Human Chordoma Research Articles

LncRNA XIST Promotes Growth of Human Chordoma Cells by Regulating miR-124-3p/iASPP Pathway.

IntroductionChordoma is a malignant primary bone tumor that is found in the spine and skull. X-inactive specific transcript (XIST) is a long non-coding RNA (lncRNA) is known to be involved in the development of various cancers, but its precise function and mechanism in human chordoma have not been elucidated. Here, we investigated the role of lncRNA XIST in chordoma progression.MethodsQuantitative real time-polymerase chain reaction (qRT-PCR) was performed to determine lncRNA XIST expression in human chordoma tissues and matched-noncancerous tissues. Western blot was used to determine protein expression. Silencing and overexpression of lncRNA XIST were carried out by RNA interference (RNAi) and lentiviral transduction, respectively. Cell Counting Kit-8 (CCK-8) assay and flow cytometry were employed to examine the effects of lncRNA XIST on growth of human chordoma cells. Lastly, the role of lncRNA XIST in vivo was explored using a xenograft model.ResultsWe found that lncRNA XIST expression was upregulated in chordoma and strongly correlated with poor patient prognosis. Moreover, lncRNA XIST promoted proliferation and inhibited apoptosis of chordoma cells. Mechanistically, upregulation of lncRNA XIST led to a decrease in miR-124-3p expression, thereby promoting the expression of the miR-124-3p target gene, inhibitor of apoptosis-stimulating protein of p53 (iASPP). Addition of miR-124-3p inhibitor or mimic reversed the effects induced by lncRNA XIST silencing or overexpression on chordoma cell proliferation. Lastly, using a xenograft mouse model, we found that silencing of lncRNA XIST decreased tumorigenicity in vivo, as shown by increased tumor cell apoptosis.ConclusionOur findings demonstrate a key role for lncRNA XIST in chordoma progression by regulating miR124-3p/iAPSS pathway.

Fabrication of a Microfluidic Device to Study the Interactions between Human Chordoma UCH-1 and Human Adipose-Derived Stem Cells

According to the National Institute of Health, 80 percent of adults suffer low back pain at some point in their lifetime. The loss of intervertebral disc elements, such as the nucleus pulposus, leads to abnormal spine mechanics, inflammatory signaling and compression of neural structures that lead to pain. Recently, undifferentiated stem cells have been used to rescue specialized cells via transfer of mitochondria and growth factors. However, it is not known if this method could be used to aid in the regeneration of human nucleus pulposus. In this work, we used a microfluidic device to co-culture human adipose-derived stem cells (ADSCs) next to human chordoma UCH-1 cells. Human chordoma derived UCH-1 cells have the same genetical and morphological properties as nucleus pulposus cells and serve as a model for in vitro studies. We made a microfluidic device that consisted of two wells separated by a channel, each well allows for culture of a specific cell type. When the channel is opened, it mixes the media from each cell line. The microfluidic device was fabricated using SLA resin 3D printing and polydimethylsiloxane (PDMS). On average we saw about a 50% increase in cell number when UCH-1 cells where cultured with ADSC cells than just UCH-1 cells alone. These results indicate that even at a distance stem cells (ADSC) are able to influence the growth of UCH-1 cells. We observed ADSCs migrate towards the chamber with UCH-1 cells, suggesting that there is a chemotactic signal attracting ADSCs to the chordoma chamber. In the future, we want to check for the growth factors that affect cell proliferation and the maximum distance between cell groups at which effects can be seen.

Development of a Novel Orthotopic Primary Human Chordoma Xenograft Model: A Relevant Support for Future Research on Chordoma.

Chordomas are slow-growing rare malignant neoplasms. The aim of this study was to establish a primary model of chordoma in the lumbosacral orthotopic area, to compare the growth rate to the subcutaneous site, and to show that this new graft site optimizes tumor growth and bony invasion. Eleven chordoma samples were transplanted subcutaneously in the flank and/or in contact with the lumbosacral region and grown into nude mice. Engraftment rate was significantly more successful in the lumbosacral environment compared with the flank at P0. Two xenografts from 2 patients showed bone invasion. One tumor was maintained through multiple rounds of serial transplantation, creating a model for study. Histological and immunostaining analysis confirmed that tumor grafts recapitulated the primary tumor from which they were derived, consisting of a myxoid chordoma expressing brachyury, cytokeratin AE1, EMA, and VEGF. Clear destruction of the bone by the tumor cells could be demonstrated. Molecular studies revealed PIK3CA and PTEN mutations involved in PI3K signaling pathway and most of the frequently reported chromosomal alterations. We present a novel orthotopic primary xenograft model of chordoma implanted for the first time in the lumbosacral area showing bone invasion, PIK3CA, and PTEN mutations that will facilitate preclinical studies.

