TRIzol and Alu qPCR-based quantification of metastatic seeding within the skeleton

Campbell, James; Mulcrone, Patrick L.; Masood, Shikohe; Karolak, Matthew R.; Merkel, Alyssa R.; Hebron, Katie E.; Zijlstra, Andries; Sterling, Julie A.; Elefteriou, Florent

doi:10.1038/srep12635

Cited by 28 publications

(24 citation statements)

References 14 publications

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“…To address this question, we reanalyzed the hindlimbs of 6‐week‐old nude mice subjected to a chronic immobilization stress (CIS) protocol that was aimed at stimulating an endogenous stress response. We also analyzed the hindlimbs of 6‐week‐old nude mice treated for 6 weeks daily with the nonselective β1/β2AR agonist isoproterenol (ISO, 3 mg/kg i.p.…”

Section: Resultsmentioning

confidence: 99%

“…cDNA for qRT‐PCR experiments was synthesized using the High Capacity cDNA Reverse Transcription Kit from ABI Applied Biosystems (#1502205). Taqman probes were used to amplify the following genes: Murine Vegf‐a (Mm00437304), Murine Hprt (Mm01545399), Murine 18s ( 4333760T), Human VEGFA (Hs00900055), and Human HPRT (Hs02800695) . Expression was analyzed by the ΔΔCt method.…”

Section: Methodsmentioning

confidence: 99%

“…Clues about the conditions driving the osteotropism of metastatic cancer cells can be obtained from retrospective studies, in which factors associated with reduced survival are identified. In that regard, chronic emotional stress has been linked to higher breast cancer recurrence, reduced survival, and poor prognosis, and β‐blockers were associated with prolonged survival in breast cancer patients when treatment was initiated at time of diagnosis . A common factor between these studies is the activity of the sympathetic nervous system (SNS) that is stimulated by chronic psychosocial stress and whose action is antagonized by β‐blockers.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Skeletal Colonization by Breast Cancer Cells Is Stimulated by an Osteoblast and β2AR-Dependent Neo-Angiogenic Switch

Mulcrone

Campbell

Clément-Demange

et al. 2017

Journal of Bone and Mineral Research

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The skeleton is a common site for breast cancer metastasis. Although significant progress has been made to manage osteolytic bone lesions, the mechanisms driving the early steps of the bone metastatic process are still not sufficiently understood to design efficacious strategies needed to inhibit this process and offer preventative therapeutic options. Progression and recurrence of breast cancer, as well as reduced survival of patients with breast cancer, are associated with chronic stress, a condition known to stimulate sympathetic nerve outflow. In this study, we show that stimulation of the beta 2-adrenergic receptor (β2AR) by isoproterenol, used as a pharmacological surrogate of sympathetic nerve activation, led to increased blood vessel density and Vegf-a expression in bone. It also raised levels of secreted Vegf-a in osteoblast cultures, and accordingly, the conditioned media from isoproterenol-treated osteoblast cultures promoted new vessel formation in two ex vivo models of angiogenesis. Blocking the interaction between Vegf-a and its receptor, Vegfr2, blunted the increase in vessel density induced by isoproterenol. Genetic loss of the β2AR globally, or specifically in type 1 collagen-expressing osteoblasts, diminished the increase in Vegf-positive osteoblast number and bone vessel density induced by isoproterenol, and reduced the higher incidence of bone metastatic lesions induced by isoproterenol following intracardiac injection of an osteotropic variant of MDA-MB-231 breast cancer cells. Inhibition of the interaction between Vegf-a and Vegfr2 with the blocking antibody mcr84 also prevented the increase in bone vascular density and bone metastasis triggered by isoproterenol. Together, these results indicate that stimulation of the β2AR in osteoblasts triggers a Vegf-dependent neo-angiogenic switch that promotes bone vascular density and the colonization of the bone microenvironment by metastatic breast cancer cells.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Skeletal Colonization by Breast Cancer Cells Is Stimulated by an Osteoblast and β2AR-Dependent Neo-Angiogenic Switch

