Dual-Modality Therapy: Synergistic Enhancement of Radio-Hyperthermia by Gold-Gold Sulfide Nanoparticles in MCF-7 Cells

Insulinoma-associated 1 (INSM1) is neuroendocrine (NE) transcription factor which temporal and spatial expression pattern is restricted to regions undergoing NE differentiation during embryogenesis. INSM1 is however re-expressed at high levels in small-cell lung cancer (SCLC) together with an array of other NE markers. The NE signature of SCLC is a central diagnostic tool for the disease but recent data pinpoints that this distinct signature might play a role in SCLC pathogenesis. The functional role of INSM1 in SCLC was here investigated by shRNA-mediated silencing of INSM1 in a panel of SCLC cell lines. Upon silencing of INSM1, significant reduction in cell viability was observed as measured by MTT assay. This reduction in cell viability was concomitant with decreased cell proliferation and increased apoptosis as measured by BrdU incorporation and cleaved caspase 3/7 levels, respectively. Accordingly with phenotypic growth changes it was demonstrated that INSM1 silencing caused reduced phosphorylation of members of the growth- and survival-promoting PI3K/Akt, MAPK and JAK-STAT signaling pathways and reduced levels of the inhibitors of apoptosis proteins (IAPs) members c-IAP1, XIAP and survivin. Importantly, it was shown that INSM1 is an upstream regulator of signatures previously shown to play a role in SCLC tumorigenesis, including achate-scute homolog 1 (ASCL1) and Sonic Hedgehog (SHh) pathway. Upon silencing of INSM1, a decrease in mRNA and protein levels of ASCL1 and downstream targets aldehyde dehydrogenase 1 (ALDH1) and delta-like ligand 3 (DLL3) was observed. Furthermore, mRNA levels of the SHh positive mediators Gli2, Gli3 and Smoothened was downregulated while the SHh inhibitor Patched was upregulated upon INSM1 silencing. Futhermore, overexpression of the Notch1 intracellular domain in SCLC cells resulted in a significant reduction in INSM1 expression and cell viability. This suggest that a master inhibitor of the NE signature, Notch1 signaling, could be an inhibitor of INSM1-driven tumorigenesis. In conclusion, INSM1 is a potential key driver of growth- and survival-promoting pathways in SCLC and warrants further investigation of INSM1 as a therapeutic target. Citation Format: Camilla L. Christensen, Takeshi Shimamura, Esra A. Akbay, Signe R. Michaelsen, Hans S. Poulsen, Kwok-kin Wong. Insulinoma-associated 1 is a key transcriptional regulator of growth and survival-promoting pathways in small cell lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3126. doi:10.1158/1538-7445.AM2013-3126

We investigated the possible anticancer mechanisms of Pteris vittata [PV] n-hexane extract on MCF-7 [breast cancer cell line]. Cultured cell lines were treated with various concentrations of this extract ± Baf-A1 [autophagic inhibitor]. Cells’ viability, apoptotic markers [caspase-7, Bax, and Bcl-2], autophagic markers [light chain 3 [LC-3] and P62/SQSTM1]], and the tumor suppressor P53 and its mRNA were checked by their corresponding methods. Treated cell lines showed significant concentration and time-dependent reductions in cell viability in response to PV-n-hexane extract and also exhibited a concomitant induction of apoptosis [increased chromatin condensation, nuclear fragmentation, and pro-apoptotic Bax, and cleaved caspase-7 levels while decreased Bcl-2 levels] and autophagy [increased autophagosomes vacuoles, and LC3B II levels while decreased P62/SQSTM1 levels]. Moreover, PV-n-hexane extract-treated cells showed significant increases in the P53 and its mRNA levels. The addition of Baf-A1 reversed the PV-n-hexane extract autophagic effects and increased apoptotic cell percentage with a much increase in the cleaved caspase-7 and P53 protein and its mRNA levels. We concluded that the PV-n-hexane extract exhibits cytotoxic effects on the MCF-7 cell line with significant reductions in cell viability and concomitant autophagy and apoptosis induction. Inhibition of autophagy in the PV-treated MCF-7 cells enhances apoptosis via a p35-dependent pathway.