RARE-29. AZD8055 ENHANCES IN VIVO EFFICACY OF AFATINIB IN CHORDOMAS

Abstract INTRODUCTION Chordomas are rare, locally aggressive bone tumors that arise in cranial base, mobile spine, and sacrum. Currently, there are no FDA-approved therapies for chordoma patients, thus there is a high unmet need to develop effective treatments. In this study, we aim to evaluate the anti-tumor efficacy of small molecule inhibitors that target crucial oncogenic pathways in chordoma, as single agents or in combination, to identify novel therapies with the greatest translation potential. METHODS A panel of small molecule compounds that had exhibited in vitro efficacy against human chordoma cell lines or target known chordoma drivers was screened in vivo against patient-derived xenograft (PDX) models of chordoma, and their efficacy was further evaluated using chordoma cell lines and xenograft models. RESULTS The in vivo activity of compounds identified in a NIH Chemical Genomics Center screen utilizing chordoma cell lines, together with inhibitors of c-MET and PDGFR, were evaluated in PDX models of chordoma that were previously described or recently established for this study. Inhibitors of EGFR (BIBX1382, erlotinib and afatinib), c-MET (crizotinib) or mTOR (AZD8055) significantly inhibited tumor growth in vivo but did not induce tumor regression. Co-inhibition of EGFR and c-MET using erlotinib and crizotinib synergistically reduced cell viability in chordoma cell lines but did not result in enhanced in vivo activity. Co-inhibition of EGFR and mTOR pathways using afatinib and AZD8055 synergistically reduced cell viability in chordoma cell lines. Importantly, co-inhibition of EGFR and mTOR also synergistically suppressed tumor growth in vivo, showing improved disease control. CONCLUSION Single inhibition of EGFR, c-MET or mTOR suppresses chordoma growth both in vitro and in vivo. Co-inhibition of EGFR and mTOR synergistically inhibits chordoma growth in a range of preclinical models. The insights gained from our study provide a novel combination therapeutic strategy for patients with chordoma.

RETRACTED ARTICLE: Silencing of TRIM11 suppresses the tumorigenicity of chordoma cells through improving the activity of PHLPP1/AKT

BackgroundTripartite motif-containing protein 11 (TRIM11), a member of RING family of E3 ubiquitin ligases, is identified as an oncogene in certain human tumors. However, the detailed biological function of TRIM11 in chordoma is still unclear. The purpose of present research is to explore the role of TRIM11 in human chordoma cells.MethodsTRIM11 was induced silencing and overexpression in human chordoma cells using RNA interference (RNAi) and lentiviral vector. qRT-PCR and western blot were used to determine gene expression in chordomas cells. Meanwhile, cell counting kit-8 (CCK-8) assay was used to examine the cell proliferation rate. Flow cytometry analysis was performed to quantify the cell apoptosis rate.ResultsWe identified that TRIM11 was upregulated in chordomas tissues. Moreover, TRIM11 presented pro-proliferation and anti-apoptosis function in chordoma cells. Further, LY294002, a specific AKT inhibitor, was utilized to examine the connection between TRIM11 and AKT in human chordoma cells. Importantly, our findings elucidated that TRIM11 promoted the growth of chordoma cells and involved in AKT signaling. Much more importantly, knockdown of TRIM11 significantly upregulated the translation of PH domain leucine-rich repeats protein phosphatase 1 (PHLPP1), whereas did not affect its transcription. Results that obtained from co-immunoprecipitation (Co-IP) and ubiquitination assay demonstrated TRIM11 interacted with PHLPP1 and promoted its ubiquitination in chordoma cells. Moreover, overexpression of PHLPP1 inhibited the phosphorylation of AKT in human chordomas cells. These results suggested that TRIM11 mediated the post-translation modification of PHLPP1 and was a novel component in PHLPP1/AKT signaling pathway in human chordoma cells.ConclusionsTaken together, the present research not only enhanced the understanding of TRIM11 but also indicated its potential target and signaling pathway in human chordoma cells.Trial registration retrospectively registered.

MiR-152 inhibits the proliferation and invasion of chordoma cells by targeting HOXC8.

ObjectiveMicroRNAs (miRNAs) are non-coding RNAs that affect the expression of their target genes by binding to the 3′-untranslated region. miR-152 has been identified as a critical modulator in tumorigenesis, but its role in chordoma has not been explored. We therefore investigated the role of miR-152 in regulating chordoma cell behavior, and examined the downstream effectors of miR-152.Materials and methodsWe examined the expression of miR-152 in two human chordoma cell lines and in a normal human embryonic kidney cell line. We also analyzed the relationship between miR-152 and homeobox C8 (HOXC8) by bioinformatics analysis and luciferase reporter assay. We determined the effects of miR-152 and HOXC8 expression on chordoma cell behavior.ResultsmiR-152 expression was downregulated in chordoma compared with normal cells. Meanwhile, miR-152 overexpression inhibited chordoma cell proliferation and invasion. The oncogene HOXC8 was a direct target of miR-152, as shown by luciferase reporter and western blot assays.ConclusionsHOXC8 acted as an effector for the suppressive role of miR-152 in chordoma, thereby providing a potential therapeutic target in patients with chordoma.

