) were analysed using Poisson and Cox regressions. RESULTS: Incidence rates (per million persons per year) for osteosarcoma were 2.5 at age 0 - 14 years; 4.5 at age 15 - 29 years and 1.0 at age 30 - 39 years. Similarly, for Ewing sarcoma the incidence rates were 2.2; 2.9; 0.4 and for chondrosarcoma rates were 0.1; 1.2; 1.8 respectively. Incidence of osteosarcoma increased at an average annual rate of 2.5% (95% CI 0.4 - 4.7; P = 0.02), but there was no change in incidence of Ewing sarcoma or chondrosarcoma. There was a marginally statistically significant improvement in survival for Ewing sarcoma (hazard ratio (HR) per annum = 0.97; 95% CI 0.94 - 1.00; P = 0.06), although patients aged 15 - 39 years (n = 93) had worse overall survival than those aged 0 - 14 (n = 73; HR = 1.46; 95% CI 0.98 - 2.17; P = 0.06). There was no significant improvement in osteosarcoma survival (HR per annum = 0.98; 95% CI 0.95 - 1.01; P = 0.18). CONCLUSIONS: Reasons for poorer survival in Ewing sarcoma patients aged 15 - 39 years and failure to significantly improve survival for osteosarcoma patients requires further investigation.

White matter microstructural abnormalities in euthymic bipolar disorder.

BACKGROUND: Abnormal diffusion parameters are reported in specific brain regions and white matter tracts in bipolar disorder. AIMS: To investigate whether these abnormalities are generalised, and thus evident in large regions of white matter. METHOD: Diffusion parameters were measured at several regions in the corpus callosum and in deep/periventricular white matter in 28 currently euthymic patients with bipolar disorder and controls. White matter hyperintensity loads were assessed. RESULTS: Comparing the whole data-sets using the sign test, in the group with bipolar disorder, mean diffusivity was greater at all 15 sites (P<0.001) and fractional anisotropy was reduced at 13 (P<0.01). The effect of diagnosis was significant for callosal mean diffusivity and fractional anisotropy and for deep/periventricular mean diffusivity (MANCOVA). Comparing individual regions (Mann-Whitney U-test), prefrontal and periventricular mean diffusivity were significantly increased; callosal and occipital fractional anisotropy were significantly reduced. Former substance use and lithium were possible confounding factors. Periventricular white matter hyperintensities were associated with significantly increased periventricular mean diffusivity in individuals with bipolar disorder. CONCLUSIONS: Generalised white matter microstructural abnormalities may exist in bipolar disorder, possibly exacerbated by past substance use and ameliorated by lithium.

Increased Expression of Interleukin-1 Receptor Characterizes Anti-estrogen-Resistant ALDH+ Breast Cancer Stem Cells.

Estrogen-receptor-positive breast tumors are treated with anti-estrogen (AE) therapies but frequently develop resistance. Cancer stem cells (CSCs) with high aldehyde dehydrogenase activity (ALDH+ cells) are enriched following AE treatment. Here, we show that the interleukin-1ß (IL-1ß) signaling pathway is activated in ALDH+ cells, and data from single cells reveals that AE treatment selects for IL-1 receptor (IL1R1)-expressing ALDH+ cells. Importantly, CSC activity is reduced by an IL1R1 inhibitor in AE-resistant models. Moreover, IL1R1 expression is increased in the tumors of patients treated with AE therapy and predicts treatment failure. Single-cell gene expression analysis revealed that at least two subpopulations exist within the ALDH+ population, one proliferative and one quiescent. Following AE therapy the quiescent population is expanded, which suggests CSC dormancy as an adaptive strategy that facilitates treatment resistance. Targeting of ALDH+IL1R1+ cells merits testing as a strategy to combat AE resistance in patients with residual disease.

Targeting STAT3 signaling using stabilised sulforaphane (SFX-01) inhibits endocrine resistant stem-like cells in ER-positive breast cancer.