Cerebral vascular deposition of the amyloid-β (Aβ) peptide, a condition known as cerebral amyloid angiopathy (CAA), is associated with intracerebral hemorrhaging and contributes to disease progression in Alzheimer's disease (AD) and vascular cognitive impairment and dementia (VCID). Familial mutations at positions 22 and 23 within the Aβ peptide lead to early onset and severe CAA pathology. Here, we evaluate the effects of fibrillar Aβ peptides on the viability of primary-cultured human cerebral smooth muscle (HCSM) cells, which are the major site of amyloid deposition in cerebral blood vessel walls. Comparisons are made of the familial E22Q (Dutch) mutant of Aβ40 with wild-type Aβ40 and Aβ42. In agreement with previous studies, we find that there is a significant reduction in cell viability when Aβ40-Dutch or Aβ42-WT peptides are added to HCSM cell cultures as monomeric Aβ, whereas Aβ40-WT is relatively nontoxic. The binding of Aβ fibrils derived from sporadic CAA or familial Dutch-type CAA brain tissue to the membrane surface of HCSM cells does not result in a significant loss of cell viability. In contrast, when Aβ40-WT monomers and sporadic CAA fibrils are coincubated in HCSM cell cultures, there is a significant reduction in HCSM cell viability that is accompanied by an increase in cell surface fibril formation. Lastly, intrathecal administration of Aβ40-Dutch fibrillar seeds promotes fibrillar amyloid accumulation in the smooth muscle of meningeal vessels in the rTg-D transgenic rat model of CAA. Together, the present findings suggest that fibrillar Aβ seeds propagate the expansion of new amyloid fibrils on cerebral vascular smooth muscle, leading to membrane disruption and cell death.

Children with Group 3 medulloblastoma (G3 MB) have a very poor prognosis, and many do not survive beyond 5 years after diagnosis. A factor that may contribute to this is the lack of available targeted therapy. Expression of protein lin‐28 homolog B (LIN28B), a regulator of developmental timing, is upregulated in several cancers, including G3 MB, and is associated with worse survival in this disease. Here, we investigate the role of the LIN28B pathway in G3 MB and demonstrate that the LIN28B–lethal‐7 (let‐7; a microRNA that is a tumor suppressor)–lymphokine‐activated killer T‐cell‐originated protein kinase (PBK; also known as PDZ‐binding kinase) axis promotes G3 MB proliferation. LIN28B knockdown in G3‐MB‐patient‐derived cell lines leads to a significant reduction in cell viability and proliferation in vitro and in prolonged survival of mice with orthotopic tumors. The LIN28 inhibitor N‐methyl‐N‐[3‐(3‐methyl‐1,2,4‐triazolo[4,3‐b]pyridazin‐6‐yl)phenyl]acetamide (1632) significantly reduces G3 MB cell growth and demonstrates efficacy in reducing tumor growth in mouse xenograft models. Inhibiting PBK using HI‐TOPK‐032 also results in a significant reduction in G3 MB cell viability and proliferation. Together, these results highlight a critical role for the LIN28B–let‐7–PBK pathway in G3 MB and provide preliminary preclinical results for drugs targeting this pathway.

Purpose: The aim of this study was to access the safety profiles of 2 fusion proteins with anti-vascular endothelial growth factor action (ziv-aflibercept and aflibercept) on retinal pigment epithelium cells and Muller-Glia cells in culture by assessing cell viability post drug exposure. Methods: Primary human retinal pigment epithelium cells (pRPE) and Muller-Glia cells (Mio-M1) were exposed to the clinical standardized concentrations of ziv-aflibercept (25 mg/mL) and aflibercept (40 mg/mL). Progressively higher concentrations of NaCl (300, 500, 1,000, 1,500, 2,000, 5,000, and 10,000 mosm/kg) were also applied to cells to assess the possibility of potentiating hyperosmotic cytotoxity effect. The study was applied to measure pRPE and Mio-M1 viability by a tetrazolium dye-reduction assay (XTT). Results: Cell viability of both pRPE and Mio-M1 presented no significant changes after exposure of ziv-aflibercept and aflibercept. Progressive NaCl concentrations did not significantly alter cell viability. The exposure to the negative control of 75 µL/mL of dimethyl sulfoxide showed significant reduction in cell viability. Conclusions: At clinical doses, neither ziv-aflibercept nor aflibercept caused any significant reduction in cell viability in vitro. Furthermore, injection solutions of NaCl with higher osmolality caused no significant reduction in cell viability.