Solamargine inhibits proliferation and promotes apoptosis of CM-319 human chordoma cells through suppression of notch pathway.

BackgroundSolamargine (SM), which represents a natural steroid alkaloid glycoside compound and a cytotoxic agent, has been proved to enhance the sensitivity of lung cancer cells to tumor necrosis factors (TNFs). In this study, we aimed to investigate the roles and mechanisms of SM in chordoma.MethodsCell viability, proliferation, apoptosis and cell cycle were measured by cell counting Kit-8 (CCK-8) assay, 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling and flow cytometry (FCM), respectively. Western blot and quantitative real-time reverse transcription PCR (qRT-PCR) assays were performed to detect the expressions of related mRNAs and proteins.ResultsThe results revealed that SM distinctly suppressed the proliferation of CM-319 cells. SM significantly induced the CM-319 cells apoptosis through up-regulating the expression levels of Caspase-3/8/9. The cell cycle of CM-319 cells was blocked by SM in G1 phase. Moreover, SM could significantly suppress the Notch pathway in CM-319 cells.ConclusionsIn conclusion, SM suppressed the proliferation and enhanced the apoptosis ability of CM-319 cells via suppressing the Notch pathway. The results suggested that SM might be a novel therapeutic agent and supported the utilization of SM in chordoma.

Solamargine inhibits proliferation and promotes apoptosis of CM-319 human chordoma cells through suppression of notch pathway

Background: Solamargine (SM), which represents a natural steroid alkaloid glycoside compound and a cytotoxic agent, has been proved to enhance the sensitivity of lung cancer cells to tumor necrosis factors (TNFs). In this study, we aimed to investigate the roles and mechanisms of SM in chordoma. Methods: Cell viability, proliferation, apoptosis and cell cycle were measured by cell counting Kit-8 (CCK-8) assay, 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling and flow cytometry (FCM), respectively. Western blot and quantitative real-time reverse transcription PCR (qRT-PCR) assays were performed to detect the expressions of related mRNAs and proteins. Results: The results revealed that SM distinctly suppressed the proliferation of CM-319 cells. SM significantly induced the CM-319 cells apoptosis through up-regulating the expression levels of Caspase-3/8/9. The cell cycle of CM-319 cells was blocked by SM in G1 phase. Moreover, SM could significantly suppress the Notch pathway in CM-319 cells. Conclusions: In conclusion, SM suppressed the proliferation and enhanced the apoptosis ability of CM-319 cells via suppressing the Notch pathway. The results suggested that SM might be a novel therapeutic agent and supported the utilization of SM in chordoma.

Abstract B165: Investigating in vivo synergistic effect of checkpoint blockade and radiation therapy against chordomas in a humanized mouse model