Estrogen receptor (ER) positive breast cancer is frequently sensitive to endocrine therapy. Multiple mechanisms of endocrine therapy resistance have been identified, including cancer stem-like cell (CSC) activity. Here we investigate SFX-01, a stabilised formulation of sulforaphane (SFN), for its effects on breast CSC activity in ER+ preclinical models. SFX-01 reduced mammosphere formation efficiency (MFE) of ER+ primary and metastatic patient samples. Both tamoxifen and fulvestrant increased MFE and aldehyde dehydrogenase (ALDH) activity of patient-derived xenograft (PDX) tumors, which was reversed by combination with SFX-01. SFX-01 significantly reduced tumor-initiating cell frequency in secondary transplants and reduced the formation of spontaneous lung micrometastases by PDX tumors in mice. Mechanistically, we establish that both tamoxifen and fulvestrant induce STAT3 phosphorylation. SFX-01 suppressed phospho-STAT3 and SFN directly bound STAT3 in patient and PDX samples. Analysis of ALDH+ cells from endocrine-resistant patient samples revealed activation of STAT3 target genes MUC1 and OSMR, which were inhibited by SFX-01 in patient samples. Increased expression of these genes after 3 months' endocrine treatment of ER+ patients (n = 68) predicted poor prognosis. Our data establish the importance of STAT3 signaling in CSC-mediated resistance to endocrine therapy and the potential of SFX-01 for improving clinical outcomes in ER+ breast cancer.

A large scale waterborne Campylobacteriosis outbreak, Havelock North, New Zealand.

BACKGROUND: We describe the investigation of a Campylobacter outbreak linked to contamination of an untreated, groundwater derived drinking water supply. METHODS: We analysed epidemiological data collected from clinician-confirmed diarrheal cases and estimated the total burden of Havelock North cases using an age-adjusted cross-sectional telephone survey. Campylobacter isolates from case fecal specimens, groundwater samples, and sheep fecal specimens from paddocks adjacent to the drinking water source were whole genome sequenced. FINDINGS: We estimate between 6260 and 8320 cases of illness including up to 2230 who lived outside the reticulation area, were linked to the contaminated water supply. Of these, 953 cases were physician reported, 42 were hospitalized, three developed Guillain-Barré syndrome, and Campylobacter infection contributed to at least four deaths. Of the 12 genotypes observed in cases, four were also observed in water, three were also observed in sheep and one was also observed in both water and sheep. INTERPRETATION: The contamination of the untreated reticulated water supply occurred following a very heavy rainfall event which caused drainage of sheep feces into a shallow aquifer. The existence of a routine clinical surveillance system for campylobacteriosis facilitated identification of the outbreak, recovery of clinical isolates, and early testing of the water for pathogens. Genotyping of the Campylobacter jejuni helped define the source of the outbreak and confirm outbreak periods and cases. Expected increases in heavy rainfall events and intensification of agriculture mean that additional safeguards are needed to protect populations from such drinking water outbreaks. FUNDING: NZ Ministry of Health, Health Research Council, ESR SSIF, Royal Society.

Epidemiology of bone tumours in children and young adults.

Although the epidemiology of malignant bone tumours in children and young adults has been explored, no definitive causation of any specific tumour has yet been identified. We performed a literature review (1970-2008) to find all papers covering possible aetiological factors involved in the development of bone tumours in children and young adults. Several associations have been reported with some consistency: the presence of hernias and Ewing sarcoma; high fluoride exposure and osteosarcoma; and parental farming and residence on a farm, younger age at puberty and family history of cancer for all bone tumours, especially osteosarcoma. Clearly further research is needed to confirm or refute these putative risk factors. It is likely that studies of gene-environment interactions may prove to be the most fruitful of future research.

The Notch Pathway Promotes Osteosarcoma Progression through Activation of Ephrin Reverse Signaling.

