Search

To assess the efficacy of Sanyrene liquid dressing (Urgo Medical) in preventing radiation dermatitis (RD) among patients with cancer after radiotherapy.The authors searched the China National Knowledge Infrastructure, SinoMed, WanFang Data, PubMed, Web of Science, EMBASE, and the Cochrane Library databases for articles published from inception to January 2021.The preliminary search identified 146 studies. After removing duplicates, applying exclusion criteria, and screening titles and abstracts, 19 studies met the inclusion criteria.A standardized form was constructed to extract data from eligible studies. Two reviewers independently screened the literature, extracted data, and assessed the risk of bias of the included studies.The authors identified a total of 19 studies involving 1,508 patients that assessed the effectiveness of Sanyrene liquid dressing in preventing RD in patients with cancer after radiotherapy. The findings suggested that Sanyrene decreases the total incidence of RD (odds ratio [OR], 5.00; 95% CI, 2.77-9.03; P.00001), as well as the incidence of RD grade 2 (OR, 0.55; 95% CI, 0.36-0.85; P = .007), grade 3 (OR, 0.22; 95% CI, 0.09-0.57; P = .002), and grade 4 (OR, 0.32; 95% CI, 0.13-0.78; P = .01). In addition, in comparison with controls, Sanyrene liquid dressing improves the cure rate (OR, 8.18; 95% CI, 4.03-16.60; P.00001) and delays the occurrence of RD (mean difference, 3.69; 95% CI, 3.03-4.36; P.00001).Sanyrene liquid dressing can decrease both the total incidence of RD and the incidence of RD above grade 2. It also improves the cure rate and delays the occurrence of RD. Thus, Sanyrene may be a superior option for preventing RD after radiotherapy. However, the findings were assessed as moderate- to low-quality evidence and more high-quality trials are needed to support this result.

Cancer initiation is orchestrated by interplay between tumor-initiating cells and their stromal/immune environment. Here, by adapted scRNAsequencing, we decipher the predicted signaling between tissue-resident hematopoietic stem/progenitor cells (HSPCs) and their neoplastic counterparts with their native niches in the human bone marrow. LEPR+stromal cells are identified as central regulators of hematopoiesis through predicted interactions with all cells in the marrow. Inflammatory niche remodeling and the resulting deprivation of critical HSPC regulatory factors is predicted to repress distinct high-output HSC subsets inNPM1-mutated AML, with relative resistance of clonal cells. Stromal gene signatures reflective of niche remodeling are associated with reduced relapse rates and favorable outcome after chemotherapy, across all genetic risk categories. Elucidation of the intercellular signaling defining human AML, thus, predicts that inflammatory remodeling of stem cell niches drives tissue repression and clonal selection, but may pose a vulnerability for relapse-initiating cells in the context of chemotherapeutic treatment.statement of significanceTumor-promoting inflammation is considered an enabling characteristic of tumorigenesis, but mechanisms remain incompletely understood. By deciphering the predicted signaling between tissue-resident stem cells and their neoplastic counterparts with their environment, we identify inflammatory remodeling of stromal niches as a determinant of normal tissue repression and clinical outcome in human AML.Key pointsA comprehensive taxonomy of the predicted interactions between LEPR+stromal niches, HSPCs and adaptive/innate immune cells in the human NBM.Inflammation-associated decline of stromal niches in AML represses residual normal hematopoiesis with relative resistance of leukemic cells.Inflammatory decline of stromal niches is associated with reduced relapse risk and favorable outcome.

Enrichement analysis for E2F.

Primers used for qPCR.

S1. Cell Sorting Workflow. S2. Effect of MCAM knockdown on RNA levels. S3. Overview of invivo study. S4. GSEA and gProfile enrichment analysis and enrichment maps. S5. Overview of in vivo study with monoclonal antiMCAM antibody administration. S6. Overview of bone morphometric images and histology. S7. Overview of kidney and bone histology.

