development-human-preclinical-model-osteoclastogenesis-from

Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We ...

establishment-primary-culture-patient-derived-soft-tissue

Establishment of a Primary Culture of Patient-derived Soft Tissue Sarcoma

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more ...

dried-blood-serum-spots-as-useful-tool-for-sample-storage-to-evaluate

Dried Blood and Serum Spots As A Useful Tool for Sample Storage to Evaluate Cancer Biomarkers

Blood sample quality is crucial to ensure accurate downstream analyses such as real-time PCR or ELISA. Correct storage of biological materials is the ...

IRCCS

Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS

Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy

Metastatic bone disease has a major impact on the morbidity and mortality of breast cancer patients, and studies on bone metastasis biology have led to the development of the most widely used drugs for bone metastases treatment: zoledronate (Zol) and denosumab (Den). The aim of the present study was to assess the effect of soluble mediators produced by breast cancer cells on human osteoclast maturation in a co-culture model. We also tested the ability of zoledronate, denosumab and 5H4, an antibody directed against CSF-1, to interfere with the osteoclastogenic potential of breast cancer. The study was performed on the triple negative cell line MDA-MB-231 and on human osteoclasts obtained from the differentiation of peripheral blood monocytes of a healthy volunteer. Osteoclastogenesis was evaluated by TRAP assay after 14days of differentiation with 10% MDA-MB-231-conditioned media or with CSF-1 and RANKL. Den, Zol and 5H4 were administered after 7days of differentiation. MDA-MB-231-conditioned media doubled the differentiation of monocytes into osteoclasts. MDA-MB-231 secreted CSF-1, especially when cells were cultured to confluence. Induced osteoclasts were sensitive to bone-targeted drugs: Den and 5H4 blocked osteoclast differentiation and survival, while Zol induced osteoclast apoptosis. Osteoclasts differentiated by breast cancer cells were less sensitive to Zol than those induced by differentiation factors, whereas sensitivity to Den was similar. Conversely, breast cancer-induced osteoclast activation resulted in a higher sensitivity to 5H4. A significant increase in CSF-1 secretion was observed in osteoclast precursors after treatment with the highest concentration of Den. Further research is ongoing to evaluate the efficacy of 5H4 combination with Den.

CSF-1 blockade impairs breast cancer osteoclastogenic potential in co-culture systems.

Bone metastasis is a complex process that needs to be better understood in order to help clinicians prevent and treat it. Xenografts using patient-derived material (PDX) rather than cancer cell lines are a novel approach that guarantees more clinically realistic results. A primary culture of bone metastasis derived from a 67-year-old patient with breast cancer was cultured and then injected into zebrafish (ZF) embryos to study its metastatic potential. In vivo behavior and results of gene expression analyses of the primary culture were compared with those of cancer cell lines with different metastatic potential (MCF7 and MDA-MB-231). The MCF7 cell line, which has the same hormonal receptor status as the bone metastasis primary culture, did not survive in the in vivo model. Conversely, MDA-MB-231 disseminated and colonized different parts of the ZF, including caudal hematopoietic tissues (CHT), revealing a migratory phenotype. Primary culture cells disseminated and in later stages extravasated from the vessels, engrafting into ZF tissues and reaching the CHT. Primary cell behavior reflected the clinical course of the patient's medical history. Our results underline the potential for using PDX models in bone metastasis research and outline new methods for the clinical application of this in vivo model.

Development of a Patient-Derived Xenograft (PDX) of Breast Cancer Bone Metastasis in a Zebrafish Model.

Sarcomas are a heterogeneous group of mesenchymal tumors arising from soft tissue or bone, with an uncertain etiology and difficult classification. Soft tissue sarcomas (STSs) account for around 1% of all adult cancers. Till date, more than 50 histologic subtypes have been identified. Adipocyte sarcoma or liposarcoma (LPS) is one of the most common STS subtypes, accounting for 15% of all sarcomas, with an incidence of 24% of all extremity STSs and 45% of all retroperitoneal STSs. The new World Health Organization classification system has divided LPS into four different subgroups: atypical lipomatous tumor/well-differentiated LPS, dedifferentiated LPS, myxoid LPS, and pleomorphic LPS. These lesions can develop at any location and exhibit different aggressive potentials reflecting their morphologic diversity and clinical behavior. Patients affected by LPS should be managed in specialized multidisciplinary cancer centers. Whereas surgical resection is the mainstay of treatment for localized disease, the benefits of adjuvant and neoadjuvant chemotherapy are still unclear. Systemic treatment, particularly chemotherapy, is still limited in metastatic disease. Despite the efforts toward a better understanding of the biology of LPS, the outcome of advanced and metastatic patients remains poor. The advent of targeted therapies may lead to an improvement of treatment options and clinical outcomes. A larger patient enrollment into translational and clinical studies will help increase the knowledge of the biological behavior of LPSs, test new drugs, and introduce new methodological studies, that is, on treatment response.