Abstract Introduction: With the advent of immunotherapy (IT) against various cancers, its applications to other cancers have been extensively investigated. However, it has been a challenge to apply IT to chordomas, due to lack of clinically translatable in vivo models. Currently, there are no well-established murine chordoma cell lines that can be injected to syngeneic mice or no transgenic mouse models that develop chordomas spontaneously, which would allow us to study the interaction between murine chordomas and murine immune cells. Hence, we aimed to develop a humanized mouse model, where human immune cells are engrafted into immunodeficient mice, to study the interaction between human immune system and human chordomas. We also sought to utilize it to investigate synergistic effect between IT and radiation therapy (RT) against chordoma. Materials and Methods: Fifteen 10-12-week-old NSG mice, which lack mouse T-cells, B cells, and NK cells as well as functional mouse macrophages, were sublethally (1.5Gy) irradiated and then implanted with fetal thymic tissue and CD34+ stem cells that had been harvested from a fetus, whose HLA-types were partially-matched with those of the U-CH1 chordoma cell line. Reconstitution of immune cells in NSG mice was confirmed eight weeks post-transplantation, and then each animal (15 humanized NSG mice and 12 naïve NSG mice) was injected with U-CH1 cell suspension bilaterally and subcutaneously. Next, they were treated for 4 weeks as follows: A) control, isotype antibodies (Abs) injection (n=3), B) anti-human-PD-1 Abs (n=4, 10 mg/kg, 3 times/week for 4 weeks), C) RT + isotype Abs (n=3, unilaterally to the left-sided tumor, 8Gy x 4), D) anti-human-PD-1 Abs and RT (n=5), E) naïve NSG mice (n=6, without the engraftment of human immune cells) + isotype, and F) naïve NSG mice (n=6) + anti-human-PD-1 Abs. During and after the treatment, anti-tumor activities were monitored via tumor size, flow cytometry, qRT-PCR, and immunohistochemistry. Results: Eight weeks after stem cell engraftment, human peripheral blood mononuclear cells (PBMCs) of 43.8% among all PBMCs (human + mouse), human T-cells of 23.4% among human PBMCs, human CD8+ T-cells of 24.3% among human T-cells, and other lymphocytes such as B cells, macrophages, and NK cells were observed in peripheral blood of humanized mice via flow cytometry, which confirmed humanization. One week after the treatment, on the irradiated side, (D) demonstrated lowest tumor volume, highest number of human PBMCs, highest % of CD8+ human (cytotoxic) T-cells, highest % of CD45RO+CD4+ human (memory) T-cells, and lowest % of PD-1+CD8+ human (exhausted) T-cells in the tumors via flow cytometry, highest IFN-gamma in the tumors via qRT-PCR, and highest CD8+ human (cytotoxic) T-cells via immunohistochemistry, compared to the other five groups with statistical significance. Of note, on the nonirradiated side, a similar trend was observed with D) harboring the smallest tumor compared to the others (P=0.09), suggesting the abscopal effect. Finally, there were no statistically significant differences amongst (A) humanized NSG mice with isotype-control antibodies, (E) naïve NSG mice with isotype-control antibodies, and (F) naïve NSG mice with anti-PD-1 antibodies on either sides, indicating that HLA-partially-mismatched immune cells derived from the fetus donor were not able to eradicate U-CH1 chordoma cells. Conclusions: We demonstrated that this humanized mouse model could be a revolutionary platform to investigate IT against rare cancers such as chordomas, where murine equivalent cell lines are not available to date, which hinders us from utilizing syngeneic or transgenic mouse models to study IT. The direct synergistic effect between IT and RT against chordoma as well as the potential abscopal effect was observed, evidenced by lowest tumor volume and highest cytotoxic T-cells and memory T-cells. Citation Format: Wataru Ishida, Kyle L. McCormick, Aayushi Mahajan, Eric Feldstein, Michael Lim, Jeffrey N. Bruce, Peter D. Canoll, Sheng-fu L Lo. Investigating in vivo synergistic effect of checkpoint blockade and radiation therapy against chordomas in a humanized mouse model [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B165.

The vacuolated morphology of chordoma cells is dependent on cytokeratin intermediate filaments.

Notochordal cells (NCs), characterized by their vacuolated morphology and coexpression of cytokeratin and vimentin intermediate filaments (IFs), form the immature nucleus pulposus (NP) of the intervertebral disc. As humans age, NCs give way to mature NP cells, which do not possess a vacuolated morphology and typically only express vimentin IFs. In light of their concomitant loss, we investigated the relationship between cytosolic vacuoles and cytokeratin IFs, specifically those containing cytokeratin-8 proteins, using a human chordoma cell line as a model for NCs. We demonstrate that the chemical disruption of IFs with acrylamide, F-actin with cytochalasin-D, and microtubules with nocodazole all result in a significant (p < 0.001) decrease in vacuolation. However, vacuole loss was the greatest in acrylamide-treated cells. Examination of the individual roles of vimentin and cytokeratin-8 IFs in the existence of vacuoles was accomplished using small interfering RNA-mediated RNA interference to knock down either vimentin or cytokeratin-8 expression. Reduction of cytokeratin-8 expression was associated with a less-vacuolated cell morphology. These data demonstrate that cytokeratin-8 IFs are involved in stabilizing vacuoles and that their diminished expression could play a role in the loss of vacuolation in NCs during aging. A better understanding of the NCs may assist in preservation of this cell type for NP maintenance and regeneration.

Friday, September 28, 2018 10:30 AM–12:00 PM abstracts: innovation, surface technology and biomechanics: 182. In vivo synergistic effect of checkpoint blockade and radiation therapy against chordomas in a humanized mouse model