Prostate Cancer is the most common cancer and the second leading cause of cancer-related death in males. When prostate cancer acquires castration resistance, incurable metastases, primarily in the bone, occur. The aim of this study is to test the applicability of targeting melanoma cell adhesion molecule (MCAM; CD146) with a mAb for the treatment of lytic prostate cancer bone metastasis. We evaluated the effect of targeting MCAM using in vivo preclinical bone metastasis models and an in vitro bone niche coculture system. We utilized FACS, cell proliferation assays, and gene expression profiling to study the phenotype and function of MCAM knockdown in vitro and in vivo. To demonstrate the impact of MCAM targeting and therapeutic applicability, we employed an anti-MCAM mAb in vivo. MCAM is elevated in prostate cancer metastases resistant to androgen ablation. Treatment with DHT showed MCAM upregulation upon castration. We investigated the function of MCAM in a direct coculture model of human prostate cancer cells with human osteoblasts and found that there is a reduced influence of human osteoblasts on human prostate cancer cells in which MCAM has been knocked down. Furthermore, we observed a strongly reduced formation of osteolytic lesions upon bone inoculation of MCAM-depleted human prostate cancer cells in animal model of prostate cancer bone metastasis. This phenotype is supported by RNA sequencing (RNA-seq) analysis. Importantly, in vivo administration of an anti-MCAM human mAb reduced the tumor growth and lytic lesions. These results highlight the functional role for MCAM in the development of lytic bone metastasis and suggest that MCAM is a potential therapeutic target in prostate cancer bone metastasis.Implications:This study highlights the functional application of an anti-MCAM mAb to target prostate cancer bone metastasis.

In vivo data are rare but essential for establishing the clinical potential of ruthenium-based photoactivated chemotherapy (PACT) compounds, a new family of phototherapeutic drugs that are activated via ligand photosubstitution. Here a novel trisheteroleptic ruthenium complex [Ru(dpp)(bpy)(mtmp)](PF6)2 ([2](PF6)2, dpp = 4,7-diphenyl-1,10-phenanthroline, bpy = 2,2'-bipyridine, mtmp = 2-methylthiomethylpyridine) was synthesized and its light-activated anticancer properties were validated in cancer cell monolayers, 3D tumor spheroids, and in embryonic zebrafish cancer models. Upon green light irradiation, the non-toxic mtmp ligand is selectively cleaved off, thereby releasing a phototoxic ruthenium-based photoproduct capable notably of binding to nuclear DNA and triggering DNA damage and apoptosis within 24-48 h. In vitro, fifteen minutes of green light irradiation (21 mW cm-2, 19 J cm-2, 520 nm) were sufficient to generate high phototherapeutic indexes (PI) for this compound in a range of cancer cell lines including lung (A549), prostate (PC3Pro4), conjunctival melanoma (CRMM1, CRMM2, CM2005.1) and uveal melanoma (OMM1, OMM2.5, Mel270) cancer cell lines. The therapeutic potential of [2](PF6)2 was further evaluated in zebrafish embryo ectopic (PC3Pro4) or orthotopic (CRMM1, CRMM2) tumour models. The ectopic model consisted of red fluorescent PC3Pro4-mCherry cells injected intravenously (IV) into zebrafish, that formed perivascular metastatic lesions at the posterior ventral end of caudal hematopoietic tissue (CHT). By contrast, in the orthotopic model, CRMM1- and CRMM2-mCherry cells were injected behind the eye where they developed primary lesions. The maximally-tolerated dose (MTD) of [2](PF6)2 was first determined for three different modes of compound administration: (i) incubating the fish in prodrug-containing water (WA); (ii) injecting the prodrug intravenously (IV) into the fish; or (iii) injecting the prodrug retro-orbitally (RO) into the fish. To test the anticancer efficiency of [2](PF6)2, the embryos were treated 24 h after engraftment at the MTD. Optimally, four consecutive PACT treatments were performed on engrafted embryos using 60 min drug-to-light intervals and 90 min green light irradiation (21 mW cm-2, 114 J cm-2, 520 nm). Most importantly, this PACT protocol was not toxic to the zebrafish. In the ectopic prostate tumour models, where [2](PF6)2 showed the highest photoindex in vitro (PI > 31), the PACT treatment did not significantly diminish the growth of primary lesions, while in both conjunctival melanoma orthotopic tumour models, where [2](PF6)2 showed more modest photoindexes (PI ∼ 9), retro-orbitally administered PACT treatment significantly inhibited growth of the engrafted tumors. Overall, this study represents the first demonstration in zebrafish cancer models of the clinical potential of ruthenium-based PACT, here against conjunctival melanoma.

