Establishment and characterisation of chemoresistant osteosarcoma cell lines by single and multi-agent induced strategies

Osteosarcoma is a rare malignant bone tumour that occurs primarily in adolescents and young adults. Prior to the adoption of chemotherapy in mid 1970s, more than 85% if post-surgery osteosarcoma patients developed metastasis. Nowadays, standard osteosarcoma treatment of osteosarcoma includes neo-adjuvant chemotherapy followed by surgical removal and adjuvant multi-drug chemotherapy. The combination of cisplatin, doxorubicin and high-dose methotrexate is the standard treatment for most patients. Surgery combined with chemotherapy has improved the survival rate for osteosarcoma patients to 60% - 70%. However, most of the patients with metastatic or recurrent osteosarcoma have poor prognosis due to the development of chemotherapeutic drug resistance. Developing drug-resistant cancer cell models is one approach to study the mechanisms of chemoresistance in cancer cells. In this study, clinically relevant chemoresistant osteosarcoma cell models were developed from the cell lines MG-63 and HOS-143B. One of the strategies used the current study includes a multiple drugs combination approach, where cisplatin, doxorubicin, and methotrexate are combined in one treatment. The purpose of this method is to simulate a similar experience with osteosarcoma patients who are receiving clinical chemotherapy treatment and therefore, to establish a clinically relevant osteosarcoma-resistant model to study the mechanisms of drug resistance. Cisplatin, doxorubicin, and methotrexate were used as single agents and in triple combination. The highest level of resistance to cisplatin was observed in MG-63/CISR8 (3.56 ± 0.43-fold; p=0.001), doxorubicin in HOS-143B/DOXR8 (1.99 ± 0.20; p=0.0002), and methotrexate in HOS-143B/MTXR8 (3.77 ± 0.90-fold; p=0.046). The MG-63/TRIR8 and HOS-143B/TRIR8 triple-resistance models showed lower levels of resistance, 2.28 ± 0.63-fold (p=0.032) and 2.17 ± 0.13-fold resistant (p=0.0004) to combination treatment; and were not resistant to the drugs individually. Apoptosis assays suggest that the resistance in MG-63/TRIR8 is mainly from cisplatin and methotrexate and not doxorubicin. In contrast, the resistance in HOS-143B/TRIR8 is mainly from doxorubicin and methotrexate instead of cisplatin. Upregulation of P-glycoprotein was seen in all resistant models except those developed with single-agent methotrexate. The P-glycoprotein inhibitor elacridar reversed the resistance of doxorubicin on MG-63/DOXR8 (0.36 ± 0.06-fold, p=0.003), MG-63/TRIR8 (0.72 ± 0.07-fold, p=0.04), HOS-143B/CISR8 (0.47 ± 0.09-fold, p=0.009), and HOS-143B/TRI (0.45 ± 0.03-fold, p=0.0005). The migration rate of the MG-63 resistant models was significantly increased by 2.12 – 2.46-fold, their invasion rate tended to increase, and RT-PCR showed a switch from epithelial to mesenchymal gene signalling. In contrast, a significant decrease in migration was seen in HOS-143B resistant models with 0.39 – 0.43-fold, their invasion rate tended to decrease and a switch from mesenchymal to epithelial gene signalling occurred.SPHK1 and HIF1A were upregulated in most of the resistant models from the PCR array analysis and SPHK1 protein level was also determined to increase in MG-63/CISR8 (2.03 ± 0.08-fold, p=0.034), MG-63/DOXR8 (1.77 ± 0.24-fold, p=0.02), and HOS-143B/CISR8 (3.55 ± 0.84-fold, p=0.0459). A strong correlation (r=0.726) was shown between the gene expression of SPHK1 and HIF1A in HOS-143B resistant sublines. Currently, there is also a lack of effective treatments for patients who experience relapsed osteosarcoma. One treatment for relapsed patients is gemcitabine and docetaxel combination chemotherapy (GEMDOX). A systematic review was performed in this study to investigate the efficacy of combination therapy of gemcitabine and docetaxel on relapsed osteosarcoma patients. The results showed the age and gender of the patients would have a prognostic effect on the GEMDOX regimen as the second-line treatment for relapsed osteosarcoma, whereas the GEMDOX therapy was determined to have a higher efficacy on male patients and with age <18. There was no difference in toxicities between different doses (675 mg/m2 and 1,000 mg/m2) of the GEMDOX regimen, age, or gender of patients. A preclinical in vitro study was performed by investigating the sensitivity of GEMDOX therapy on the established resistant sublines. The established resistant osteosarcoma sublines were used to investigate the efficacy of the GEMDOX treatment in the relapsed setting. Out of 8 of the resistant models, MG-63/DOXR8 was significantly resistant to gemcitabine (2.44 ± 0.26-fold, p=0.001) compared to MG-63 and HOS-143B/MTXR8 was significantly resistant to docetaxel (2.32 ± 0.17-fold, p=0.005) compared to HOS-143B. These two resistant sublines were also significantly resistant to the combination of gemcitabine and docetaxel with 2.50 ± 0.53-fold (p=0.04) and 2.09 ± 0.32-fold (p=0.017) respectively. However, the rest of the 6 resistant sublines were not resistant to GEMDOX treatment, which indicates GEMDOX regimen as a potential therapeutic treatment for relapsed osteosarcoma. This project is the first to develop chemoresistant osteosarcoma cell lines with a triple combination of drugs. The characteristics of these resistant models also provide a better understanding of the resistant mechanisms in osteosarcoma cells. Lastly, these developed single and multi-agents induced clinically-relevant osteosarcoma cell lines could act as an invaluable tool for future studies of drug resistant mechanisms in osteosarcoma cells