Current classification, treatment options, and new perspectives in the management of adipocytic sarcomas.

Metastatic bone disease has a major impact on morbidity of breast cancer (BC) patients. Alterations in mTOR signaling are involved both in cancer progression and in osteoclast differentiation. The purpose of this study was to assess the role of mTOR inhibitor Everolimus (Eve) on osteoclastogenesis induced by triple negative BC cells. To this aim, we developed an in vitro human model of osteoclastogenesis from peripheral blood monocytes co-cultured with the triple negative SCP2 and the hormonal receptor positive MCF7 cell lines. Osteoclastogenesis was evaluated by TRAP staining, evaluation of F actin rings and Calcitonin Receptor expression. Eve significantly reduced differentiation induced by cancer cells and resulted more effective when evaluated in combination with Denosumab and Zoledronic Acid (Zol). Combination with Zol showed a total abrogation of osteoclast differentiation induced by the triple negative cell line, not by MCF7. Finally, we observed that Eve was active in the inhibition of the crosstalk between cancer cells and osteoclasts reproduced by our model, highlighting a new therapeutic choice for the subsetting of triple negative BC patients. We observed a difference in the response to bone-targeted therapy with respect to BC subtypes. Our model may represent a valid platform for preclinical trials on bone-targeted drugs and for the study of the interplay of BC with bone stromal cells.

The Effect of Everolimus in an In Vitro Model of Triple Negative Breast Cancer and Osteoclasts.

Patient-derived specimens are an invaluable resource to investigate tumor biology. However, in vivo studies on primary cultures are often limited by the small amount of material available, while conventional in vitro systems might alter the features and behavior that characterize cancer cells. We present our data obtained on primary dedifferentiated liposarcoma cells cultured in a 3D scaffold-based system and injected into a zebrafish model. Primary cells were characterized in vitro for their morphological features, sensitivity to drugs and biomarker expression, and in vivo for their engraftment and invasiveness abilities. The 3D culture showed a higher enrichment in cancer cells than the standard monolayer culture and a better preservation of liposarcoma-associated markers. We also successfully grafted primary cells into zebrafish, showing their local migratory and invasive abilities. Our work provides proof of concept of the ability of 3D cultures to maintain the original phenotype of ex vivo cells, and highlights the potential of the zebrafish model to provide a versatile in vivo system for studies with limited biological material. Such models could be used in translational research studies for biomolecular analyses, drug screenings and tumor aggressiveness assays.

Innovative approaches to establish and characterize primary cultures: an ex vivo 3D system and the zebrafish model.

Eribulin mesylate is a novel, non-taxane, synthetic microtubule inhibitor showing antitumor activity in a wide range of tumors including soft tissue sarcomas (STS). Eribulin has been recently approved for the treatment of metastatic liposarcoma (LPS) patients previously treated with anthracyclines. This work investigated the mechanism of action of this innovative antitubulin agent in well-differentiated/dedifferentiated LPS (ALT/DDLPS) which represents one of the most common adipocytic sarcoma histotypes. A primary culture of ALT/DDLPS from a 54-year-old patient was established. The anticancer activity of eribulin on the patient-derived primary culture was assessed by MTT and tunel assays. Eribulin efficacy was compared to other drugs approved for the treatment of STS. Cell migration and morphology were examined after exposure to eribulin to better understand the drug mechanism of action. Finally, Western blot analysis of apoptosis and migration proteins was performed. The results showed that eribulin exerts its antiproliferative effect by the arrest of cell motility and induction of apoptosis. Our results highlighted the activity of eribulin in the treatment of ALT/DDLPS patients.

Activity of Eribulin in a Primary Culture of Well-Differentiated/Dedifferentiated Adipocytic Sarcoma.