Skin tear knowledge is an important predictor of the decreased incidence and management of skin tears, and the knowledge level among Chinese nurses is unknown so far. A validated instrument for measuring skin tear knowledge is urgent.To culturally adapt the skin tear knowledge assessment instrument (OASES) into Chinese and verify its validity and reliability in the Chinese context.The cultural adaptation process for OASES into Chinese was established on Beaton's translation model. Content validity was determined by the 8-expert group in wound care. A nationwide psychometric validation study was performed on a convenience sample of 3333 nurses from 113 tertiary hospitals, of whom 98 nurses finished the test-retest procedure for reliability analysis. Item validity (item difficulty and discriminating index) and construct validity (known-groups technique) were tested.The content validity index was 0.88-1.00. The item validity was as follows: Item difficulty ranged from 0.16 to 0.86, with an average value of 0.52; the discriminating index varied between 0.05 and 0.61. The known-group technique demonstrated excellent construct validity with a significant difference between predefined groups with theoretically expected higher knowledge scores and theoretically expected lower knowledge scores (P 0.001). For the test-retest reliability, the Intraclass correction coefficient (ICC) during a 14-day interval for the overall tool was 0.79 (95% CI = 0.71-0.86), and Cohen's kappa value for each item varied from 0.17 to 0.62.The Chinese version of OASES was validated to be suitable for skin tear knowledge assessment with acceptable psychometric properties, through which the knowledge and training priorities of skin tear among Chinese nurses can be quantified.