BACKGROUND CONTEXT With the advent of immunotherapy (IT) against various cancers, its applications to other cancers have been extensively investigated. However, it has been a challenge to apply IT to chordomas, due to lack of clinically-translatable in vivo models. Unlike othercancers that have been extensively studied from the immunotherapeutic standpoint such as melanomas, currently, there are no well-established murine chordoma cell lines that can be injected to syngeneic mice or transgenic mouse models that develop chordomas spontaneously, which prevents us from investigating the interaction between murine chordomas and murine immune cells. Meanwhile, in the field of immunology, the development of humanized mouse models, where a human immune system is incorporated into immunodeficient mice by engrafting them with human hematopoietic stem cells and fetal thymus, has emerged as a state-of-the-art technique, which allows researchers to investigate human immune system in vivo in the context of several immunological disorders including cancer. To scrutinize IT against chordoma, the development of this model is imperative. PURPOSE We aimed to develop a humanized mouse model engrafted with chordoma cells to study the interaction between humanimmune system and human chordomas. We also sought to utilize it to investigate synergistic effect between IT and radiation therapy (RT) against chordoma. STUDY DESIGN/SETTING Animal experiments. OUTCOME MEASURES Tumor size, cell subpopulations of tumor infiltratinglymphocytes, and cytokine levels in blood and tumors in each treatment group. METHODS Fifteen 10-12-week-old NSG mice, which lacks mouse T cells, B cells, and NK cells as well as functional mouse macrophages, were sub-lethally (1.5Gy) irradiated and then implanted with human fetal thymic tissue and CD34+ stem cells that had been harvested from a fetus, whose HLA-type is partially-matched with that of the U-CH1 chordoma cell line. Reconstitution of immune cells in NSG mice was confirmed 8 weeks posttransplantation and then each animal (15 humanized NSG mice and 12 naive NSG mice) was injected with U-CH1 cell suspension bilaterally and subcutaneously. Next, they were treated for 4 weeks as follows: (A) control, isotype antibodies (Abs) injection (n=3), (B) anti-human-PD-1 Abs (n=4), (C) RT + isotype Abs (n=3, unilaterally to the left-sided tumor, 8Gy × 4), (D) anti-human-PD-1 Abs and RT (n=5), (E) naive NSG mice (n=6, without the engraftment of human immune cells) + isotype, and (F) naive NSG mice (n=6) + anti-human-PD-1 Abs. During and after the treatment, anti-tumor activities were monitored via tumor size, flow cytometry, qRT-PCR, and immunohistochemistry. The data were statistically analyzed and p l 0.05 was set as statistically significant. RESULTS On average, human peripheral blood mononuclear cells (PBMCs) of 43.8% among all PBMCs (human + mouse), human T cells of 23.4% among human PBMCs, human CD8+ T cells of 24.3% among human T cells, and other lymphocytes such as B cells, macrophages, and NK cells were observed in peripheral blood of humanized mice via flow cytometry. One week after the treatment, on the irradiated side, (D) demonstrated lowest tumor volume ((A) 2220.0mm 3 , (B) 615.8mm 3 , (C) 1259.0mm 3 , (D)144.0mm 3 , p CONCLUSIONS We demonstrated that this humanized mouse model could be a revolutionary platform to investigate IT against rare cancers such as chordomas, where murine equivalent cell lines are not available to date, which hinders us from utilizing syngeneic or transgenic mouse models to study IT against cancers. The direct synergistic effect between IT and RT against chordoma as well as the potential abscopal effect was observed.

Characterization of the endolysosomal system in human chordoma cell lines: is there a role of lysosomes in chemoresistance of this rare bone tumor?

Chordoma is a rare tumor of the bone derived from remnants of the notochord with pronounced chemoresistance. A common feature of the notochord and chordoma cells is distinct vacuolization. Recently, the notochord vacuole was described as a lysosome-related organelle. Since lysosomes are considered as mediators of drug resistance in cancer, we were interested whether they may also play a role in chemoresistance of chordoma. We characterized the lysosomal compartment in chordoma cell lines by cytochemistry, electron microscopy (ELMI) and mutational analysis of genes essential for the physiology of lysosomes. Furthermore, we tested for the first time the cytotoxicity of chloroquine, which targets lysosomes, on chordoma. Cytochemical stainings clearly demonstrated a huge mass of lysosomes in chordoma cell lines with perinuclear accumulation. Also vacuoles in chordoma cells were positive for the lysosomal marker LAMP1 but showed no acidic pH. Genetic analysis detected no apparent mutation associated with known lysosomal pathologies suggesting that vacuolization and the huge lysosomal mass of chordoma cell lines is rather a relict of the notochord than a result of transformation. ELMI investigation of chordoma cells confirmed the presence of large vacuoles, lysosomes and autophagosomes with heterogeneous ultrastructure embedded in glycogen. Interestingly, chordoma cells seem to mobilize cellular glycogen stores via autophagy. Our first preclinical data suggested no therapeutically benefit of chloroquine for chordoma. Even though, chordoma cells are crammed with lysosomes which are according to their discoverer de Duve "cellular suicide bags". Destabilizing these "suicide bags" might be a promising strategy for the treatment of chordoma.

Targeting the Cohesive Cluster Phenotype in Chordoma via β1 Integrin Increases Ionizing Radiation Efficacy

