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The use of three-dimensional (3D) bioprinting has remained at the forefront of tissue engineering and has recently been employed for generating bioprinted solid tumors to be used as cancer models to test therapeutics. In pediatrics, neural crest-derived tumors are the most common type of extracranial solid tumors. There are only a few tumor-specific therapies that directly target these tumors, and the lack of new therapies remains detrimental to improving the outcomes for these patients. The absence of more efficacious therapies for pediatric solid tumors, in general, may be due to the inability of the currently employed preclinical models to recapitulate the solid tumor phenotype. In this study, we utilized 3D bioprinting to generate neural crest-derived solid tumors. The bioprinted tumors consisted of cells from established cell lines and patient-derived xenograft tumors mixed with a 6% gelatin/1% sodium alginate bioink. The viability and morphology of the bioprints were analyzed via bioluminescence and immunohisto chemistry, respectively. We compared the bioprints to traditional two-dimensional (2D) cell culture under conditions such as hypoxia and therapeutics. We successfully produced viable neural crest-derived tumors that retained the histology and immunostaining characteristics of the original parent tumors. The bioprinted tumors propagated in culture and grew in orthotopic murine models. Furthermore, compared to cells grown in traditional 2D culture, the bioprinted tumors were resistant to hypoxia and chemotherapeutics, suggesting that the bioprints exhibited a phenotype that is consistent with that seen clinically in solid tumors, thus potentially making this model superior to traditional 2D culture for preclinical investigations. Future applications of this technology entail the potential to rapidly print pediatric solid tumors for use in high-throughput drug studies, expediting the identification of novel, individualized therapies.

Cancer continues to be a significant cause of non-traumatic pediatric mortality. Diagnosis of pediatric solid tumors is paramount to prescribing the correct treatment regimen. Recent efforts have focused on non-invasive methods to obtain tumor tissues, but one of the challenges encountered is the ability to obtain an adequate amount of viable tissue. In this study, a wireless, inductor-capacitor (LC) sensor was employed to detect relative permittivity of pediatric tumor tissues. There is a comparison of resonant frequencies of tumor tissues between live versus dead tissues, the primary tumor tissue versus tissue from the organs of origin or metastasis, and treated versus untreated tumors. The results show significant shifts in resonant frequencies between the comparison groups. Dead tissues demonstrated a significant shift in resonant frequencies compared to alive tissues. There were significant differences between the resonant frequencies of normal tissues versus tumor tissues. Resonant frequencies were also significantly different between primary tumors compared to their respective metastases. These data indicate that there are potential clinical applications of LC technology in the detection and diagnosis of pediatric solid tumors.

Patients presenting with metastatic hepatoblastoma have limited treatment options and survival rates as low as 25%. We previously demonstrated that Proviral Integration site in Maloney murine leukemia virus 3 (PIM3) kinase promotes tumorigenesis and cancer cell stemness in hepatoblastoma. In this study, we assessed the role of PIM3 kinase in promoting hepatoblastoma metastasis. We utilized a tail vein injection model of metastasis to evaluate the effect of CRISPR/Cas9-mediated PIM3 knockout, stable overexpression of PIM3, and pharmacologic PIM inhibition on the formation of lung metastasis. In vivo studies revealed PIM3 knockout impaired the formation of lung metastasis: 5 out of 6 mice injected with wild type hepatoblastoma cells developed lung metastasis while none of the 7 mice injected with PIM3 knockout hepatoblastoma cells developed lung metastasis. PIM3 overexpression in hepatoblastoma increased the pulmonary metastatic burden in mice and mechanistically, upregulated the phosphorylation and cell surface expression of CXCR4, a key receptor in the progression of cancer cell metastasis. CXCR4 blockade with AMD3100 decreased the metastatic phenotype of PIM3 overexpressing cells, indicating that CXCR4 contributed to PIM3's promotion of hepatoblastoma metastasis. Clinically, PIM3 expression correlated positively with CXCR4 expression in primary hepatoblastoma tissues. In conclusion, we have shown PIM3 kinase promotes the metastatic phenotype of hepatoblastoma cells through upregulation of CXCR4 cell surface expression and these findings suggest that targeting PIM3 kinase may provide a novel therapeutic strategy for metastatic hepatoblastoma.