Prostate Cancer is the most common cancer and the second leading cause of cancer-related death in males. When prostate cancer acquires castration resistance, incurable metastases, primarily in the bone, occur. The aim of this study is to test the applicability of targeting melanoma cell adhesion molecule (MCAM; CD146) with a mAb for the treatment of lytic prostate cancer bone metastasis. We evaluated the effect of targeting MCAM using in vivo preclinical bone metastasis models and an in vitro bone niche coculture system. We utilized FACS, cell proliferation assays, and gene expression profiling to study the phenotype and function of MCAM knockdown in vitro and in vivo. To demonstrate the impact of MCAM targeting and therapeutic applicability, we employed an anti-MCAM mAb in vivo. MCAM is elevated in prostate cancer metastases resistant to androgen ablation. Treatment with DHT showed MCAM upregulation upon castration. We investigated the function of MCAM in a direct coculture model of human prostate cancer cells with human osteoblasts and found that there is a reduced influence of human osteoblasts on human prostate cancer cells in which MCAM has been knocked down. Furthermore, we observed a strongly reduced formation of osteolytic lesions upon bone inoculation of MCAM-depleted human prostate cancer cells in animal model of prostate cancer bone metastasis. This phenotype is supported by RNA sequencing (RNA-seq) analysis. Importantly, in vivo administration of an anti-MCAM human mAb reduced the tumor growth and lytic lesions. These results highlight the functional role for MCAM in the development of lytic bone metastasis and suggest that MCAM is a potential therapeutic target in prostate cancer bone metastasis. Implications: This study highlights the functional application of an anti-MCAM mAb to target prostate cancer bone metastasis.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. Despite increasing treatment options for this disease, prognosis remains poor. CRIPTO (TDGF1) protein is expressed at high levels in several human tumours and promotes oncogenic phenotype. Its expression has been correlated to poor prognosis in HCC. In this study, we aimed to elucidate the basis for the effects of CRIPTO in HCC. We investigated CRIPTO expression levels in three cohorts of clinical cirrhotic and HCC specimens. We addressed the role of CRIPTO in hepatic tumourigenesis using Cre-loxP-controlled lentiviral vectors expressing CRIPTO in cell line-derived xenografts. Responses to standard treatments (sorafenib, doxorubicin) were assessed directly on xenograft-derived ex vivo tumour slices. CRIPTO-overexpressing patient-derived xenografts were established and used for ex vivo drug response assays. The effects of sorafenib and doxorubicin treatment in combination with a CRIPTO pathway inhibitor were tested in ex vivo cultures of xenograft models and 3D cultures. CRIPTO protein was found highly expressed in human cirrhosis and hepatocellular carcinoma specimens but not in those of healthy participants. Stable overexpression of CRIPTO in human HepG2 cells caused epithelial-to-mesenchymal transition, increased expression of cancer stem cell markers, and enhanced cell proliferation and migration. HepG2-CRIPTO cells formed tumours when injected into immune-compromised mice, whereas HepG2 cells lacking stable CRIPTO overexpression did not. High-level CRIPTO expression in xenograft models was associated with resistance to sorafenib, which could be modulated using a CRIPTO pathway inhibitor in ex vivo tumour slices. Our data suggest that a subgroup of CRIPTO-expressing HCC patients may benefit from a combinatorial treatment scheme and that sorafenib resistance may be circumvented by inhibition of the CRIPTO pathway. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Metastasis is a main cause of death in prostate cancer (PCa). To dissect the molecular cues from cancer cell-microenvironment interaction that drive metastatic cascade, bone metastatic PCa cells were intravenously implanted into zebrafish embryos and mice tibia forming metastatic lesions. Transcriptomic analysis showed an elevated expression of stemness genes, pro-inflammatory cytokines and TGF-β family member Activin A in the cancer cells at metastatic onset in both animal models. Consistently, analysis of clinical datasets revealed that the expression of Activin A is specifically elevated in metastases and correlates with poor prognosis in stratified high-risk PCa patients. It is further unveiled that the microenvironment induced Activin A expression by NF-κB activation. The elevated level of Activin A enhanced the invasive ALDHhi CSC-like phenotypes and PCa proliferation by activation of Smad and ERK1/2 signaling driving metastasis. Suppression of Activin A or Activin receptor significantly reduced the CSC-like subpopulation, invasion, metastatic growth, and bone lesion formation in zebrafish and mice xenografts, suggesting a functional role of NF-κB-dependent Activin A in PCa metastasis. Overall, our study demonstrates that human PCa cells can display a comparable response with the microenvironment in zebrafish and mice xenografts. Combining both animal models, we uncovered the microenvironment-dependent activin signaling as an essential driver in PCa metastasis with therapeutic potential.

Figure S2. miR-221-5p exerts tumor suppressive function on PCa cell lines in vitro. (a) Cell cycle of wild-type PC-3 M-Pro4luc2 and C4–2 cells was analysed by PI staining at 0 h, 16 h and 48 h after release starvation. The frequency of cells in G1, S and G2/M phase was quantified by Dean-Jett-Fox model. Results of n = 2 technical replicates were analysed by unpaired, two-tailed t-test. * p

Table S1. Body weight and tumor size of mouse in vivo experiment. Table S2: Primer sequences. (DOCX 16 kb)

Figure S1. miR-221 is downregulated during PCa progression in patient samples. (a) microRNA dataset analysis in GSE21036 dataset. Expression of miR-221-3p in 113 PCa samples compared to 28 normal tissue samples. Fold change (FC = 0.85) was calculated and data was analysed by t-test. (b) Analysis of miR-221-3p expression in metastatic PCa samples compared to primary PCa tissue in GSE21036 dataset. Fold change (FC = 0.74) was calculated and data analysed by t-test. (c) Data of GSE21036 was grouped according to the indicated Gleason score (GS) and miR-221-3p expression analysed. Adjusted p-value was calculated by one-way ANOVA. (d) miR-221-3p expression (GSE21036) was analysed in samples grouped for pathological stage (T). Data was analysed by one-way ANOVA. (e) Analysis of miR-221-5p expression in samples grouped for clinical stage (T) in GSE21036 dataset. Data was analysed by one-way ANOVA. (f) Analysis of miR-221-3p expression in sample groups according to clinical stage (T). Data of GSE21036 was analysed by one-way ANOVA. (PDF 280 kb)