Chordoma is a rare, radiation-resistant, skull-base and spinal tumor with high local recurrence containing mixed cell-adhesion phenotypes. We characterized DNA damage response (DDR) signaling (γH2AX, pKAP1, pATM) and survival response to ionizing radiation (IR) in human chordoma samples (42 resections, 23 patients) to test if blocking cell adhesion sensitizes U-CH1 tumor cells to IR. U-CH1 cells expressed brachyury, YAP, and laminin adhesion receptors (CD49c, CD49f, CD44), and approximately 15% to 20% of U-CH1 cells featured an α6 integrin-dependent (CD49f) cohesive cluster phenotype, which confers therapeutic resistance and aids metastasis. DDR to IR in U-CH1 cells was compared to normal prostate epithelial (PrEC) and tumor cells (DU145). Flow cytometry showed a dose- and time-dependent increase in γH2AX and pKAP1 expression in all cell lines. However, nearly 50% of U-CH1 cells exhibited nonresponsive phenotype to IR (measured by γH2AX and pKAP1) independent of cell cycle status. Immunofluorescence microscopy verified that only 15% of U-CH1 clustered cells were γH2AX or pKAP1 positive (versus 80% of nonclustered cells) 2 hours following 2-Gy IR. Conversely, both tumor cell lines were uniformly defective in pATM response. HYD1, a synthetic ECM ligand, inhibited DDR through an unresolved γH2AX response. β1 integrin-blocking antibody (AIIB2) decreased cell survival 50% itself and approximately doubled the IR-induced cell kill at all IR doses observed at 2 and 4 weeks posttreatment. These results suggest that a heterogeneity of DDR to IR exists within a chordoma population. Blocking integrin function alone and/or as an adjuvant to IR may eradicate chordomas containing the cohesive cluster phenotype.

5-Aminolevulinic acid-based photodynamic therapy of chordoma: In vitro experiments on a human tumor cell line

BackgroundChordomas are very rare tumors of the skull base and the sacrum. They show infiltrating and destructive growth and are known to be chemo- and radio-resistant. After surgical resection, the recurrence rate is high and overall survival limited. As current adjuvant treatments are ineffective, new treatment concepts are urgently needed. 5-aminolevulinic acid-based photodynamic therapy (5-ALA based PDT) showed promising results for malignant gliomas. However, it is unknown so far, whether chordomas accumulate protoporphyrin IX (PPIX) after application of 5-ALA and whether they are sensitive to subsequent 5-ALA based PDT. MethodsThe immortalized human chordoma cells U-CH2 were used as in vitro model. After incubation for 4h or 6h with different 5-ALA concentrations, PPIX accumulation was determined by flow cytometry. To assess sensitivity to PDT, chordoma cells were incubated at 30.000cells/well (high cell density) or 15.000cells/well (low cell density) with graded doses of 5-ALA (0–50μg/ml) in 96-well plates and subsequently exposed to laser light of 635nm wavelength (18.75J/cm2). Cell survival was measured 24h after exposure to laser light using the WST-1 assay. ResultsU-CH2 cells dose-dependently accumulated PPIX (ANOVA; p<0.0001). PPIX fluorescence was significantly higher, when cells were incubated with 5-ALA for 6h compared to 4h at higher 5-ALA concentrations (ANOVA/Bonferroni; p≤0.05 for≥30μg/ml 5-ALA). For both cell densities, a 5-ALA dose-dependent decline in viability was observed (ANOVA; p<0.0001). Viability was significantly lower at higher 5-ALA concentrations, when 30.000 cells/wells were treated compared to 15.000cells/well (ANOVA/Bonferroni; p≤0.001 for≥30μg/ml 5-ALA). LD50 was 30.25μg/ml 5-ALA. ConclusionThe human UCH-2 cell line was a very useful in vitro model to study different effects of 5-ALA based PDT. For the first time, it could be shown that human chordoma cells may be destroyed by 5-ALA/PDT.

Effects of primary and recurrent sacral chordoma on the motor and nociceptive function of hindlimbs in rats: an orthotopic spine model.

OBJECTIVE Chordoma is a slow-growing, locally aggressive cancer that is minimally responsive to conventional chemotherapy and radiotherapy and has high local recurrence rates after resection. Currently, there are no rodent models of spinal chordoma. In the present study, the authors sought to develop and characterize an orthotopic model of human chordoma in an immunocompromised rat. METHODS Thirty-four immunocompromised rats were randomly allocated to 4 study groups; 22 of the 34 rats were engrafted in the lumbar spine with human chordoma. The groups were as follows: UCH1 tumor-engrafted (n = 11), JHC7 tumor-engrafted (n = 11), sham surgery (n = 6), and intact control (n = 6) rats. Neurological impairment of rats due to tumor growth was evaluated using open field and locomotion gait analysis; pain response was evaluated using mechanical or thermal paw stimulation. Cone beam CT (CBCT), MRI, and nanoScan PET/CT were performed to evaluate bony changes due to tumor growth. On Day 550, rats were killed and spines were processed for H & E-based histological examination and immunohistochemistry for brachyury, S100β, and cytokeratin. RESULTS The spine tumors displayed typical chordoma morphology, that is, physaliferous cells filled with vacuolated cytoplasm of mucoid matrix. Brachyury immunoreactivity was confirmed by immunostaining, in which samples from tumor-engrafted rats showed a strong nuclear signal. Sclerotic lesions in the vertebral body of rats in the UCH1 and JHC7 groups were observed on CBCT. Tumor growth was confirmed using contrast-enhanced MRI. In UCH1 rats, large tumors were observed growing from the vertebral body. JHC7 chordoma-engrafted rats showed smaller tumors confined to the bone periphery compared with UCH1 chordoma-engrafted rats. Locomotion analysis showed a disruption in the normal gait pattern, with an increase in the step length and duration of the gait in tumor-engrafted rats. The distance traveled and the speed of rats in the open field test was significantly reduced in the UCH1 and JHC7 tumor-engrafted rats compared with controls. Nociceptive response to a mechanical stimulus showed a significant (p < 0.001) increase in the paw withdrawal threshold (mechanical hypalgesia). In contrast, the paw withdrawal response to a thermal stimulus decreased significantly (p < 0.05) in tumor-engrafted rats. CONCLUSIONS The authors developed an orthotopic human chordoma model in rats. Rats were followed for 550 days using imaging techniques, including MRI, CBCT, and nanoScan PET/CT, to evaluate lesion progression and bony integrity. Nociceptive evaluations and locomotion analysis were performed during follow-up. This model reproduces cardinal signs, such as locomotor and sensory deficits, similar to those observed clinically in human patients. To the authors' knowledge, this is the first spine rodent model of human chordoma. Its use and further study will be essential for pathophysiology research and the development of new therapeutic strategies.