Despite significant advancements in the diagnosis and treatment of osteosarcoma, the molecular mechanisms underpinning disease progression remain unclear. This work presents strong clinical and experimental evidence demonstrating that Notch signaling contributes to osteosarcoma progression. First, using a cohort of 12 patients, Notch genes were upregulated in tumors compared with adjacent normal tissue, and high tumor expression of Notch1 intercellular domain (NICD1) and the Notch target gene Hes1 correlated with poor chemotherapy response. Data mining of publicly available datasets confirmed that expression of Notch pathway genes is related to poor prognosis in osteosarcoma. On the basis of in vitro analysis, Notch signaling promoted osteosarcoma proliferation, enhanced chemoresistance, facilitated both migration and invasion, and upregulated stem cell-like characteristics. Xenograft models demonstrated that Notch signaling promotes primary tumor growth and pulmonary metastasis, and Notch inhibition is effective in reducing tumor size and preventing metastasis. Mechanistically, activated Notch signaling induces the expression of ephrinB1 and enhances the tumor-promoting ephrin reverse signaling. Overall, these findings provide functional evidence for Notch pathway genes as candidate biomarkers to predict prognosis in patients with osteosarcoma, and suggest a mechanistic rationale for the use of Notch inhibitors to treat osteosarcoma. IMPLICATIONS: The study provides preclinical evidence for Notch pathway as a molecular marker to predict osteosarcoma prognosis and as a therapeutic target against osteosarcoma. In addition, we identified a novel mechanism that ephrin reverse signaling acts as a key mediator of Notch pathway.

Microenvironmental IL1ß promotes breast cancer metastatic colonisation in the bone via activation of Wnt signalling.

Dissemination of tumour cells to the bone marrow is an early event in breast cancer, however cells may lie dormant for many years before bone metastases develop. Treatment for bone metastases is not curative, therefore new adjuvant therapies which prevent the colonisation of disseminated cells into metastatic lesions are required. There is evidence that cancer stem cells (CSCs) within breast tumours are capable of metastasis, but the mechanism by which these colonise bone is unknown. Here, we establish that bone marrow-derived IL1ß stimulates breast cancer cell colonisation in the bone by inducing intracellular NFkB and CREB signalling in breast cancer cells, leading to autocrine Wnt signalling and CSC colony formation. Importantly, we show that inhibition of this pathway prevents both CSC colony formation in the bone environment, and bone metastasis. These findings establish that targeting IL1ß-NFKB/CREB-Wnt signalling should be considered for adjuvant therapy to prevent breast cancer bone metastasis.

Cisplatin selects for stem-like cells in osteosarcoma by activating Notch signaling.

Notch signaling regulates normal stem cells and is also thought to regulate cancer stem cells (CSCs). Recent data indicate that Notch signaling plays a role in the development and progression of osteosarcoma, however the regulation of Notch in chemo-resistant stem-like cells has not yet been fully elucidated. In this study we generated cisplatin-resistant osteosarcoma cells by treating them with sub-lethal dose of cisplatin, sufficient to induce DNA damage responses. Cisplatin-resistant osteosarcoma cells exhibited lower proliferation, enhanced spheroid formation and more mesenchymal characteristics than cisplatin-sensitive cells, were enriched for Stro-1+/CD117+ cells and showed increased expression of stem cell-related genes. A similar effect was observed in vivo, and in addition in vivo tumorigenicity was enhanced during serial transplantation. Using several publicly available datasets, we identified that Notch expression was closely associated with osteosarcoma stem cells and chemotherapy resistance. We confirmed that cisplatin-induced enrichment of osteosarcoma stem cells was mediated through Notch signaling in vitro, and immunohistochemistry showed that cleaved Notch1 (NICD1) positive cells were significantly increased in a relapsed xenograft which had received cisplatin treatment. Furthermore, pretreatment with a γ-secretase inhibitor (GSI) to prevent Notch signalling inhibited cisplatin-enriched osteosarcoma stem cell activity in vitro, including Stro-1+/CD117+ double positive cells and spheroid formation capacity. The Notch inhibitor DAPT also prevented tumor recurrence in resistant xenograft tumors. Overall, our results show that cisplatin induces the enrichment of osteosarcoma stem-like cells through Notch signaling, and targeted inactivation of Notch may be useful for the elimination of CSCs and overcoming drug resistance.

Anti-estrogen Resistance in Human Breast Tumors Is Driven by JAG1-NOTCH4-Dependent Cancer Stem Cell Activity.