Figure S6. Differential EMT marker expression at early and late time point post transfection. Expression of selected EMT markers in PC-3 M-Pro4luc2 cells at 72 h and 2 weeks post transfection. LOG difference was calculated as LOG(2-ΔΔCt) normalised to scrambled. Results of n = 3 technical triplicates are shown and data were analysed by unpaired, two-tailed t-test. * p

Figure S4. Orthotopic tumor growth is reduced by miR-221-5p overexpression in vivo. (a) miR-221-5p expression in PC-3 M-Pro4luc2 cells 48 h post transfection, prior to intraprostatic injection in mice. Data are shown as LOG difference (LOG(2-ΔΔCt)) to scrambled and were analysed by unpaired, two-tailed t-test. **** p

Figure S3. miR-221-5p overexpression affects plasticity of PCa cells and EMT marker expression. (a) EMT marker expression in PC-3 M-Pro4luc2 cell overexpressing miR-221-5p or negative scrambled control. Data of three independent experiments (n = 3) are shown as LOG difference to control (LOG(2-ΔΔCt)) and were analysed by paired, two-tailed t-test. * p

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. Despite increasing treatment options for this disease, prognosis remains poor. CRIPTO (TDGF1) protein is expressed at high levels in several human tumours and promotes oncogenic phenotype. Its expression has been correlated to poor prognosis in HCC. In this study, we aimed to elucidate the basis for the effects of CRIPTO in HCC. We investigated CRIPTO expression levels in three cohorts of clinical cirrhotic and HCC specimens. We addressed the role of CRIPTO in hepatic tumourigenesis using Cre-loxP-controlled lentiviral vectors expressing CRIPTO in cell line-derived xenografts. Responses to standard treatments (sorafenib, doxorubicin) were assessed directly on xenograft-derived ex vivo tumour slices. CRIPTO-overexpressing patient-derived xenografts were established and used for ex vivo drug response assays. The effects of sorafenib and doxorubicin treatment in combination with a CRIPTO pathway inhibitor were tested in ex vivo cultures of xenograft models and 3D cultures. CRIPTO protein was found highly expressed in human cirrhosis and hepatocellular carcinoma specimens but not in those of healthy participants. Stable overexpression of CRIPTO in human HepG2 cells caused epithelial-to-mesenchymal transition, increased expression of cancer stem cell markers, and enhanced cell proliferation and migration. HepG2-CRIPTO cells formed tumours when injected into immune-compromised mice, whereas HepG2 cells lacking stable CRIPTO overexpression did not. High-level CRIPTO expression in xenograft models was associated with resistance to sorafenib, which could be modulated using a CRIPTO pathway inhibitor in ex vivo tumour slices. Our data suggest that a subgroup of CRIPTO-expressing HCC patients may benefit from a combinatorial treatment scheme and that sorafenib resistance may be circumvented by inhibition of the CRIPTO pathway. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John WileySons, Ltd.

CRIPTO (CR-1, TDGF1) is a cell surface/secreted oncoprotein actively involved in development and cancer. Here, we report that high expression of CRIPTO correlates with poor survival in stratified risk groups of prostate cancer (PCa) patients. CRIPTO and its signaling partner glucose-regulated protein 78 (GRP78) are highly expressed in PCa metastases and display higher levels in the metastatic ALDH(high) sub-population of PC-3M-Pro4Luc2 PCa cells compared with non-metastatic ALDH(low). Coculture of the osteotropic PC-3M-Pro4Luc2 PCa cells with differentiated primary human osteoblasts induced CRIPTO and GRP78 expression in cancer cells and increases the size of the ALDH(high) sub-population. Additionally, CRIPTO or GRP78 knockdown decreases proliferation, migration, clonogenicity and the size of the metastasis-initiating ALDH(high) sub-population. CRIPTO knockdown reduces the invasion of PC-3M-Pro4Luc2 cells in zebrafish and inhibits bone metastasis in a preclinical mouse model. These results highlight a functional role for CRIPTO and GRP78 in PCa metastasis and suggest that targeting CRIPTO/GRP78 signaling may have significant therapeutic potential.