Introduction Retinoic acid (RA) is a differentiating agent utilized as maintenance therapy for high-risk neuroblastoma (NB), but associated toxicities limit its use. We have previously shown that a non-toxic, novel rexinoid, 9-cis-UAB30 (UAB30), decreased NB cell proliferation and in vivo tumor growth. A second generation, mono-methylated compound, 6-Methyl-UAB30 (6-Me), has been recently designed having greater potency compared with UAB30. In the current study, we hypothesized that 6-Me would inhibit NB cell proliferation and survival and induce differentiation and cell-cycle arrest. Methods Proliferation and viability were measured in four human NB cell lines following treatment with UAB30 or 6-Me. Cell-cycle was analyzed and tumor cell stemness was evaluated with extreme limiting dilution assays and immunoblotting for expression of stem cell markers. A xenograft murine model was utilized to study the effects of 6-Me in vivo. Results Treatment with 6-Me led to decreased proliferation and viability, induced cell cycle arrest, and increased neurite outgrowth, indicating differentiation of surviving cells. Furthermore, treatment with 6-Me decreased tumorsphere formation and expression of stem cell markers. Finally, inhibition of tumor growth and increased animal survival was observed in vivo following treatment with 6-Me. Conclusion These results indicate a potential therapeutic role for this novel rexinoid in neuroblastoma treatment.

Cancer is the leading cause of death by disease in children, and over 15% of pediatric cancer-related mortalities are due to neuroblastoma. Current treatment options for neuroblastoma remain suboptimal as they often have significant toxicities, are associated with long-term side effects, and result in disease relapse in over half of children with high-risk disease. There is a dire need for new therapies, and oncolytic viruses may represent an effective solution. Oncolytic viruses attack tumor cells in two ways: direct infection of tumor cells leading to cytolysis, and production of a debris field that stimulates an anti-tumor immune response. Our group has previously shown that M002, an oncolytic herpes simplex virus (oHSV), genetically engineered to express murine interleukin-12 (mIL-12), was effective at targeting and killing long term passage tumor cell lines. In the current study, we investigated M002 in three neuroblastoma patient-derived xenografts (PDXs). PDXs better recapitulate the human condition, and these studies were designed to gather robust data for translation to a clinical trial. We found that all three PDXs expressed viral entry receptors, and that the virus actively replicated in the cells. M002 caused significant tumor cell death in 2D culture and 3D bioprinted tumor models. Finally, the PDXs displayed variable susceptibility to M002, with a more profound effect on high-risk neuroblastoma PDXs compared to low-risk PDX. These findings validate the importance of incorporating PDXs for preclinical testing of oncolytic viral therapeutics and showcase a novel technique, 3D bioprinting, to test therapies in PDXs. Collectively, our data indicate that oHSVs effectively target high-risk neuroblastoma, and support the advancement of this therapy to the clinical setting.

BACKGROUND/PURPOSE: Metastatic hepatoblastoma continues to pose a significant treatment challenge, primarily because the precise mechanisms involved in metastasis are not fully understood, making cell lines and preclinical models that depict the progression of disease and metastasis-related biology paramount. We aimed to generate and characterize a metastatic hepatoblastoma cell line to create a model for investigation of the molecular mechanisms associated with metastasis. MATERIALS/METHODS: Using a murine model of serial tail vein injections of the human hepatoblastoma HuH6 cell line, non-invasive bioluminescence imaging, and dissociation of metastatic pulmonary lesions, we successfully established and characterized the metastatic human hepatoblastoma cell line, HLM_3. RESULTS: The HLM_3 cells exhibited enhanced tumorigenicity and invasiveness, both in vitro and in vivo compared to the parent HuH6 cell line. Moreover, HLM_3 metastatic hepatoblastoma cells exhibited a stem cell-like phenotype and were more resistant to the standard chemotherapeutic cisplatin. CONCLUSION: This newly described metastatic hepatoblastoma cell line offers a novel tool to study mechanisms of tumor metastasis and evaluate new therapeutic strategies for metastatic hepatoblastoma.

The archaeological rescue excavations occasioned by the construction of the Lot no. 1 of the Highway no. 10, Sebeș-Turda, highlighted an unexpected Bronze Age necropolis belonging to Wietenberg Culture. The Bronze Age necropolis was delimited between 6+600 and 6+650 kilometers, and located on the southwestern part of the Site no. 6, right above the early Vinča settlement defensive trenches (Fig. 1-2). With 74 excavated funerary complexes, the partially excavated Wietenberg cemetery is one of the largest in Transylvania. According the radiocarbon data presented in this study, the necropolis was used in the Middle Bronze Age (1780-1690 cal. BC), associated with the Wietenberg culture (phase II), and is contemporary with the end of the cemetery from Sebeș-Între Răstoace located approximately 7.5 km South - South-West, on the Sebeș River terraces.