IntroductionGold nanoparticles (GNPs) have drawn immense attention in cancer diagnosis and therapy during the past few years. Nanoparticles preferably leak and accumulate inside the tumour due to many reasons including high vascular density, increased vessel permeability, and defective lymphatic draining of tumours via a process called the enhanced permeability and retention (EPR) effect. Very small particles (<30 nm), although can enter the tumour tissue by the EPR effect, they also may leave it again by passive diffusion. This size-dependent cellular uptake thus limits the use of very small nanoparticles in cancer therapy. This study was conducted to improve the retention of small nanoparticles and overcome its size limitation by linking it to a noncoding DNA fragment.Material and methodsA noncoding DNA fragment was amplified by PCR using a 5’ thiol-labelled forward primer and an unmodified 3’ reverse primer. After amplification, the PCR product was purified then conjugate to citrate stabilized10 nm GNPs. MCF-7 cells were treated with GNP-DNA conjugate using metafectene as a transfection agent for cytotoxicity studies. The GNP-DNA conjugate chemosensitization of MCF-7 cells to doxorubicin (Adriamycin ) was also tested.Results and discussionsTreatment of MCF-7 cells with GNP-DNA conjugate resulted in a significant reduction in cell viability when compared to both control and unmodified GNP groups. The cell toxicity tests revealed that the conjugation of GNP with DNA lead to a 69.4-fold decrease in the IC 50 of GNP in MCF-7 cells. Furthermore, GNP-DNA conjugate showed a synergistic effect with doxorubicin leading to a significant reduction in cell viability at concentrations as low as 70 µM GNP-DNA and 1.6 µM doxorubicin.ConclusionThe results of this study shed light on the marked effect of the modification of small GNP with DNA fragment on the effect of GNP in cancer cells and hence enables studying the effects of GNP size on cellular chemosensitivity without being restricted by GNP size-related differences in uptake efficiency.

Many epithelial cancers have been shown to overexpress the enzyme cyclooxygenase-2 (COX-2), an enzyme responsible for metabolizing anandamide (AEA) to prostamides. AEA has demonstrated cytotoxicity in COX-2 overexpressing cancers via its metabolism to novel J-series prostamides, namely 15d-PMJ2. Fatty acid amide hydrolase (FAAH) degrades AEA into arachidonic acid and ethanolamine (EA), limiting the cytotoxic capability of AEA. Cell lines with high FAAH expression have demonstrated resistance to AEA. By understanding the metabolic characteristics of FAAH, we can design AEA analogs which circumvent FAAH breakdown. To examine the effects of altering polarity, steric bulk, and functional groups on AEA-mediated cytotoxicity, we investigated known AEA derivatives which possess these properties. Arvanil, Arachidonoyl Diethanolamine (ADA), Arachidonoyl Serinol (AS), and R1-methanandamide (m-AEA) add steric bulk to the molecule via aromatic rings, extra EA arms and additional alcohol/methyl functional groups respectively. Arachidonoyl glycine (NAGly) substitutes the terminal EA alcohol with a carboxylic acid increasing polarity. Arachidonoyl-2’-chloroethylamine (AC) substitutes the terminal EA alcohol with a highly soluble chlorine. Furthermore, it is known that Arvanil, ADA, AS, and m-AEA resist degradation of the molecule by FAAH and NAGly is a known substrate of COX-2. Therefore our goal was to determine which structural modifications improve AEA-mediated cytotoxicity. To determine this, JWF2 tumorigenic keratinocytes were exposed to differing concentrations of the AEA analogs for 24 hours and cell viability was measured by conducting MTS assays. Arvanil demonstrated a 90% reduction in cell viability, NAGly demonstrated a 70% reduction in cell viability, and m-AEA demonstrated a 100% reduction in cell viability at 20 µM, the optimal dosage of AEA. Due to the fact that ADA, AS, and AC did not show a significant reduction in cell viability these molecules were not further examined. MTS assays were conducted in other epithelial cancer cell lines with varying expressions of COX-2 and FAAH. Each cell line was exposed to varying concentrations of either NAGly, Arvanil, or both for 24 hours. NAGly demonstrated a 40% reduction in cell viability in HT-29 colon cancer cells (low COX-2, high FAAH). Arvanil demonstrated a 60% reduction in A431 tumorigenic keratinocytes. NAGly demonstrated a 60% reduction in cell viability and Arvanil demonstrated a 100% reduction in cell viability in patient-derived primary melanoma. These findings suggest that modulation and substitute to the core structure of AEA will result in decreased susceptibility to FAAH degradation and enhanced antineoplastic activity. Citation Format: Andrew Morris, Eman Soliman, Rukiyah Van Dross, Colin Burns. Structural modification of the chemotherapeutic anandamide: Designing anti-cancer agents and investigating their COX-2 metabolic products [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2193. doi:10.1158/1538-7445.AM2017-2193