As total vitamin D levels are often lower in black than in white Americans, the former are frequently classified as vitamin D-deficient. To fully understand African vitamin D (25(OH)D) status, other factors should be considered, e.g. vitamin D blood carrier, vitamin D-binding protein (DBP), vitamin D receptor (VDR) and DBP polymorphisms. A prospective study on an indigenous black Tanzanian and a Caucasian Italian population was performed on 50 healthy donors from both populations and 35 Caucasian and 18 African breast cancer patients. 25(OH)D and DBP serum levels were analyzed by ELISA. A1012G, Cdx2 and Fok1 VDR polymorphisms and DBP polymorphisms rs4588 and rs7041 were genotyped by real-time PCR. Vitamin D and DBP levels were lower in healthy African donors than in Caucasians. Africans had a significantly higher frequency of AA and CC for Cdx2 and Fok1 polymorphisms, respectively. These allelic variants were related to a higher transcription of VDR gene and a higher activity of VDR receptor. With regard to polymorphism distribution, Africans showed innate higher levels and activity of VDR. We conclude that a strengthening of the vitamin D pathway could have a protective role against the development of breast cancer in the African population.

Vitamin D receptor polymorphisms or serum levels as key drivers of breast cancer development? The question of the vitamin D pathway.

CORRECTION: Innovative approaches to establish and characterize primary cultures: an ex vivo 3D system and the zebrafish model.

Although bone metastases represent a major challenge in the natural history of breast cancer (BC), the complex interactions involved have hindered the development of robust in vitro models. The aim of this work is the development of a preclinical model of cancer and bone stromal cells to mimic the bone microenvironment. We studied the effects on osteoclastogenesis of BC cells and Mesenchymal stem cells (MSC) cultured alone or in combination. We also analyzed: (a) whether the blockade of the Epithelial Growth Factor Receptor (EGFR) pathway modified their influence on monocytes towards differentiation, and (b) the efficacy of bone-targeted therapy on osteoclasts. We evaluated the osteoclastogenesis modulation of human peripheral blood monocytes (PBMC) indirectly induced by the conditioned medium (CM) of the human BC cell line SCP2, cultured singly or with MSC. Osteoclastogenesis was evaluated by TRAP analysis. The effect of the EGFR blockade was assessed by treating the cells with gefitinib, and analyzed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Western Blot (WB). We observed that SCP2 co-cultured with MSC increased the differentiation of PBMC. This effect was underpinned upon pre-treatment of the co-culture with gefitinib. Co-culture of SCP2 with MSC increased the expression of both the bone-related marker Receptor Activator of Nuclear Factor κB (RANK) and EGFR in BC cells. These upregulations were not affected by the EGFR blockade. The effects of the CM obtained by the cells treated with gefitinib in combination with the treatment of the preosteoclasts with the bone-targeted agents and everolimus enhanced the inhibition of the osteoclastogenesis. Finally, we developed a fully human co-culture system of BC cells and bone progenitor cells. We observed that the interaction of MSC with cancer cells induced in the latter molecular changes and a higher power of inducing osteoclastogenesis. We found that blocking EGFR signaling could be an efficacious strategy for breaking the interactions between cancer and bone cells in order to inhibit bone metastasis.

Tumor-Stroma Crosstalk in Bone Tissue: The Osteoclastogenic Potential of a Breast Cancer Cell Line in a Co-Culture System and the Role of EGFR Inhibition.

The use of patient-derived primary cell cultures in cancer preclinical assays has increased in recent years. The management of resected tumor tissue remains complex and a number of parameters must be respected to obtain complete sample digestion and optimal vitality yield. We provide an overview of the benefits of correct primary cell culture management using different preclinical methodologies, and describe the pros and cons of this model with respect to other kinds of samples. One important advantage is that the heterogeneity of the cell populations composing a primary culture partially reproduces the tumor microenvironment and crosstalk between malignant and healthy cells, neither of which is possible with cell lines. Moreover, the use of patient-derived specimens in innovative preclinical technologies, such as 3D systems or bioreactors, represents an important opportunity to improve the translational value of the results obtained. In vivo models could further our understanding of the crosstalk between tumor and other tissues as they enable us to observe the systemic and biological interactions of a complete organism. Although engineered mice are the most common model used in this setting, the zebrafish (Danio rerio) species has recently been recognized as an innovative experimental system. In fact, the transparent body and incomplete immune system of zebrafish embryos are especially useful for evaluating patient-derived tumor tissue interactions in healthy hosts. In conclusion, ex vivo systems represent an important tool for cancer research, but samples require correct manipulation to maximize their translational value.

Management and potentialities of primary cancer cultures in preclinical and translational studies.

Leiomyosarcoma (LMS) is a malignant sarcoma that can occur in different anatomic sites, including the bone, showing similar histological characteristics but heterogeneous clinical behavior and prognosis. Primary bone LMS was first described in 1965. It is a very rare sarcoma, accounting for <0.7% of all primary malignant bone tumors.