Highlights • Rare, high grade neuroendocrine-like pediatric tumor has TYRO3 and ALK mutations. • Crizotinib downregulates STAT3 and ERK1/2 signaling in tumor. • In vivo analysis demonstrated crizotinib resistance. • Crizotinib resistant cells have upregulation of PDGFRß signaling. • PDGFRß inhibition overrides resistance., Patient-derived xenografts provide significant advantages over long-term passage cell lines when investigating efficacy of treatments for solid tumors. Our laboratory encountered a high-grade, metastatic, neuroendocrine-like tumor from a pediatric patient that presented with a unique genetic profile. In particular, mutations in TYRO3 and ALK were identified. We established a human patient-derived xenoline (PDX) of this tumor for use in the current study. We investigated the effect of crizotinib, a chemotherapeutic known to effectively target both TYRO3 and ALK mutations. Crizotinib effectively decreased viability, proliferation, growth, and the metastatic properties of the PDX tumor through downregulation of STAT3 signaling, but expression of PDGFRß was increased. Sunitinib is a small molecule inhibitor of PDGFRß and was studied in this PDX independently and in combination with crizotinib. Sunitinib alone decreased viability, proliferation, and growth in vitro and decreased tumor growth in vivo. In combination, sunitinib was able to overcome potential crizotinib-induced resistance through downregulation of ERK 1/2 activity and PDGFRß receptor expression; consequently, tumor growth was significantly decreased both in vitro and in vivo. Through the use of the PDX, it was possible to identify crizotinib as a less effective therapeutic for this tumor and suggest that targeting PDGFRß would be more effective. These findings may translate to other solid tumors that present with the same genetic mutations.

As archaeologists expand the accessibility of legacy data, they have an opportunity to use these datasets to design future research. We argue that legacy data can be a critical resource to help predict characteristics of sites and socioeconomic systems. In this article, we present a combined geographic information system (GIS) and network analysis methodology that turns site location data into testable hypotheses about site characteristics and the organization of regional settlement systems. We demonstrate the utility of this approach with a case study: Bronze Age (2700–1100 BC) settlement patterns in the mining region of Hunedoara in southwest Transylvania, Romania. We leverage unsystematically collected site location information in legacy datasets to develop testable predictions about sites, regional networks, and socioeconomic systems that can be evaluated through future systematic surveys and large-scale excavations. Such testable hypotheses can inform archaeological research design by providing a quantitative basis for determining where to focus research efforts and can also help secure funding and fieldwork permits. The method developed here can be applied in diverse archaeological contexts to reinvigorate legacy data as part of future archaeological research design.

Aggressive therapies for patients with metastatic Wilms tumor (WT) with subsequent severe late effects warrant the search for novel therapies. The role of focal adhesion kinase (FAK), a non-receptor tyrosine kinase important in pediatric solid tumor development and progression, has not been examined in metastatic WT. Using a novel patient-derived xenograft (PDX) of a primary and matched, isogenic, metastatic WT, the hypothesis of the current study was that FAK would contribute to metastatic WT and small molecule inhibition would decrease tumor growth. Immunohistochemical staining, immunoblotting, cell viability and proliferation assays, cell cycle analysis, and cellular motility and attachment-independent growth assays were performed. FAK was present and phosphorylated in both WT PDXs and in the human samples from which they were derived. FAK inhibition decreased cellular survival, proliferation, and cell cycle progression in both PDXs but only significantly decreased migration, invasion, and attachment-independent growth in the primary WT PDX. Kinomic profiling revealed that platelet-derived growth factor receptor beta (PDGFRβ) may be affected by FAK inhibition in WT. Pharmacologic inhibition of FAK and PDGFRβ was synergistic in primary WT PDX cells. These findings broaden the knowledge of metastatic WT and support further investigations on the potential use of FAK and PDGFRβ inhibitors.

Hafting is an important part of lithic technology that can increase our understanding of socioeconomic behavior in the past. In this article, we develop a holistic approach to studying hafting by using the concept of curation within a broader assessment of lithic technological organization in early villages. Early villages were loci of socioeconomic transformation as part of the shift from mobile foraging to more sedentary cultivation lifeways. We suggest that an examination of hafting can provide new insights into how early villagers negotiated technological requirements, economic decision making, and social interactions in these novel contexts. As a case study, we develop a curation index and apply it to an archaeological context of hafted and unhafted pointed tools from the early Neolithic village of Dhra’, Jordan. This curation index allows for a discussion of the technological, economic, and social dimensions of hafting strategies at Dhra’. The presence of multiple hafting traditions within early Neolithic villages of Southwest Asia is evidence of persistent social segmentation despite food storage and ritual practices that emphasized communal integration. Through the lens of lithic technological organization, we demonstrate that hafting and curation patterns can increase our understanding of technological, economic, and social strategies in early villages.

