NSG Mice Facilitate ex vivo Characterization of Ewing Sarcoma Lung Metastasis Using the PuMA Model

Ewing sarcoma (EwS) is a highly malignant bone and soft tissue tumor primarily affecting children and young adults. While most patients initially respond well to conventional front-line therapy, frequent metastasis results in poor 5-year overall survival rates for this disease. Accordingly, there is a critical need to develop better models to understand EwS metastasis. We and others previously used the ex vivo pulmonary metastasis assay (PuMA) to study lung metastasis in solid tumors including osteosarcoma (OS), but this technique has to date not been achievable for EwS. PuMA involves tail vein injection of fluorescent tumor cells into NOD-SCID mice, followed by their visualization in long-term cultures of tumor-bearing lung explants. Here we demonstrate successful implementation of PuMA for EwS cells using NOD-SCID-IL2 receptor gamma null (NSG) immunocompromised mice, which demonstrated high engraftment of EwS cell lines compared to NOD-SCID mice. This may be linked to immune permissiveness required by EwS cells, as increased basal cytotoxicity of EwS cells was observed in NOD-SCID compared to NSG lung sections, possibly due to the absence of natural killer (NK) cell activity in the latter. Together, our data demonstrate the utility of NSG mice for PuMA modeling of EwS lung metastasis.

Pleiotropic Properties of Amphiphilic Dihydropyridines, Dihydropyridones, and Aminovinylcarbonyl Compounds

Three groups of synthetic lipids are chosen for studies: (1) 1,4-dihydropyridines (1,4-DHPs) containing two cationic moieties and their analogues; (2) 3,4-dihydro-2(1H)-pyridones containing a cationic moiety; and (3) acyclic, open-chain analogues, i.e., 2-amino-3-alkoxycarbonylalkylammonium derivatives. 1,4-DHPs possessing dodecyl alkyl chains in the ester groups in positions 3 and 5 and cationic nitrogen-containing groups in positions 2 and 6 have high cytotoxicity in cancer cells HT-1080 (human lung fibrosarcoma) and MH-22A (mouse hepatoma), but low cytotoxicity in the noncancerous NIH3T3 cells (mouse embryonic fibroblast). On the contrary, similar compounds having short (methyl, ethyl, or propoxyethyl) chains in the ester groups in positions 3 and 5 lack cytotoxicity in the cancer cells HT-1080 and MH-22A even at high doses. Inclusion of fluorine atoms in the alkyl chains in positions 3 and 5 of the DHP cycle decreases the cytotoxicity of the mentioned compounds. Structurally related dihydropyridones with a polar head group are substantially more toxic to normal and cancerous cells than the DHP analogues. Open-chain analogues of DHP lipids comprise the same conjugated aminovinylcarbonyl moiety and possess anticancer activity, but they also have high basal cytotoxicity. Electrochemical oxidation data demonstrate that oxidation potentials of selected compounds are in the range of 1.6–1.7 V for cationic 1,4-DHP, 2.0–2.4 V for cationic 3,4-dihydropyridones, and 1.2–1.5 V for 2-amino-3-alkoxycarbonylalkylammonium derivatives. Furthermore, the tested cationic 1,4-DHP amphiphiles possess antiradical activity. Molecular topological polar surface area values for the tested compounds were defined in accordance with the main fragments of compound structures. The determined log P values were highest for dodecyl ester groups in positions 3 and 5 of the 1,4-DHP and lowest for short alkyl chain-containing amphiphiles.

Contribution of Molecular Structure to Self-Assembling and Biological Properties of Bifunctional Lipid-Like 4-(N-Alkylpyridinium)-1,4-Dihydropyridines

The design of nanoparticle delivery materials possessing biological activities is an attractive strategy for the development of various therapies. In this study, 11 cationic amphiphilic 4-(N-alkylpyridinium)-1,4-dihydropyridine (1,4-DHP) derivatives differing in alkyl chain length and propargyl moiety/ties number and position were selected for the study of their self-assembling properties, evaluation of their cytotoxicity in vitro and toxicity on microorganisms, and the characterisation of their interaction with phospholipids. These lipid-like 1,4-DHPs have been earlier proposed as promising nanocarriers for DNA delivery. We have revealed that the mean diameter of freshly prepared nanoparticles varied from 58 to 513 nm, depending upon the 4-(N-alkylpyridinium)-1,4-DHP structure. Additionally, we have confirmed that only nanoparticles formed by 4-(N-dodecylpyridinium)-1,4-DHP derivatives 3 and 6, and by 4-(N-hexadecylpyridinium)-1,4-DHP derivatives 10 and 11 were stable after two weeks of storage. The nanoparticles of these compounds were found to be homogenous in size distribution, ranging from 124 to 221 nm. The polydispersity index (PDI) values of 1,4-DHPs samples 3, 6, 10, and 11 were in the range of 0.10 to 0.37. We also demonstrated that the nanoparticles formed by 4-(N-dodecylpyridinium)-1,4-DHP derivatives 3, 6, and 9, and 4-(N-hexadecylpyridinium)-1,4-DHP derivatives 10 and 11 had zeta-potentials from +26.07 mV (compound 6) to +62.80 mV (compound 11), indicating a strongly positive surface charge and confirming the relative electrostatic stability of these nanoparticle solutions. Transmission electron microscopy (TEM) images of nanoaggregates formed by 1,4-DHPs 3 and 11 confirmed liposome-like structures with diameters around 70 to 170 nm. The critical aggregation concentration (CAC) value interval for 4-(N-alkylpyridinium)-1,4-DHP was from 7.6 µM (compound 11) to 43.3 µM (compound 6). The tested 4-(N-alkylpyridinium)-1,4-DHP derivatives were able to quench the fluorescence of the binary 1,6-diphenyl-1,3,5-hexatriene (DPH)—1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) system, demonstrating hydrophobic interactions of 1,4-DHPs with phospholipids. Thus, 4-(N-dodecylpyridinium)-1,4-DHP derivative 3 quenched the fluorescence of the DPH–DPPC system more efficiently than the other 4-(N-alkylpyridinium)-1,4-DHP derivatives. Likewise the compound 3, also 4-(N-dodecylpyridinium)-1,4-DHP derivative 9 interacted with the phospholipids. Moreover, we have established that increasing the length of the alkyl chain at the quaternised nitrogen of the 4-(N-alkylpyridinium)-1,4-DHP molecule or the introduction of propargyl moieties in the 1,4-DHP molecule significantly influences the cytotoxicity on HT-1080 (human fibrosarcoma) and MH-22A (mouse hepatocarcinoma) cell lines, as well as the estimated basal cytotoxicity. Additionally, it was demonstrated that the toxicity of the 4-(N-alkylpyridinium)-1,4-DHP derivatives on the Gram-positive and Gram-negative bacteria species and eukaryotic microorganism depended on the presence of the alkyl chain length at the N-alkyl pyridinium moiety, as well as the number of propargyl groups. These lipid-like compounds may be proposed for the further development of drug formulations to be used in cancer treatment.