Primary leiomyosarcoma of the bone: a case report and a review of the literature.

Undifferentiated pleomorphic sarcoma (UPS) is an aggressive mesenchymal neoplasm with no specific line of differentiation. Eribulin, a novel synthetic microtubule inhibitor, has shown anticancer activity in several tumors, including soft tissue sarcomas (STS). We investigated the molecular biology of UPS, and the mechanisms of action of this innovative microtubule-depolymerizing drug. A primary culture from a patient with UPS was established and characterized in terms of gene expression. The activity of eribulin was also compared with that of other drugs currently used for STS treatment, including trabectedin. Finally, Western blot analysis was performed to better elucidate the activity of eribulin. Our results showed an upregulation of epithelial mesenchymal transition-related genes, and a downregulation of epithelial markers. Furthermore, genes involved in chemoresistance were upregulated. Pharmacological analysis confirmed limited sensitivity to chemotherapy. Interestingly, eribulin exhibited a similar activity to that of standard treatments. Molecular analysis revealed the expression of cell cycle arrest-related and pro-apoptotic-related proteins. These findings are suggestive of aggressive behavior in UPS. Furthermore, the identification of chemoresistance-related genes could facilitate the development of innovative drugs to improve patient outcome. Overall, the results from the present study furnish a rationale for elucidating the role of eribulin for the treatment of UPS.

Primary Culture of Undifferentiated Pleomorphic Sarcoma: Molecular Characterization and Response to Anticancer Agents.

Myxofibrosarcoma (MFS), formerly considered as a myxoid variant of malignant fibrous histiocytoma, is the most common sarcoma of the extremities in adults and is characterized by a high frequency of local recurrence. The clinical behavior of MFS is unpredictable and the efficacy of chemotherapy is still not well documented. Furthermore, given the relatively recent recognition of MFS as a distinct pathologic entity its cellular and molecular biology has still not been extensively studied in patient-derived preclinical models. We examined the molecular biology and treatment outcomes of high-grade, patient-derived MFS primary cultures.

ArticleTotal : 3	Year
Publication title Cited by 6	2017
Publication title Cited by 16	2018
Publication title Cited by 9	2018

Article	Year
CSF-1 blockade impairs breast cancer osteoclastogenic potential in co-culture systems. Bone\| PubMed ID: 24956020	2014
Development of a Patient-Derived Xenograft (PDX) of Breast Cancer Bone Metastasis in a Zebrafish Model. International journal of molecular sciences\| PubMed ID: 27556456	2016
Current classification, treatment options, and new perspectives in the management of adipocytic sarcomas. OncoTargets and therapy\| PubMed ID: 27785071	2016
The Effect of Everolimus in an In Vitro Model of Triple Negative Breast Cancer and Osteoclasts. International journal of molecular sciences\| PubMed ID: 27809291	2016
Innovative approaches to establish and characterize primary cultures: an ex vivo 3D system and the zebrafish model. Biology open\| PubMed ID: 27895047	2017
Activity of Eribulin in a Primary Culture of Well-Differentiated/Dedifferentiated Adipocytic Sarcoma. Molecules (Basel, Switzerland)\| PubMed ID: 27918490	2016
Vitamin D receptor polymorphisms or serum levels as key drivers of breast cancer development? The question of the vitamin D pathway. Oncotarget\| PubMed ID: 28061456	2017
CORRECTION: Innovative approaches to establish and characterize primary cultures: an ex vivo 3D system and the zebrafish model. Biology open\| PubMed ID: 28202474	2017
Tumor-Stroma Crosstalk in Bone Tissue: The Osteoclastogenic Potential of a Breast Cancer Cell Line in a Co-Culture System and the Role of EGFR Inhibition. International journal of molecular sciences\| PubMed ID: 28758931	2017
Management and potentialities of primary cancer cultures in preclinical and translational studies. Journal of translational medicine\| PubMed ID: 29116016	2017
Primary leiomyosarcoma of the bone: a case report and a review of the literature. Medicine\| PubMed ID: 29137065	2017
Primary Culture of Undifferentiated Pleomorphic Sarcoma: Molecular Characterization and Response to Anticancer Agents. International journal of molecular sciences\| PubMed ID: 29292724	2017
Myxofibrosarcoma primary cultures: molecular and pharmacological profile. Therapeutic advances in medical oncology\| PubMed ID: 29449896	2017

Chiara Spadazzi

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