Simple Summary Neuroblastoma is the most common extra-cranial tumor in children and despite medical advancements in cancer treatment, five-year survival for high-risk neuroblastoma remains less than 50%. Investigation of the mechanisms responsible for aggressive disease is necessary to identify novel therapeutic targets and improve survival. Serine-threonine kinase receptor associated protein (STRAP) is upregulated in several malignancies and plays an important role in tumor growth and metastasis. The role of STRAP in pediatric malignancies and specifically in neuroblastoma has not been explored. We sought to determine whether STRAP functions assisted to promote the malignant phenotype in neuroblastoma and could provide a potential target for future therapies. Abstract Background: Serine-threonine kinase receptor-associated protein (STRAP) plays an important role in neural development but also in tumor growth. Neuroblastoma, a tumor of neural crest origin, is the most common extracranial solid malignancy of childhood and it continues to carry a poor prognosis. The recent discovery of the role of STRAP in another pediatric solid tumor, osteosarcoma, and the known function of STRAP in neural development, led us to investigate the role of STRAP in neuroblastoma tumorigenesis. Methods: STRAP protein expression was abrogated in two human neuroblastoma cell lines, SK-N-AS and SK-N-BE(2), using transient knockdown with siRNA, stable knockdown with shRNA lentiviral transfection, and CRISPR-Cas9 genetic knockout. STRAP knockdown and knockout cells were examined for phenotypic alterations in vitro and tumor growth in vivo. Results: Cell proliferation, motility, and growth were significantly decreased in STRAP knockout compared to wild-type cells. Indicators of stemness, including mRNA abundance of common stem cell markers Oct4, Nanog, and Nestin, the percentage of cells expressing CD133 on their surface, and the ability to form tumorspheres were significantly decreased in the STRAP KO cells. In vivo, STRAP knockout cells formed tumors less readily than wild-type tumor cells. Conclusion: These novel findings demonstrated that STRAP plays a role in tumorigenesis and maintenance of neuroblastoma stemness.

Despite increasing incidence, treatment for hepatoblastoma has not changed significantly over the past 20 years. Chemotherapeutic strategies continue to rely on cisplatin, as it remains the most active single agent against hepatoblastoma. However, chemoresistance remains a significant challenge with 54–80% of patients developing resistance to chemotherapy after 4–5 cycles of treatment. Stem cell-like cancer cells (SCLCCs) are a subset of cells thought to play a role in chemoresistance and disease recurrence. We have previously demonstrated that Proviral Integration site for Moloney murine leukemia virus (PIM) kinases, specifically PIM3, play a role in hepatoblastoma cell proliferation and tumor growth and maintain the SCLCC phenotype. Here, we describe the development of a cisplatin-resistant hepatoblastoma xenograft model of the human HuH6 cell line and a patient-derived xenograft, COA67. We provide evidence that these cisplatin-resistant cells are enriched for SCLCCs and express PIM3 at higher levels than cisplatin-naïve cells. We demonstrate that PIM inhibition with AZD1208 sensitizes cisplatin-resistant hepatoblastoma cells to cisplatin, enhances cisplatin-mediated apoptosis, and decreases the SCLCC phenotype seen with cisplatin resistance. Together, these findings indicate that PIM inhibition may be a promising adjunct in the treatment of hepatoblastoma to effectively target SCLCCs and potentially decrease chemoresistance and subsequent disease relapse.

Simple Summary Children with high-risk neuroblastoma have limited therapeutic options poor survival rates. The neuroblastoma tumor microenvironment contributes the lack of response to many interventions so innovative methods are needed to study the effects of the tumor microenvironment on new therapies. In this manuscript, we review the current literature related to the components of the tumor microenvironment and to the use of three-dimensional printing as modality to study cancer. This review highlights the potential for using three-dimensional printing to create an artificial tumor microenvironment in the presence of neuroblastoma to provide improved preclinical testing of novel therapies. Abstract In the quest to advance neuroblastoma therapeutics, there is a need to have a deeper understanding of the tumor microenvironment (TME). From extracellular matrix proteins to tumor associated macrophages, the TME is a robust and diverse network functioning in symbiosis with the solid tumor. Herein, we review the major components of the TME including the extracellular matrix, cytokines, immune cells, and vasculature that support a more aggressive neuroblastoma phenotype and encumber current therapeutic interventions. Contemporary treatments for neuroblastoma are the result of traditional two-dimensional culture studies and in vivo models that have been translated to clinical trials. These pre-clinical studies are costly, time consuming, and neglect the study of cofounding factors such as the contributions of the TME. Three-dimensional (3D) bioprinting has become a novel approach to studying adult cancers and is just now incorporating portions of the TME and advancing to study pediatric solid. We review the methods of 3D bioprinting, how researchers have included TME pieces into the prints, and highlight present studies using neuroblastoma. Ultimately, incorporating the elements of the TME that affect neuroblastoma responses to therapy will improve the development of innovative and novel treatments. The use of 3D bioprinting to achieve this aim will prove useful in developing optimal therapies for children with neuroblastoma.

