(W1030-07-39) Mesenchymal Stem Cell-Based Combination Therapy for Enhanced Efficacy Against Patient-Derived Lung Cancer Xenograft Model

Purpose: Lung cancer is the third most common type of cancer, with the highest death estimated in 2024. The lack of efficient strategies to achieve cytotoxic concentrations of chemotherapeutics in the tumor tissue and the tumor's innate resistance to chemotherapy are significant barriers to improving treatment outcomes for lung cancer. Therefore, tumor-targeted drug delivery can improve therapeutic effectiveness while minimizing chemotherapy-associated nonspecific toxicity. Additionally, combining chemotherapeutic agents can enhance therapeutic efficacy compared to the monotherapy approach since it simultaneously targets multiple key pathways synergistically or additively while reducing drug resistance and metastatic potential. Herein, we used a combination of paclitaxel (PTX) and entinostat (ENT, an HDAC inhibitor), where ENT can increase the PTX sensitivity towards lung cancer.

Methods: Initially, A549 and H1299 cells were treated with various concentrations of PTX (0.1– 100 nM) and ENT (0.1 – 100 µM) solutions as monotherapy and combination therapy for 3 days, and cell viability was measured using an MTS assay. The viability data was fed into combenefit software to determine synergistic/antagonistic interactions. We look at the mechanism of synergism by determining HDAC, survivin, and Cleaved Caspase 3 expression in treated cells. The drugs were individually encapsulated into dibenzyl cyclooctane (DBCO)-functionalized poly lactic-co-glycolic acid (PLGA) nanoparticles using the solvent emulsification evaporation method. Nanoparticles were characterized for particle size, zeta potential, drug loading, and drug release behavior. Nanocarriers are loaded into MSCs via endocytosis and surface conjugation. Surface anchoring was performed by forming covalent bonds using click chemistry between the DBCO functionalities on the nanocarrier and the artificial azide group on the MSC (MSC-Az) surface. MSCs were treated with 25 µM of N-azidoacetylmannosamine-tetraacylated (Ac₄ManNAz) sugar for 3 days to generate artificial azide on their surface. MSC-Az was incubated separately with PTX-NP or ENT-NP suspension (0.1 mg/mL) in a CO₂ incubator for 4 hours to generate nanoparticle-loaded MSCs (nano-MSCs). In the next step, we characterized nano-MSCs for drug loading capacity, drug release behavior, differentiation capacity, and phenotypes. In vitro cytotoxicity of PTX or ENT-loaded nano-MSCs, or their combinations, was performed against A549 and H1299 cells using trans-well plates. The antitumor efficacy of nano-MSCs was evaluated in a patient-derived tumor xenograft (PDX) lung cancer model in athymic nude mice.

Results: We observed synergistic killings of A549 and H1299 cells at multiple PTX and ENT concentration combinations. As expected, ENT treatment enhanced acetylated histone H3 and H4 expression in treated cells, which was even augmented in the presence of PTX. Similarly, cells treated with a combination therapy showed higher expression of cleaved caspase 3 and increased apoptosis compared to monotherapy. On the other hand, combination therapy suppressed survivin expression, which is responsible for drug resistance. The nanoparticles of PTX (size: 276 nm, surface charge: -17.6 mV, drug loading: 21.5 w/w%) and ENT (size: 288.8 nm, surface charge: -18.7 mV, and drug loading 15.2% w/w) exhibited sustained drug release profiles. The nano-MSCs possess a loading capacity of 56 pg/cell and 41 pg/cell for PTX and ENT, respectively. Furthermore, the loading of nanocarriers on MSCs did not alter the MSC's differentiation ability and phenotype. The nano-MSCs exhibited efficient in vitro cytotoxicity against A549 and H1299 cells. Most importantly, nano-MSCs mediated PTX and ENT combination exhibited enhanced antitumor efficacy and superior survival than other treatment groups.

Conclusion: We reported a novel therapeutic strategy against lung cancer by combining a novel HDAC inhibitor ENT with PTX. The combination therapy exhibited synergistic cell killing against lung cancer cells over various drug concentrations. The synergistic cytotoxicity could be attributed to higher HDAC inhibition, greater extent of apoptosis, and suppression of survivin expression by the combination treatment. To enhance the therapeutic ability of this drug combination, both PTX and ENT were encapsulated in MSCs. The nano-MSCs exhibited enhanced antitumor efficacy against in vitro and in vivo models of lung cancers.

Figure: Synergy analysis by the Loewe model for paclitaxel and entinostat combination using Combenefit® software. (A) A549 cells and (B) H1299 cells. (n = 4).


Drug uptake by MSCs when incubated with nanoparticle suspension at a concentration of 0.1 mg/ml at different periods. (A) PTX uptake (B) ENT uptake. MSC-Az-PTX NP: Azide-expressed MSCs incubated with PTX NP. MSC-PTX: Control MSCs incubated with PTX NP. MSC-Az-ENT: Azide-expressed MSCs incubated with entinostat nanoparticles. MSC-ENT: Control MSCs incubated with ENT NP. Data represents mean ± SD (n = 3).

Antitumor efficacy of a combination of PTX NP-loaded nanoMSCs and ENT NP-loaded nanoMSCs against lung cancer mice model. Patient-derived xenograft lung cancer mice were either treated with saline, MSC (2 × 106 MSC), paclitaxel loaded nanocarriers (equivalent to 5mg kg−1 PTX, “PTX NP”), entinostat loaded nanocarriers (equivalent to 5mg kg−1 ENT, “ENTNP”), PTX NP + ENT NP (equivalent to 5mg kg−1 PTX + equivalent to 5mg kg−1 ENT), PTX NP loaded nanoMSCs (2 × 106 MSC, “MSC-PTX-NP”), ENT NP loaded nanoMSCs (2 × 106 MSC, “MSC-ENT-NP”), MSC-PTX-NP+MSC-ENT-NP (2 × 106 MSC-PTX-NP + 2 × 106 MSC-ENT-NP). All animals received respective treatments every 7 days. Tumor size was determined weekly using vernier caliper (mean ± SEM; n = 6; 3 male and 3 female).