Mulcrone

Campbell

Clément-Demange

et al. 2017

Journal of Bone and Mineral Research

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“…Total RNA was extracted from pork samples using a modified TRizol method (Preston Campbell and others ). Three milliliters of the above‐mentioned suspension was centrifuged for 1 min at 12000 × g to obtain the precipitate.…”

Section: Methodsmentioning

confidence: 99%

High‐Throughput Sequencing of Viable Microbial Communities in Raw Pork Subjected to a Fast Cooling Process

Yang

Che

et al. 2016

Journal of Food Science

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This study aimed to investigate the effect of the fast cooling process on the microbiological community in chilled fresh pork during storage. We established a culture-independent method to study viable microbes in raw pork. Tray-packaged fresh pork and chilled fresh pork were completely spoiled after 18 and 49 d in aseptic bags at 4 °C, respectively. 16S/18S ribosomal RNAs were reverse transcribed to cDNA to characterize the activity of viable bacteria/fungi in the 2 types of pork. Both cDNA and total DNA were analyzed by high-throughput sequencing, which revealed that viable Bacteroides sp. were the most active genus in rotten pork, although viable Myroides sp. and Pseudomonas sp. were also active. Moreover, viable fungi were only detected in chilled fresh pork. The sequencing results revealed that the fast cooling process could suppress the growth of microbes present initially in the raw meat to extend its shelf life. Our results also suggested that fungi associated with pork spoilage could not grow well in aseptic tray-packaged conditions.

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“…Quantitative qPCR for the human ALU sequence was carried out as previously described [44]. Briefly, lungs were homogenized, and DNA was isolated using a DNA purification kit (Cat#: 69504, Qiagen; Hilden, Germany).…”

Section: Quantitative Pcr (Qpcr) and Quantitative Rt-pcr (Qrt-pcr)mentioning

confidence: 99%

Triple-Negative Primary Breast Tumors Induce Supportive Premetastatic Changes in the Extracellular Matrix and Soluble Components of the Lung Microenvironment

Medeiros

Goodale

Postenka

et al. 2020

Cancers

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The lung is one of the deadliest sites of breast cancer metastasis, particularly in patients with triple-negative (TN) disease. We hypothesized that the presence of a TN primary breast tumor induces changes in the extracellular matrix (ECM) and soluble components of the lung microenvironment that support metastatic behavior. SUM159 (TN) and MCF7 (luminal A) breast cancer cells were injected into mice, and primary breast tumors were established prior to assessing metastatic niche changes. We observed increased CD117+ hematopoietic progenitor cells in the bone marrow of SUM159 mice versus MCF7 or control mice (p < 0.05). Relative to mice bearing MCF7 tumors and non-tumor controls, mice bearing SUM159 tumors demonstrated enhanced expression of ECM proteins in the lung (fibronectin, tenascin-c and periostin), with similar changes observed in lung fibroblasts treated with extracellular vesicles (EVs) from TN breast cancer cells (p < 0.05). Exposure to lung-conditioned media (LCM) from SUM159 tumor-bearing mice resulted in increased migration/proliferation of both SUM159 and MCF7 cells relative to the control (p < 0.05). In contrast, LCM from MCF-7 tumor-bearing mice had no such effect. LCM from SUM159 tumor-bearing mice contained 16 unique proteins relative to other LCM conditions, including the metastasis-associated proteins CCL7, FGFR4, GM-CSF, MMP3, thrombospondin-1 and VEGF. These findings suggest for the first time that the TN breast cancer molecular subtype may be an important determinant of premetastatic changes to both the ECM and soluble components of the lung, potentially mediated via breast cancer-derived EVs.

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TRIzol and Alu qPCR-based quantification of metastatic seeding within the skeleton

Cited by 28 publications

References 14 publications

Skeletal Colonization by Breast Cancer Cells Is Stimulated by an Osteoblast and β2AR-Dependent Neo-Angiogenic Switch

Skeletal Colonization by Breast Cancer Cells Is Stimulated by an Osteoblast and β2AR-Dependent Neo-Angiogenic Switch

High‐Throughput Sequencing of Viable Microbial Communities in Raw Pork Subjected to a Fast Cooling Process

Triple-Negative Primary Breast Tumors Induce Supportive Premetastatic Changes in the Extracellular Matrix and Soluble Components of the Lung Microenvironment

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