Background: Current chemotherapy regimens for hepatoblastoma include platinum-based combinations that have remained virtually unchanged over the last twenty years. The overall survival for children with advanced stage or recurrent disease utilizing these regimens remains poor. We have previously shown that Proviral Integration site for Moloney murine leukemia virus (PIM) kinases act as oncoproteins promoting hepatoblastoma cell survival and the cancer stem cell phenotype, which may contribute to chemoresistance. We hypothesized that PIM inhibition utilizing …

Background: Serine-threonine kinase receptor associated protein (STRAP) is a scaffolding protein that has been implicated in tumor growth and metastasis. The role of STRAP in neuroblastoma (NB) has not been explored. Stem cell-like cancer cells (SCLCCs) are believed to be responsible for tumor cell self-renewal and cancer recurrence. We sought to determine the role of STRAP in neuroblastoma SCLCCs maintenance. …

In this introduction to the thematic issue Living and Dying in Mountain Landscapes, we develop an analytical framework for the bioarchaeology and mortuary archaeology of highland landscapes. We highlight new theoretical, methodological, and comparative contributions to the anthropological study of upland spaces. Theoretical contributions include examining identity, connectivity, and adaptation from an explicitly biocultural perspective. By bridging the biological anthropological focus on the somatic with an archaeological focus on the long term, bioarchaeology allows for the development of an embodied understanding of “marginal” highland environments, investigating how such landscapes shape and are shaped by human action over time. Recent advances in bioarchaeological methods, including isotopic analyses of mobility and diet and ancient DNA studies of kinship and relatedness, are combined with traditional osteological examinations of age, sex, ancestry, and disease to reconstruct the lifeways of mountain communities. These methodological advances take advantage of the topographical, geological, and ecological diversity of mountain landscapes. Finally, a comparative bioarchaeology of upland and lowland communities across space and time provides a deeper understanding of highland adaptations and identities. The papers share a number of unifying themes, including the impact of mountain landscapes on channeling resource control, creating or mediating diverse identities, and the importance of interdisciplinary investigations for developing an understanding of the relationship between people and place. As this issue demonstrates, the study of human remains must be situated within a holistic bioarchaeological approach to life and death in order to understand the dynamic relationships between people and the highland environments they occupy. En esta introducción a la cuestión temática 'Vivir y morir en paisajes de montaña’ desarrollamos un marco analítico para la bioarqueología y la arqueología mortuoria de los paisajes serranos. Destacamos un conjunto de nuevas contribuciones teóricas, metodológicas, y comparativas al estudio antropológico de estos paisajes. Entre ellas destacan los análisis de la identidad, la conectividad y la adaptación, todos aproximados desde una perspectiva explícitamente biocultural. Al unir el énfasis bioantropológico en lo somático con el interés arqueológico en la larga duración, la bioarqueología favorece una aproximación corporizada a los ambientes “marginales” de las tierras altas, investigando cómo estos paisajes moldean y son moldeados por la acción humana a lo largo del tiempo. Los recientes avances en métodos bioarqueológicos, como los análisis isotópicos de movilidad y dieta o los estudios de ADN antiguo sobre parentesco y otros principios de existencia compartida, se combinan con los tradicionales exámenes osteológicos de edad, sexo, ascendencia y enfermedad para reconstruirlas formas de vida de las comunidades de montaña. Estos avances metodológicos aprovechan la diversidad topográfica, geológica y ecológica de los entornos serranos. Finalmente, una comprensión más profunda de las adaptaciones e identidades de las serranías requiere de una aproximación bioarqueológica comparativa de las comunidades serranas y de las que habitan las tierras bajas a través del tiempo y del espacio. Todos estos trabajos comparten una serie de temas comunes: la manera en la que los paisajes de montaña canalizan las formas de control de los recursos, la creación o mediación de distintas identidades y la importancia de las investigaciones interdisciplinares para desarrollar una comprensión de la relación entre personas y lugares. Como se demuestra a lo largo de este número, el estudio de los restos humanos requiere de un enfoque bioarqueológico holístico de la vida y la muerte que permita comprender las relaciones dinámicas que se desarrollaron entre las personas y sus respectivos entornos serranos.