In vitro cytotoxic potential of extracts from Aristolochia foetida Kunth against MCF-7 and bMECs cell lines.

CRLF2 Rearrangement Status in Ph-like ALL Predicts Intrinsic Glucocorticoid Resistance In Vitro that is Reversible with Targeted MAPK and PI3K Pathway Inhibition

Anandamide (AEA) and 2‐arachidonoylglycerol (2‐AG) are compounds naturally produced in humans and animals that interact with the cannabinoid system. These compounds activate specific cannabinoid receptors on cells to alter the neurotransmitter release from presynaptic neuron. There are studies which claim that endocannabinoids are selective towards tumor cells without affecting the functioning of normal cells, mainly by regulating different cell‐death pathways and downstream signaling molecules. The purpose of this study is to evaluate the antiproliferative activity of AEA and 2‐AG in U87‐MG glioblastoma cells. A cell viability assay utilizing 3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium (MTS) was performed to evaluate the antiproliferative effects. The results demonstrated that AEA at concentrations 10 and 30 μM decreased cell viability as compared to control (P≤ 0.001). 2‐arachidonoylglycerol at concentrations 10, 30 and 100 μM decreased cell viability as compared to control (P≤ 0.001). Interestingly, a combination of AEA and 2‐AG at concentrations 10 and 30 μM, respectively reduced cell viability as compared to their individual effects suggesting a synergistic approach. Moreover, a significant reduction in cell viability was observed with a combination of OL‐135, a reversible inhibitor of fatty acid amide hydrolase (FAAH) and AEA as compared to AEA alone. Similarly, a significant reduction in cell viability was observed with a combination of JZL‐184, an irreversible inhibitor for monoacylglycerol lipase (MAGL) and 2‐AG as compared to 2‐AG alone. These results suggest that by inhibiting the enzymes FAAH and MAGL which are involved in the degradation of AEA and 2‐AG, respectively a further reduction in cell viability could be observed, suggesting a strategy to inhibit endocannabinoid degrading enzymes and use of endocannabinoids as potential anti‐cancer agents. Further assays will be performed to determine the mechanism of anti‐cancer activity demonstrated by these compounds.

In order to determine the effects of a variety of flavonoids, we applied differing amounts of several flavonoids to human breast cancer cells. Kaempferol treatment resulted in significant reduction of cell viability in the MCF-7 cells, although it exerted only minor effect on the cell viability of MDA-MB-231 or mammary epithelial HC-11 cells. Kaempferol was demonstrated to induce sustained ERK activation concomitantly with MEK1 and ELK1 activation, and this kaempferol-induced apoptosis was suppressed by treatment with PD98059, the overexpression of a kinase-inactive ERK mutant, or ERK siRNA. Kaempferol treatment was shown to profoundly induce the generation of fluorescent DCF in the MCF-7 cells, and treatment with N-acetyl cysteine suppressed kaempferol-induced PARP cleavage. Moreover, because breast cancer is associated with increased collagen synthesis and accumulation, we utilized a collagen-based 3D culture method. Under the 3-dimensional culture condition employed herein, kaempferol treatment was shown to result in a significant reduction in cell viability, an effect which occurred in a dose-dependent manner. Compared with what was observed under conventional 2D culture condition, we observed more evident apoptotic cell death and ERK activation as the result of kaempferol treatment in a collagen-based 3D culture environment. Similar to the case of conventional 2D cultured cells, the addition of PD98059 significantly suppressed intracellular ROS production. Collectively, these results show that the sustained activation of the ERK signaling pathway is markedly involved in kaempferol-induced apoptosis of breast cancer MCF-7 cells, and that this effect is more evident under 3D culture condition.