Immunotherapy with LAK Cells and Anti-CD20 Monoclonal Antibodies for Follicular Lymphoma: Enhanced Antibody-Dependent Cell Cytotoxicity of LAK Cells in Association with GA101 Rather Than Rituximab

Abstract 4881 Background The current gold standard treatment for follicular lymphoma (FL) consists in the association of rituximab, that is an anti-CD20 monoclonal antibody (mAb), and one among many possible chemotherapy regimens, followed by maintenance with the same mAb. However, LF is still considered incurable. Hence, it is necessary to keep investigating new strategies aiming at improving the overall clinical results. As an anti-tumor mechanism, antibody-dependent cellular cytotoxicity (ADCC) depends on both the immune system effector arm and the antibody structure. The culture of peripheral blood lymphocytes with IL-2 activates killer cell subpopulations (LAK cells), mainly consisting of natural killer (NK) cells and cytotoxic T lymphocytes (CTL), both with significant cytotoxic capacity. The combination of LAK cells with a mAb could achieve a synergistic effect by enhancing ADCC and potentially achieving clinical efficacy. As structural modifications in mABs may alter their biological activity, new anti-CD20 with structural changes in the Fc portion of the immunoglobulin have been synthesized in order to obtain a greater therapeutic effect. GA101 is a new anti-CD20 mAB that binds with higher affinity to the CD20 type II epitope, increasing the ADCC effect as compared to to rituximab. Currently, no data support the notion of a possible synergistic effect of GA101 and LAK cells. Aim The objective of this work was to assess the cytotoxic capacity of LAK cells generated from the peripheral blood of patients with FL. In addition, we aimed at ascertaining whether the combination of rituximab and LAK could improve the functional activity of LAK alone. Finally, we conducted a comparative study of two anti-CD20 antibodies structurally different, namely rituximab and GA101, used in combination with LAK cells. Material and methods LAK cells were expanded in vitro from 39 peripheral blood samples of patients with FL. Cytotoxicity studies were performed using chromium release assays in which LAK cells targeted K562 (as NK target), Daudi (as LAK cells target) and CRL-1596 (CD20 positive) cells. Overall, mononuclear cells were isolated and basal cytotoxicity was measured in 39 peripheral blood samples from patients with FL. The ability of two anti-CD20 antibody (rituximab and GA101) to enhance LAK cell activity was evaluated against the CRL-1596 cell line. As a control, an irrelevant antibody (cetuximab) was used. To perform these studies, target cells were incubated with effector cells and antibody at 10 mg/ml concentration. Results Results are expressed as mean percentages. Basal cytotoxicity against K562, Daudi and CRL-1596 cells was 19.4%, 11.9% and 1.5% respectively. After culture with IL-2, the cytotoxicity activity against the same cell lines was reassessed and in all cases a statistically significant increase was observed (p<0,05). In particular, cytotoxicity against K562, Daudi and CRL-1596 was 39.5%, 39.6% and 21.1%, respectively. Furthermore, we studied the rituximab effect on the cytotoxic capacity against CRL-1596. The observed cytotoxicity of LAK cells with rituximab was 39.9% vs 23.6% for LAK cells alone (p<0,001). In parallel, the GA101 effect on the cytotoxic capacity against CRL-1596 was also evaluated. The cytotoxicity of LAK cells with GA101 was 52.3% vs 23.6% for LAK cells alone (p<0,001). Vice versa, no difference was observed in the cytotoxic activity of LAK cells against CRL1596 cell line when the effector cells were incubated with the irrelevant antibody cetuximab (26.0% vs 23.6%). Finally, when comparing the ability of both anti-CD20 antibodies to enhance ADCC, we observed a significant difference in favor of GA101 (GA101: 52.3%, rituximab: 39.9%; p<0,001). Conclusions LAK cells generated from peripheral blood lymphocytes by culture with IL-2 in patients with LF show a higher cytotoxic activity than naive lymphocytes. The observed cytotoxic capacity of these LAK cells against a CD20 positive cell line (CRL-1596) is enhanced by the addition of anti-CD20 mAbs. Interestingly, GA101 was consistently more effective than rituximab in enhancing the cytotoxic capacity of LAK cells. Disclosures: No relevant conflicts of interest to declare.