The Apuseni Mountains of southwestern Transylvania (Romania) are home to the richest gold and copper deposits in Europe, key resources that fueled the development of social complexity during the Bronze Age (ca. 2700–800 B.C.E.). This landscape encompasses a significant amount of topographic and ecological diversity, with upland landscapes incorporating major mineral deposits, forests, pastures, and salt springs, and lowland agropastoral landscapes abutting the major interregional Mureș River corridor. Local Early Bronze Age (ca. 2700–2000 B.C.E.) communities typically buried their dead in stone-covered tumuli in the uplands, though there are also examples of burial in lowland settlements. The relationship between upland and lowland mortuary contexts is an enduring question within the regional archaeological record. In this paper we present a case study that compares individuals from two sites: the lowland settlement of Alba Iulia-Pârâul Iovului and the upland cemetery of Meteș-La Meteșel. We ask whether there were differences between the uplands and the lowlands in terms of mortuary practices and eligibility for burial, or differences in the lived experience of pathology or trauma. Our results show that there are few significant differences between the two samples. Adults and subadults, as well as males and females, are represented at both sites, and levels of skeletal pathology are low, while dental insults are more frequent. We conclude by outlining a strategy for developing a regional bioarchaeology that will incorporate multiple lines of archaeological and bioarchaeological evidence and enhance our understanding of the biocultural dynamics of the region. Localizaţi în sud-vestul Transilvaniei (România), Munţii Apuseni adăpostesc cele mai bogate zăcăminte de aur şi cupru din Europa, resurse vitale care au alimentat dezvoltarea complexităţii sociale pe parcursul epocii bronzului (cca. 2700-800 BC). Acest peisaj încorporează o remarcabilă diversitate topografică şi ecologică, cu zonele înalte adăpostind principalele zăcăminte metalifere, păduri, păşuni montane, zonele agro-pastorale mai joase învecinându-se cu principalul coridor interregional al văii Mureşului. Comunităţile locale ale Bronzului timpuriu (cca. 2700-2000 BCE) din zona muntoasă îşi îngropau de obicei morţii în tumuli cu manta de piatră , dar există şi exemple de înmormântări în aşezările din zonele mai joase. Relaţia dintre contextele funerare din zonele înalte şi cele din zonele joase de relief rămâne o întrebare de durată în contextual arheologic regional. Studiul de faţă prezintă un studiu de caz care compară indivizi din două situri: aşezarea din zona joasă de la Alba Iulia-Pârâul Iovului şi cimitirul din zona muntoasă de la Meteş-La Meteşel. Sunt puse întrebări care privesc existenţa unor diferenţe între practicile funerare din zonele joase şi cele înalte sau în ceea ce priveşte experienţa trăită a traumelor şi patologiei. Rezultatele noastre indică puţine diferenţe semnificative între cele două loturi de probe. Adulţi şi subadulţi, de sex masculine sau feminin, sunt reprezentaţi în ambele situri, nivelurile de patologie osoasă fiind joase, în vreme ce afecţiunile dentare sunt mai frecvente. Concluzionăm prin evidenţierea unei strategii pentru dezvoltarea unei bioarheologii regionale, care va încorpora multiple linii de dovezi arheologice şi bioarheologice şi va înbunătăţi înţelegerea dinamicii bioculturale a regiunii.

Introduction The poor therapeutic efficacy seen with current treatments for neuroblastoma may be attributed to stem cell-like cancer cells (SCLCCs), a subpopulation of cancer cells associated with poor prognosis and disease recurrence. Retinoic acid (RA) is a differentiating agent used as maintenance therapy for high-risk neuroblastoma but nearly half of children treated with RA relapse. We hypothesized that 6-Methyl-UAB30 (6-Me), a second-generation rexinoid recently developed with a favorable toxicity profile compared to RA, would reduce cancer cell stemness in human neuroblastoma patient-derived xenografts (PDXs). Methods Cells from three neuroblastoma PDXs were treated with 6-Me and proliferation, viability, motility, and cell-cycle progression were assessed. CD133 expression, sphere formation, and mRNA abundance of stemness and differentiation markers were evaluated using flow cytometry, in vitro extreme limiting dilution analysis, and real-time PCR, respectively. Results Treatment with 6-Me decreased proliferation, viability, and motility, and induced cell-cycle arrest and differentiation in all three neuroblastoma PDXs. In addition, 6-Me treatment led to decreased CD133 expression, decreased sphere-forming ability, and decreased mRNA abundance of Oct4, Nanog, and Sox2, indicating decreased cancer cell stemness. Conclusions 6-Me decreased oncogenicity and reduced cancer cell stemness of neuroblastoma PDXs, warranting further exploration of 6-Me as potential novel therapy for neuroblastoma.