Breast cancers (BCs) typically express estrogen receptors (ERs) but frequently exhibit de novo or acquired resistance to hormonal therapies. Here, we show that short-term treatment with the anti-estrogens tamoxifen or fulvestrant decrease cell proliferation but increase BC stem cell (BCSC) activity through JAG1-NOTCH4 receptor activation both in patient-derived samples and xenograft (PDX) tumors. In support of this mechanism, we demonstrate that high ALDH1 predicts resistance in women treated with tamoxifen and that a NOTCH4/HES/HEY gene signature predicts for a poor response/prognosis in 2 ER+ patient cohorts. Targeting of NOTCH4 reverses the increase in Notch and BCSC activity induced by anti-estrogens. Importantly, in PDX tumors with acquired tamoxifen resistance, NOTCH4 inhibition reduced BCSC activity. Thus, we establish that BCSC and NOTCH4 activities predict both de novo and acquired tamoxifen resistance and that combining endocrine therapy with targeting JAG1-NOTCH4 overcomes resistance in human breast cancers.

Enrichment of human osteosarcoma stem cells based on hTERT transcriptional activity.

Telomerase is crucial for the maintenance of stem/progenitor cells in adult tissues and is detected in most malignant cancers, including osteosarcoma. However, the relationship between telomerase expression and cancer stem cells remains unknown. We observed that sphere-derived osteosarcoma cells had higher telomerase activity, indicating that telomerase activity might be enriched in osteosarcoma stem cells. We sorted subpopulations with high or low telomerase activity (TEL) using hTERT transcriptional promoter-induced green fluorescent protein (GFP). The TELpos cells showed an increased sphere and tumor propagating capacity compared to TELneg cells, and enhanced stem cell-like properties such as invasiveness, metastatic activity and resistance to chemotherapeutic agents both in vitro and in vivo. Furthermore, the telomerase inhibitor MST312 prevented tumorigenic potential both in vitro and in vivo, preferentially targeting the TELpos cells. These data support telomerase inhibition as a potential targeted therapy for osteosarcoma stem-like cells.

The temporal and spatial expression patterns of ABCG2 in the developing human heart.

BACKGROUND: The discovery that the adult heart is not a terminally differentiated organ and contains stem/progenitor cells has important implications for the development of cellular therapeutics to treat heart disease. Moreover the discovery of cardiac stem cells might be important in furthering our understanding of both normal and abnormal cardiac development and yet little is known about these cell populations in the developing human heart, which we have focused on in this study. METHODS: The presence of ABCG2 and islet-1 expressing cells in human heart was determined using immunohistochemistry and RT-PCR (and western blotting for ABCG2). Cardiac SP cells were isolated using FACS. Co-localisation immunohistochemistry was used to determine if ABCG2 positive cells expressed other known stem/progenitor cell, endothelial markers or cardiac markers. RESULTS: We observed that ABCG2 expressing cells show a difference in both their temporal and spatial patterns of expression from Islet-1 expressing cardiac progenitors. We identified rare cells that expressed both ABCG2 and markers of other cell lineages including CD31, CD34 and alpha-actinin. We also noted the presence of cells that only expressed ABCG2. We isolated cardiac SP cells and confirmed the SP cell phenotype. CONCLUSIONS: Our results suggest that the developing human heart contains at least two distinct cardiac stem/progenitor cell populations one of which, the ABCG2 positive cells, can be readily isolated, suggesting that this tissue could be a useful source of cardiac stem cells.

Community participation in a rural community health trust: the case of Lawrence, New Zealand.

Since the mid-1980s, the New Zealand health sector has been in a state of continual change. The most radical changes were in the early-1990s, with the creation of an internal market system for public health care delivery. Rural health services, seen to be unviable, were given the option of establishing themselves as 'community trusts', owning and running their own services. Community trusts have since become a feature of rural health care in New Zealand. An expectation was that community trusts would facilitate community participation. This article reports on a study of participation in a rural community health trust. The 'pentagram model' of Rifkin and coworkers, with its five dimensions of participation-needs assessment, leadership, resource mobilization, management and organization-was applied. High levels of participation were found across each of these dimensions. The research revealed additional dimensions that could be added to the framework, including 'sustainability of participation', 'equity in participation' and 'the dynamic socio-political context'. In this regard, it supports recent theoretical work by Laverack (2001) and Laverack and Wallerstein (2001). Finally, the article comments on the future of rural health trusts in the current round of health sector restructuring.