Validation of reference genes in human chordoma.

Background:Chordoma are rare slow-growing tumors of the axial skeleton, which are thought to arise from remnants of the notochord. Little is known about the underlying mechanisms that drive this tumor. However, the assessment of gene expression levels by quantitative real-time polymerase chain reaction (qRT-PCR) is hampered due to a lack of validated reference genes. Using an unstable reference gene in qRT-PCR may lead to irreproducible results.Methods:The expression of 12 candidate reference genes (ACTB, B2M, T, EF1a, GAPDH, HPRT, KRT8, KRT19, PGK1, RS27a, TBP, and YWHAZ) was analyzed by qRT-PCR in flash frozen chordoma samples from 18 patients. GeNorm and NormFinder algorithms were used to rank the stability of the genes.Results:From most to least stably expressed, the top six genes found by geNorm were PGK1, YWHAZ, ACTB, HPRT, EF1A, and TBP. When analyzed by NormFinder, the top six genes were ACTB, YWHAZ, PGK1, B2M, TBP, and HPRT. GAPDH alone, which is often used as a reference gene in chordoma gene expression studies, is not stable enough for reliable results.Conclusion:In gene expression studies of human chordomas, PGK1, ACTB, and YWHAZ are more stably expressed, and therefore, are preferred reference genes over the most often used reference gene so far, GAPDH.

MiR-219-5p inhibits proliferation and clonogenicity in chordoma cells and is associated with tumor recurrence

Chordoma is a rare malignant bone tumor that is usually localized to the skull base, vertebral column and sacrum. The transcription factor brachyury, which is encoded by the T gene, has a critical role in the development and progression of chordoma, although the mechanisms underlying brachyury regulation remain unclear. The aim of the current study was to identify and characterize microRNAs (miRs) that regulate brachyury expression in chordoma. MicroRNAs that target brachyury were predicted using miRanda and TargetScan. Using reverse transcription-quantitative polymerase chain reaction, miR-219-5p was shown to be significantly downregulated in chordoma tissues and the U-CH2 chordoma cell lines. A dual-luciferase reporter assay was used to validate the inhibitory effect of miR-219-5p on brachyury mRNA expression. The expression level of brachyury was downregulated in U-CH2 cells following transfection with miR-219-5p mimics and upregulated following transfection with the miR-219-5p inhibitor. The effects of miR-219-5p on the proliferation and clonogenicity of chordoma cells were assessed using cell counting kit-8, EdU and clone formation assays. These in vitro results indicated that miR-219-5p may have an important role in regulating the cell proliferation and clonogenicity of human chordoma cells, potentially by targeting brachyury. Furthermore, the associations between the expression levels of miR-219-5p and various clinicopathological factors were analyzed, and miR-219-5p expression was shown to correlate with tumor extent and recurrence. These results suggested that miR-219-5p functions as a tumor suppressor in chordoma and, therefore, that miR-219-50 may be a potential target for therapeutic intervention.

Preclinical Characterization of Novel Chordoma Cell Systems and Their Targeting by Pharmocological Inhibitors of the CDK4/6 Cell-Cycle Pathway.