Background Novel therapies are needed for patients with hepatoblastoma because of an increasing incidence of disease and poor prognosis for advanced, refractory, and recurrent disease. PIM kinases promote tumorigenesis in hepatoblastoma. A novel PIM inhibitor, PIM447, has shown promise in inhibiting oncogenesis in hematologic and lymphoid malignancies. We hypothesized that PIM inhibition with PIM447 would result in decreased tumorigenesis in hepatoblastoma. Methods The effects of PIM447 on hepatoblastoma viability, proliferation, motility, apoptosis, and tumor cell stemness were assessed in HuH6, a human hepatoblastoma cell line, and COA67, a human hepatoblastoma patient-derived xenograft. Results PIM447 significantly decreased the viability, proliferation, and motility of HuH6 and COA67 cells. Apoptosis significantly increased following PIM447 treatment. PIM447 had a significant impact on tumor cell stemness as evidenced by decreased expression of CD133 and reduced ability of HuH6 and COA67 cells to form tumorspheres. Furthermore, combining PIM447 with cisplatin resulted in a significant decrease in cell viability compared to either treatment alone. Conclusion We showed that PIM447 inhibits oncogenesis and potentiates the effects of cisplatin in hepatoblastoma and, therefore, warrants further investigation as a potential therapeutic agent for hepatoblastoma.

Retinoic acid (RA) therapy has been utilized as maintenance therapy for high-risk neuroblastoma, but over half of patients treated with RA relapse. Neuroblastoma stem cell-like cancer cells (SCLCCs) are a subpopulation of cells characterized by the expression of the cell surface marker CD133 and are hypothesized to contribute to drug resistance and disease relapse. A novel rexinoid compound, 9-cis-UAB30 (UAB30), was developed having the same anti-tumor effects as RA but a more favorable toxicity profile. In the current study, we investigated the efficacy of UAB30 in neuroblastoma patient-derived xenografts (PDX). Two PDXs, COA3 and COA6, were utilized and alterations in the malignant phenotype were assessed following treatment with RA or UAB30. UAB30 significantly decreased proliferation, viability, and motility of both PDXs. UAB30 induced cell-cycle arrest as demonstrated by the significant increase in percentage of cells in G1 (COA6: 33.7 ± 0.7 vs. 43.3 ± 0.7%, control vs. UAB30) and decrease in percentage of cells in S phase (COA6: 44.7 ± 1.2 vs. 38.6 ± 1%, control vs. UAB30). UAB30 led to differentiation of PDX cells, as evidenced by the increase in neurite outgrowth and mRNA abundance of differentiation markers. CD133 expression was decreased by 40% in COA6 cells after UAB30. The ability to form tumorspheres and mRNA abundance of known stemness markers were also significantly decreased following treatment with UAB30, further indicating decreased cancer cell stemness. These results provide evidence that UAB30 decreased tumorigenicity and cancer cell stemness in neuroblastoma PDXs, warranting further exploration as therapy for high-risk neuroblastoma.

e22010 Background: The outcomes for children with osteosarcoma and synovial sarcoma remain poor and are even worse for metastatic or relapsed disease. Those who do survive frequently suffer from long-term toxicities from current standard-of-care therapies. It is evident novel therapeutics are warranted to improve the outcomes for patients with these difficult tumors. Stem cell-like cancer cells (SCLCCs) are a subpopulation of tumor cells thought to be responsible for treatment resistance, development of metastases and tumor recurrence, making the targeting of this cell population critical. We sought to evaluate the effect of lerociclib, a CDK4/6 inhibitor, on pediatric sarcoma cell stemness. Methods: We investigated two established human osteosarcoma cells lines (U2-OS and MG-63), two metastatic synovial sarcoma patient-derived xenografts (PDXs) (COA-30 and COA-79) and a metastatic epithelioid sarcoma PDX (COA-171). Cells were treated with the CDK4/6 inhibitor, lerociclib, at concentrations below the known LD50. Synovial sarcoma stemness markers Octamer-binding transcription factor 4 (Oct4), homeobox protein Nanog, SOX 2 and nestin were evaluated by qPCR. CD117, a marker of stemness in osteosarcoma cells, was examined by flow cytometry. Finally, long-term passaged U2-OS and MG-63 cells were placed in low attachment serum-free conditions, and tumorsphere formation was evaluated using extreme limiting dilution assay (ELDA) in all cell lines. Results: Lerociclib treatment significantly decreased abundance of Oct4 (by 54%), Nanog (by 29%), SOX 2 (by 60%) and nestin (by 63%) mRNA in COA-30 synovial sarcoma cells. Cell surface expression of CD117 decreased from 17% to 11% in MG-63 cells when treated with lerociclib 2μM. Treatment of all cell types with lerociclib led to significantly decreased tumorsphere formation (Table). Conclusions: Treatment with lerociclib led to a decrease in mRNA abundance in known synovial sarcoma stem cell markers, a decrease in CD117 cell surface expression in osteosarcoma cells and decreased the ability of cells to form tumorspheres. These findings indicate lerociclib leads to decreased sarcoma cell stemness, which plays a key role in tumor progression and recurrence and should be further investigated for potential translation to the clinical setting.[Table: see text]