Most synovial sarcomas contain a chromosomal translocation t(X;18), which results in the formation of an oncoprotein SS18-SSX critical to the viability of synovial sarcoma. We (1) established and characterized three novel synovial sarcoma cell lines and asked (2) whether inhibition of SS18-SSX1 decreases cell viability in these cell lines; and (3) whether reduction in viability after SS18-SSX1 knockdown is caused by apoptosis. After identifying a specific posttranscriptional splice variant in our cell lines, we asked (4) whether this provides a survival benefit in synovial sarcoma. Cells lines were characterized. SS18-SSX1 knockdown was achieved using a shRNA system. Cell viability was assessed by WST-1 analysis and apoptosis examined by caspase-3 activity. We confirmed the SS18-SSX1 translocation in all cell lines and identified a consistent splicing variant. We achieved successful knockdown of SS18-SSX1 and with this saw a significant reduction in cell viability. Decreased viability was a result of increased apoptosis. Reintroduction of the exon 8 sequence into cells reduced cell viability in all cell lines. We confirmed the presence of the SS18-SSX1 translocation in our cell lines and its importance in the survival of synovial sarcoma. We have also demonstrated that reduction in cell viability is related to an increase in apoptosis. In addition, we have identified a potential mediator of SS18-SSX function in exon 8. SS18-SSX represents a tumor-specific target in synovial sarcoma. Exploitation of SS18-SSX and its protein partners will allow us to develop potent tumor-specific therapeutic agents.

Junctional Adhesion Molecule-A (JAM-A) is a transmembrane protein with important physiological functions in regulating cell-cell adhesion. Pathophysiologically, its high expression in breast tumour tissue has been shown to correlate with that of Human Epidermal Growth Factor Receptor-2 (HER2), whilst JAM-A knockdown in breast cancer cells has been shown to reduce HER2 expression and signaling. Although HER2 has been successfully targeted in the oncology setting for several years, the problem of clinical drug resistance to HER2-targeted therapies (among other factors) has recently put the spotlight on other HER family members and their upstream regulators as potential drug targets. HER3 is the most potent binding partner of HER2 in activating tumor growth signaling, and accumulating evidence suggests that HER3 plays an important role in resistance to anti-HER2 therapies. Furthermore HER3 is frequently overexpressed in HER2-negative breast cancers, and, along with other HER family members, may drive HER2-independent tumorigenic mechanisms. Since JAM-A levels have been reported to regulate HER2 expression (Brennan et al, Oncogene 2013 32(22):2799-804), we hypothesised that JAM-A also regulates expression of HER3 in breast cancer cells. Results from our study showed that stable overexpression of JAM-A in MCF7 breast cancer cells (MCF7-JAM) increased both mRNA and protein expression of HER3. Correspondingly, transient gene silencing of JAM-A reduced the mRNA and protein expression of HER3 in MCF7 and MCF7-JAM cells. JAM-A silencing also reduced HER3 expression in HER2-positive BT474 breast cancer cells. As the cell lines tested were all Estrogen Receptor-α(ERα)-positive, we examined whether ERα was required to permit JAM-dependent regulation of HER3. However concomitant silencing of ERα in MCF7 cells did not alter the capacity of JAM-A silencing to reduce HER3 protein levels. In MCF7 and MCF7-JAM cells, JAM-A gene silencing phenocopied that of HER3 gene silencing by reducing protein expression of the HER downstream effectors phospho-AKT and phospho-ERK, in parallel with significant reductions in cell viability (measured by Alamar Blue assay). To begin exploring the mechanism whereby JAM-A regulates HER3 expression, we focused on the HER3 transcription factor FOXA1. JAM-A knockdown reduced expression of FOXA1 in MCF7 and MCF7-JAM cells, and knockdown of FOXA1 was sufficient to reduce HER3 expression in the same cells. Taken together, our data provide novel evidence of a direct relationship between levels of JAM-A, FOXA1 and HER3 in breast cancer cells. The relationship appears to be uni-directional, since silencing of HER3 did not alter JAM-A expression in any cell line tested. In conclusion, we suggest that JAM-A merits investigation as a novel target to inhibit HER3-dependent tumorigenic signaling in breast cancer. Our ongoing investigations will determine the pharmacological value of inhibiting JAM-A signaling in breast cancer models, and its potential significance in the setting of resistance to HER2-targeted therapies. Citation Format: Cruz RGB, Hopkins AM. Junctional adhesion molecule-A is a novel upstream regulator of human epidermal growth factor receptor-3 signaling in breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-03-07.