Lethal and incurable bone metastasis is one of the main causes of death in multiple types of cancer. A small subpopulation of cancer stem/progenitor-like cells (CSCs), also known as tumor-initiating cells from heterogenetic cancer is considered to mediate bone metastasis. Although over the past decades numerous studies have been performed in different types of cancer, it is still difficult to track small numbers of CSCs during the onset of metastasis. With use of noninvasive high-resolution imaging, transparent zebrafish embryos can be employed to dynamically visualize cancer progression and reciprocal interaction with stroma in a living organism. Recently we established a zebrafish CSC-xenograft model to visually and functionally analyze the role of CSCs and their interactions with the microenvironment at the onset of metastasis. Given the highly conserved human and zebrafish genome, transplanted human cancer cells are able to respond to zebrafish cytokines, modulate the zebrafish microenvironment, and take advantage of the zebrafish stroma during cancer progression. This chapter delineates the zebrafish CSC-xenograft model as a useful tool for both CSC biological study and anticancer drug screening.

The xenograft model, using the early life stages of the zebrafish, allows imaging of tumor cell behavior both on a single cell and whole organism level, over time, within a week. This robust and reproducible assay can be used as an intermediate step between in vitro techniques and the expensive, and time consuming, murine models of cancer invasion and metastasis.In this chapter, a detailed protocol to inject human cancer cells into the blood circulation of a zebrafish embryo is described; the engraftment procedure is then followed by visualization and quantification methods of tumor cell proliferation, invasion, and micrometastasis formation during subsequent larval development. Interaction with the host microenvironment is also considered.

Zebrafish embryos can be obtained for research purposes in large numbers at low cost and embryos develop externally in limited space, making them highly suitable for high-throughput cancer studies and drug screens. Non-invasive live imaging of various processes within the larvae is possible due to their transparency during development, and a multitude of available fluorescent transgenic reporter lines.To perform high-throughput studies, handling large amounts of embryos and larvae is required. With such high number of individuals, even minute tasks may become time-consuming and arduous. In this chapter, an overview is given of the developments in the automation of various steps of large scale zebrafish cancer research for discovering important cancer pathways and drugs for the treatment of human disease. The focus lies on various tools developed for cancer cell implantation, embryo handling and sorting, microfluidic systems for imaging and drug treatment, and image acquisition and analysis. Examples will be given of employment of these technologies within the fields of toxicology research and cancer research.

Incurable bone metastasis is a main cause of death in prostate cancer. Metastasis is believed to be initiated by a small subpopulation of cancer cells termed tumor initiating cells (TICs) or cancer stem-like cells (CSCs). One of the key steps of the metastatic cascade is metastatic colonization of circulating tumor cells/clusters (CTCs) in a distant niche from circulation. From clinical database analysis, a significant up regulation of genes related to cell-ECM interaction was observed in prostate cancer patients derived CSCs, implying that the enhanced focal adhesion ability and its correlated cytoskeleton remodeling of CSCs plays a critical role in regulating metastatic colonization. However, due to a lack of tools to visualize the low amount of disseminated CSCs at high imaging resolution, it remains unknown how those cells behave when they interact with the stromal compartment at the inception of metastatic colonization. Hereby, we intravenously injected osteotropic prostate cancer cells with fluorescently-labeled F-actin into transparent zebrafish embryos to monitor single cell dynamics during metastatic colonization. In this model we observed a highly dynamic actin-based cytoskeletal remodeling in ALDHhi CSCs that controls extravasation and metastatic colonization. Transcriptome analysis revealed that this cytoskeleton remodeling was regulated by the focal adhesion factors: Integrinβ1 and integrin linked kinase (ILK). Genetic targeting of Integrinβ1 and ILK significantly inhibited expression of pluripotency genes, ALDH activity, metastatic colonization and outgrowth, suggesting the Integrinβ1-ILK axis as a key metastatic regulator that drives stem-like properties of CSCs during metastasis. Further in vitro and in vivo analysis showed that the Integrinβ1-ILK axis controls prostate cancer stem plasticity by generating contractile force through small GTPase-CDC42 and invadopodia regulator-N-wasp when the cells physically interact with the ECM. This mechanical transduction further activates mechanical sensor YAP/TAZ in a hippo-independent manner and promotes the expression of pluripotency genes that support metastatic initiation. Interference with this process by blocking CDC42-N-wasp signaling significantly attenuated mechanical transduction, impaired YAP/TAZ nuclear translocation and eventually inhibited metastatic initiation. Taken together, this study indicates a fundamental role of the mechanical transduction between ECMs and CSCs mediated by integrin/ILK dependent cytoskeleton remodeling at metastatic onset and outgrowth. Pharmacological targeting of this process may be a potent approach to attenuate the formation of prostate cancer metastasis on clinic. Citation Format: Lanpeng Chen, Stefano Coppola, Nick Landman, Arwin Groenewoud, Thomas Schmidt, B.Ewa Snaar-Jagalska. Mechanical transduction mediated by Integrin-ILK dependent actin dynamics drives stem-plasticity leading experimental metastatic colonization of prostate cancer leading experimental metastatic colonization of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 175.