Hepatoblastoma remains one of the most difficult childhood tumors to treat and is alarmingly understudied. We previously demonstrated that Proviral Insertion site in Maloney murine leukemia virus (PIM) kinases, specifically PIM3, are overexpressed in human hepatoblastoma cells and function to promote tumorigenesis. We aimed to use CRISPR/Cas9 gene editing with dual gRNAs to introduce large inactivating deletions in the PIM3 gene and achieve stable PIM3 knockout in the human hepatoblastoma cell line, HuH6. PIM3 knockout of hepatoblastoma cells led to significantly decreased proliferation, viability, and motility, inhibited cell-cycle progression, decreased tumor growth in a xenograft murine model, and increased animal survival. Analysis of RNA sequencing data revealed that PIM3 knockout downregulated expression of pro-migratory and pro-invasive genes and upregulated expression of genes involved in apoptosis and differentiation. Furthermore, PIM3 knockout decreased hepatoblastoma cancer cell stemness as evidenced by decreased tumorsphere formation, decreased mRNA abundance of stemness markers, and decreased cell surface expression of CD133, a marker of hepatoblastoma stem cell-like cancer cells. Reintroduction of PIM3 into PIM3 knockout cells rescued the malignant phenotype. Successful CRISPR/Cas9 knockout of PIM3 kinase in human hepatoblastoma cells confirmed the role of PIM3 in promoting hepatoblastoma tumorigenesis and cancer cell stemness.

Investigation of the mechanisms responsible for aggressive neuroblastoma and its poor prognosis is critical to identify novel therapeutic targets and improve survival. Enhancer of Zeste Homolog 2 (EZH2) is known to play a key role in supporting the malignant phenotype in several cancer types and knockdown of EZH2 has been shown to decrease tumorigenesis in neuroblastoma cells. We hypothesized that the EZH2 inhibitor, GSK343, would affect cell proliferation and viability in human neuroblastoma. We utilized four long-term passage neuroblastoma cell lines and two patient-derived xenolines (PDX) to investigate the effects of the EZH2 inhibitor, GSK343, on viability, motility, stemness and in vivo tumor growth. Immunoblotting confirmed target knockdown. Treatment with GSK343 led to significantly decreased neuroblastoma cell viability, migration and invasion, and stemness. GSK343 treatment of mice bearing SK-N-BE(2) neuroblastoma tumors resulted in a significant decrease in tumor growth compared to vehicle-treated animals. GSK343 decreased viability, and motility in long-term passage neuroblastoma cell lines and decreased stemness in neuroblastoma PDX cells. These data demonstrate that further investigation into the mechanisms responsible for the anti-tumor effects seen with EZH2 inhibitors in neuroblastoma cells is warranted.

The Bronze Age was a period of significant socio-economic transformation that gave rise to the first complex regional polities with institutionalized inequality in Europe. Communities in southwest Transylvania, a major source of gold, copper, and salt, played a critical role in this transformation. This article examines how socio-economic changes affected how people situated settlements in resource procurement zones during the Early and Middle Bronze Age (2700–1500 cal. BCE). Taking advantage of the heterogeneous distribution of natural resources across the landscape, a GIS catchment analysis of the orientation of settlements toward particular constellations of resources is presented. Our results show increased preference for access to high quality agricultural land and access to interregional trade through the Mureș River corridor over the course of the Bronze Age. Despite the increased importance of metal within Bronze Age economies, there is no evidence that Transylvanian communities placed their settlements to maximize their ability to contest or secure access to the metal ore sources in the Apuseni Mountains. The organization of settlement systems in the Bronze Age demonstrates that Transylvanian communities prioritized socio-economic institutions beyond metal procurement. This study demonstrates that tracing how humans situate themselves in variable landscapes can provide new insights into the conditions and mechanisms of social change.