Laser anneal of oxycarbosilane low-k film

Submilisecond laser anneal has been experimentally investigated for porogen removal and its ability to improve the mechanical strength in oxycarbosilane ultra low-k films compromised due to the introduction of porosity. We report the occurrence of extensive bond rearrangements inferred from Fourier-transform infra-red (FTIR) spectroscopy, elastic recoil detection (ERD) and spectroscopic ellipsometry (SE) in the energy range of 1.4-8 eV. The laser anneal affects most notably the organic content of the organosilicate matrix leading to depletion and reorganization. Nevertheless, the tested conditions reveal a processing window which allows for 13% improvement of Young’s modulus as compared to the reference film, annealed in a conventional furnace at 400°C for 2 h, while not impacting the relative dielectric constant of 2.25

On the lowest energy excitations of one-dimensional strongly correlated electrons

It is proven that the lowest excitations $E_{low}(k)$ of one-dimensional half-integer spin generalized Heisenberg models and half-filled extended Hubbard models are $\pi$-periodic functions. For Hubbard models at fractional fillings $E_{low}{(k+ 2 k_f)} = E_{low}(k)$, where $2 k_f= \pi n$, and $n$ is the number of electrons per unit cell. Moreover, if one of the ground states of the system is magnetic in the thermodynamic limit, then $E_{low}(k) = 0$ for any $k$, so the spectrum is gapless at any wave vector. The last statement is true for any integer or half-integer value of the spin.Comment: 6 Pages, Revtex, final versio

Model-independent low momentum nucleon interaction from phase shift equivalence

We present detailed results for the model-independent low momentum nucleon-nucleon interaction V_{low k}. By introducing a cutoff in momentum space, we separate the Hilbert space into a low momentum and a high momentum part. The renormalization group is used to construct the effective interaction V_{low k} in the low momentum space, starting from various high precision potential models commonly used in nuclear many-body calculations. With a cutoff in the range of $\Lambda ~ 2.1 fm^{-1}$, the new potential V_{low k} is independent of the input model, and reproduces the experimental phase shift data for corresponding laboratory energies below $E_{lab} ~ 350 MeV$, as well as the deuteron binding energy with similar accuracy as the realistic input potentials. The model independence of V_{low k} demonstrates that the physics of nucleons interacting at low momenta does not depend on details of the high momentum dynamics assumed in conventional potential models. V_{low k} does not have momentum components larger than the cutoff, and as a consequence is considerably softer than the high precision potentials. Therefore, when V_{low k} is used as microscopic input in the many-body problem, the high momentum effects in the particle-particle channel do not have to be addressed by performing a Brueckner ladder resummation or short-range correlation methods. By varying the cutoff, we study how the model independence of V_{low k} is reached in different partial waves. This provides numerical evidence for the separation of scales in the nuclear problem, and physical insight into the nature of the low momentum interaction.Comment: 32 pages, 19 figure