Increasing evidence suggests that unknown collagen remodeling mechanisms in the sclera underlie myopia development. We are proposing a novel organ culture system in combination with two-photon fluorescence imaging to quantify collagen remodeling at the tissue- and lamella-level. Tree shrew scleral shells were cultured up to 7 days in serum-free media and cellular viability was investigated under: (i) minimal tissue manipulations; (ii) removal of intraocular tissues; gluing the eye to a washer using (iii) 50 μL and (iv) 200 μL of cyanoacrylate adhesive; (v) supplementing media with Ham's F-12 Nutrient Mixture; and (vi) culturing eyes subjected to 15 mmHg intraocular pressure in our new bioreactor. Two scleral shells of normal juvenile tree shrews were fluorescently labeled using a collagen specific protein and cultured in our bioreactor. Using two-photon microscopy, grid patterns were photobleached into and across multiple scleral lamellae. These patterns were imaged daily for 3 days, and tissue-/lamella-level strains were calculated from the deformed patterns. No significant reduction in cell viability was observed under conditions (i) and (v). Compared to condition (i), cell viability was significantly reduced starting at day 0 (condition (ii)) and day 3 (conditions (iii, iv, vi)). Tissue-level strain and intralamellar shear angel increased significantly during the culture period. Some scleral lamellae elongated while others shortened. Findings suggest that tree shrew sclera can be cultured in serum-free media for 7 days with no significant reduction in cell viability. Scleral fibroblasts are sensitive to tissue manipulations and tissue gluing. However, Ham's F-12 Nutrient Mixture has a protective effect on cell viability and can offset the cytotoxic effect of cyanoacrylate adhesive. This is the first study to quantify collagen micro-deformations over a prolonged period in organ culture providing a new methodology to study scleral remodeling in myopia.

Objectives Targeted inhibitors of the PI3 kinase (PI3K) pathway have shown promising but incomplete antitumor activity in preclinical chordoma models. The aim of this study is to advance methodology for a high-throughput drug screen using chordoma models to identify new combination therapies for chordoma. Study Design Present work is an in vitro study. Setting The study conducted at an academic research laboratory. Materials and Methods An in vitro study on automated high-throughput screening of chordoma cells was performed using a library of 1,406 drugs as both mono- and combination therapies with PI3K inhibitors. Combination indices were determined for dual therapies and synergistic outliers were identified as potential therapeutic agents. T (brachyury) siRNA knockdown in combination with PI3K pathway inhibition was also assessed. Results Fifty-nine combination therapies were identified as having potential therapeutic efficacy. Effective combinations included PI3K inhibitors with GSK1838705A (ALK/IGF-1R inhibitor), LY2874455 (VEGFR/FGFR inhibitor), El1 (selective Ezh2 inhibitor), and (-)-p-bromotetramisole oxalate (alkaline phosphatase inhibitor). The top ranking targets identified included ALK, PDGFR, VEGFR, aurora kinase, and BCL-2. T (brachyury) inhibition produced significant reduction in cell viability and growth; however PI3K inhibition in combination with T (brachyury) knockdown did not result in further reduction in growth and viability in vitro. Conclusion High throughput with in vitro combination screening is feasible with chordoma cells and allows for rapid identification of synergistic dual-therapies. Potential combination therapies and targetable pathways were identified. T (brachyury) knockdown produced significant reduction in cell viability, but did not show additional benefit with PI3K pathway inhibition in this model. Further in vitro and in vivo validation of these therapeutic combinations is warranted.