Metastatic triple negative breast cancer (TNBC) can be considered as incurable endpoint in the progression of TNBC, prevention or treatment of metastasis therefore is of paramount importance. New insights into intra-tumoral heterogeneity have cast light on the existence of multiple functionally distinct cellular entities within one tumor, among others these cells can be divided based on their metastatic potential. The zebrafish provides us with the unique opportunity to study the first steps of metastatic initiation in great detail both microscopically and transcriptionally, on the side of both the host and cancer cells. Organotropic TNBC lines engrafted into this model recapitulate the tropism found in higher vertebrates. Using our zebrafish TNBC xenograft model we study these cellular sub-populations in vivo during metastatic initiation. These small metastatic lesions can be further analyzed ex vivo through the re-isolation of novel zebrafish specific organotropic cell lines. Re-engraftment of these greatly enhances the tumorigenic potential, and transcriptional analysis reveals increased EMT plasticity and an induction of stemness markers. Engraftment of these cells into murine tibia shows a significantly increased tumorigenic and osteolytic potential, further underscoring the critical role of this sub-population in cancer initiation. To determine the genetic drivers of this process we performed whole exome sequenced (NGS) of both, in vitro cultured cancer cells (maternal and re-isolated) and micro-dissected tissue samples derived from in situ zebrafish xenograft material. We are currently in the process of evaluating the candidate genes underpinning the enhanced tumorigenic potential in both the zebrafish and murine models used. Citation Format: B.Ewa Snaar-Jagalska, Arwin Groenewoud, Letizia Astrologo, Lanpeng Chen, Marianna Kruithof-de Julio. Triple negative breast cancer cell heterogeneity in zebrafish-host microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 42.

Zebrafish embryos can be obtained for research purposes in large numbers at low cost and embryos develop externally in limited space, making them highly suitable for high-throughput cancer studies and drug screens. Non-invasive live imaging of various processes within the larvae is possible due to their transparency during development, and a multitude of available fluorescent transgenic reporter lines.

The xenograft model, using the early life stages of the zebrafish, allows imaging of tumor cell behavior both on a single cell and whole organism level, over time, within a week. This robust and reproducible assay can be used as an intermediate step between in vitro techniques and the expensive, and time consuming, murine models of cancer invasion and metastasis.In this chapter, a detailed protocol to inject human cancer cells into the blood circulation of a zebrafish embryo is described; the engraftment procedure is then followed by visualization and quantification methods of tumor cell proliferation, invasion, and micrometastasis formation during subsequent larval development. Interaction with the host microenvironment is also considered.