Chordomas are tumors that arise at vertebral bodies and the base of the skull. Although rare in incidence, they are deadly owing to slow growth and a lack of effective therapeutic options. In this study, we addressed the need for chordoma cell systems that can be used to identify therapeutic targets and empower testing of candidate pharmacologic drugs. Eight human chordoma cell lines that we established exhibited cytology, genomics, mRNA, and protein profiles that were characteristic of primary chordomas. Candidate responder profiles were identified through an immunohistochemical analysis of a chordoma tissue bank of 43 patients. Genomic, mRNA, and protein expression analyses confirmed that the new cell systems were highly representative of chordoma tissues. Notably, all cells exhibited a loss of CDKN2A and p16, resulting in universal activation of the CDK4/6 and Rb pathways. Therefore, we investigated the CDK4/6 pathway and responses to the CDK4/6-specific inhibitor palbociclib. In the newly validated system, palbociclib treatment efficiently inhibited tumor cell growth in vitro and a drug responder versus nonresponder molecular signature was defined on the basis of immunohistochemical expression of CDK4/6/pRb (S780). Overall, our work offers a valuable new tool for chordoma studies including the development of novel biomarkers and molecular targeting strategies.

Abstract 5149: An orthotopic model of human chordoma in rats

Abstract Introduction. Chordoma (CHO) is a relatively rare, slow-growing, malignant, locally aggressive cancer that constitutes 1% to 4% of all primary bone tumors. It arises from notochordal remnants along the neuraxis at developmentally active sites. CHO causes significant damage through local bone destruction and compromise of surrounding neurologic and neurovascular structures. CHO in the lumbar spine results in devastating effects that include pain, paralysis, bowel/bladder dysfunction, and decreased ambulation. These effects are minimally responsive to conventional chemotherapy and radiotherapy and as such, surgical resection is the mainstay of treatment. Pre-clinical models that better recapitulate the clinical symptoms observed in patients diagnosed with CHO are needed to increase the understanding of this disease and to develop strategies to develop new therapeutic strategies. Here we developed the first orthotopic animal model of human CHO in the lumbar spine of immunocompromised rats. Methods. 11 immunocompromised rats were implanted in the lumbar spine with human CHO tumor tissue. A group of 6 sham rats (surgical approach without tumor implantation) and another with 6 intact rats were used as control groups. This tumor was obtained from a donor rat that was previously injected with UCH1CHO cells in the flank. Nociceptive response was conducted every week after the first neurological deficits were present using Randal-Sellito. Locomotion gait analysis was used to evaluate gait impairment. Rats were clinically observed for neurological impairment in the lumbar spine and/or hind limbs. Spines were collected after euthanasia, and inspected for signs of spinal cord compression followed by H&amp;E staining to evaluate the histology. Results. In CHO-implanted rats, the threshold for a mechanic nociceptive response was significantly reduced in time and pressure applied. Locomotion gait analysis showed a disruption in the normal pattern of the rats’ gait. At D25-35 after tumor implantation rats showed impairment in the flexion and extension of the knees, ankles, and toes. At the gross morphology level, the spinal cord showed signs of compression at upper and lower levels related with the implanted CHO tumor. Histology revealed tumor invasion in the spinal canal that was related with deficit in the flexion and extension of the toes. The tumor showed typical CHO morphology: physaliferous cells filled with vacuolated cytoplasm of mucoid matrix. Conclusions. We have developed a novel orthotopic human CHO model in rats. This model reproduces cardinal clinical signs such as locomotor and sensory deficits present in human patients. To our knowledge, this is the first orthotopic animal model of human CHO. Its use and further study will be essential for pathophysiology research and the development of new therapeutic strategies. Citation Format: Rachel Sarabia-Estrada, Alejandro Ruiz-Valls, Courtney R. Goodwin, Sagar Sha, Alfredo Quiñones-Hinojosa, Ziya L. Gokaslan, Daniel M. Sciubba. An orthotopic model of human chordoma in rats. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5149. doi:10.1158/1538-7445.AM2015-5149

Expression of programmed cell death ligand 1 (PD-L1) and prevalence of tumor-infiltrating lymphocytes (TILs) in chordoma

Chordomas are primary malignant tumors of the notochord that are resistant to conventional chemotherapy. Expression of programmed cell death ligand 1 (PD-L1), prevalence of tumor-infiltrating lymphocytes (TILs), and their clinical relevance in chordoma remain unknown. We evaluated PD-L1 expression in three chordoma cell lines and nine chordoma tissue samples by western blot. Immunohistochemical staining was performed on a chordoma tissue microarray (TMA) that contained 78 tissue specimens. We also correlated the expression of PD-L1 and TILs with clinical outcomes. PD-L1 protein expression was demonstrated to be induced by IFN-γ in both UCH1 and UCH2 cell lines. Across nine human chordoma tissue samples, PD-L1 protein was differentially expressed. 94.9% of chordoma samples showed positive PD-L1 expression in the TMA. The expression score of PD-L1 for metastatic chordoma tumors was significant higher as compared with non-metastatic chordoma tumors. Expression of PD-L1 protein significantly correlates with the presence of elevated TILs, which correlates with metastasis. In summary, our study showed high levels of PD-L1 are expressed in chordoma, which is correlated with the prevalence of TILs. The current study suggests targeting PD-L1 may be a novel immunotherapeutic strategy for chordoma clinical trials.