Mechanistic Study Of Plasma Damage Of Low k Dielectric Surfaces

Plasma damage to low k dielectric materials was investigated from a mechanistic point of view. Low k dielectric films were treated by plasma Ar, O-2, N-2/H-2, N-2 and H-2 in a standard RIE chamber and the damage was characterized by Angle Resolved X-ray Photoelectron Spectroscopy (ARXPS), X-Ray Reflectivity (XRR), Fourier Transform Infrared Spectroscopy (FTIR) and Contact Angle measurements. Both carbon depletion and surface densification were observed on the top surface of damaged low k materials while the bulk remained largely unaffected. Plasma damage was found to be a complicated phenomenon involving both chemical and physical effects, depending on chemical reactivity and the energy and mass of the plasma species. A downstream hybrid plasma source with separate ions and atomic radicals was employed to study their respective roles in the plasma damage process. Ions were found to play a more important role in the plasma damage process. The dielectric constant of low k materials can increase up to 20% due to plasma damage and we attributed this to the removal of the methyl group making the low k surface hydrophilic. Annealing was generally effective in mitigating moisture uptake to restore the k value but the recovery was less complete for higher energy plasmas. Quantum chemistry calculation confirmed that physisorbed water in low k materials induces the largest increase of dipole moments in comparison with changes of surface bonding configurations, and is primarily responsible for the dielectric constant increase.Microelectronics Research Cente

Investigation of ultra-thin Al₂O₃ film as Cu diffusion barrier on low-k (k=2.5) dielectrics

Ultrathin Al(2)O(3) films were deposited by PEALD as Cu diffusion barrier on low-k (k=2.5) material. The thermal stability and electrical properties of the Cu/low k system with Al(2)O(3) layers with different thickness were studied after annealing. The AES, TEM and EDX results revealed that the ultrathin Al(2)O(3) films are thermally stable and have excellent Cu diffusion barrier performance. The electrical measurements of dielectric breakdown and TDDB tests further confirmed that the ultrathin Al(2)O(3) film is a potential Cu diffusion barrier in the Cu/low-k interconnects system

Pairing Matrix Elements and Pairing Gaps with Bare, Effective and Induced Interactions

The dependence on the single-particle states of the pairing matrix elements of the Gogny force and of the bare low-momentum nucleon-nucleon potential $v_{low-k}$ is studied in the semiclassical approximation for the case of a typical finite, superfluid nucleus ($^{120}$Sn). It is found that the matrix elements of $v_{low-k}$ follow closely those of $v_{Gogny}$ on a wide range of energy values around the Fermi energy $e_F$, those associated with $v_{low-k}$ being less attractive. This result explains the fact that around $e_F$ the pairing gap $\Delta_{Gogny}$ associated with the Gogny interaction (and with a density of single-particle levels corresponding to an effective $k$-mass $m_k\approx 0.7 m$) is a factor of about 2 larger than $\Delta_{low-k}$,being in agreement with $\Delta_{exp}$= 1.4 MeV. The exchange of low-lying collective surface vibrations among pairs of nucleons moving in time-reversal states gives rise to an induced pairing interaction $v_{ind}$ peaked at $e_F$. The interaction $(v_{low-k}+ v_{ind})Z_{\omega}$ arising from the renormalization of the bare nucleon-nucleon potential and of the single-particle motion ($\omega-$mass and quasiparticle strength $Z_{\omega}$) due to the particle-vibration coupling leads to a value of the pairing gap at the Fermi energy $\Delta_{ren}$ which accounts for the experimental value