Tumor angiogenesis and metastasis are key steps of cancer progression. In vitro and animal model studies have contributed to partially elucidating the mechanisms involved in these processes and in developing therapies. Besides the improvements in fundamental research and the optimization of therapeutic regimes, cancer still remains a major health threatening condition and therefore the development of new models is needed. The zebrafish is a powerful tool to study tumor angiogenesis and metastasis, because it allows the visualization of fluorescently labelled tumor cells inducing vessel remodeling, disseminating and invading surrounding tissues in a whole transparent embryo. The embryo model has also been used to address the contribution of the tumor stroma in sustaining tumor angiogenesis and spreading. Simultaneously, new anti-angiogenic drugs and compounds affecting malignant cell survival and migration can be tested by simply adding the compound into the water of living embryos. Therefore the zebrafish model offers the opportunity to gain more knowledge on cancer angiogenesis and metastasis in vivo with the final aim of providing new translational insights into therapeutic approaches to help patients.

e23008 Background: Despite the advances in cancer therapy, when Prostate Cancer (PCa) progress to castration resistant phase, patients develop incurable bone metastases. Understanding the processes that regulate homing and survival of metastatic cancer cells in the bone is crucial for the identification of new therapies. TGF-β signaling plays a major role in bone remodeling and according to the “vicious cycle hypothesis” is a master regulator of maintenance of prostate cancer cells in lytic bone lesions. microRNAs (miRs) are a class of small non-coding RNAs that regulates many biological process. miR-221 expression has been previously associated with prostate cancer progression. Here we studied the effect of miR-221 on TGF-β signaling and the impact of miR-221 on tumor growth in vivo Methods: miR-221 was overexpressed in PC3 and RWPE-1 cells and PMEPA was monitored by RT-qPCR and western blot. Expression of miR-221 in PC3 and RWPE-1 cells was assessed by RT-qPCR. Luciferase assay was used to investigate the interaction between miR-221 and PMEPA1. For zebrafish experiment, fluorescently labelled PC-3M-Pro4 cells overexpressing miR-221 were injected in the Duct of Cuvier (DC) of zebrafish embryos. Results: miR-221 overexpression resulted in decreased PMEPA1 mRNA and protein in PC-3 cells. Luciferase reporter assay indicated that miR-221 can directly interact with PMEPA1 3’ UTR. We show an enhancement of the proliferative effect of TGF-β in PC-3 cells, following miR-221 overexpression. In conclusion, we observed increased Smad2 activation upon TGF-β treatment in miR-221 overexpressing PC-3 cells. Inoculation of PC-3M-Pro4 cells overexpressing miR-221 in the DC of zebrafish embryos resulted reduced tumor burden compared to control. Finally, we observed inverse correlation between miR-221 and PMEPA1 expression in normal vs. tumor tissue collected from PCa patients. Conclusions: Our results indicate that miR-221 is a regulator of TGF-β signaling via modulation of PMEPA1 and miR-221 overexpression can reduce tumor growth in vivo.

Figure S2. miR-221-5p exerts tumor suppressive function on PCa cell lines in vitro. (a) Cell cycle of wild-type PC-3 M-Pro4luc2 and C4–2 cells was analysed by PI staining at 0 h, 16 h and 48 h after release starvation. The frequency of cells in G1, S and G2/M phase was quantified by Dean-Jett-Fox model. Results of n = 2 technical replicates were analysed by unpaired, two-tailed t-test. * p

Figure S5. Morphology of orthotopically grown PC-3 M-Pro4luc2 tumors is not affected by miR-221-5p overexpression. H&E and immunofluorescence imaging of all dissected tumors. The expression of proliferation marker PCNA (green), apoptosis marker cl. casp-3 (red), tumor cell marker panCK (red) and stroma marker α-SMA (green) was analysed by immunofluorescence staining. Representative images of each mouse are shown. (PDF 988 kb)

Figure S5. Morphology of orthotopically grown PC-3 M-Pro4luc2 tumors is not affected by miR-221-5p overexpression. H&E and immunofluorescence imaging of all dissected tumors. The expression of proliferation marker PCNA (green), apoptosis marker cl. casp-3 (red), tumor cell marker panCK (red) and stroma marker α-SMA (green) was analysed by immunofluorescence staining. Representative images of each mouse are shown. (PDF 988 kb)