Category: Preclinical Development
Purpose: Conventionally used in vitro cancer models such as cancer cells cultured as a monolayer, are not representative of the in vivo environment. Systematic scaffold-based generation of cancer spheroids (CS) represent cell-cell or cell-extracellular matrix interactions features of solid tumors  formed in humans and hence can be used for accurate early screening of promising drugs. Here, we propose to develop porous PLGA (poly (lactic-co-glycolic acid)) microspheres (PPMS) using a novel alginate microspheres (AMS) porogen-based controlled pore formation method. The PPMS were subsequently used to develop scaffold-based lung cancer spheroids using co-culture of lung adenocarcinoma cells (A549) and human lung fibroblasts (HLF) cell lines, with uniform size and morphology.
Methods: The AMS porogen was developed by water-in-oil emulsion technique as described by Zhu et al.  with minor modification. PPMS was developed by double emulsion solvent evaporation technique using AMS as the porogen whereas non-porous PLGA microspheres (PLGAMS) were formulated by conventional single emulsion method . Formulated microspheres were then treated with ethylenediaminetetraacetic acid (EDTA) to digest AMS followed by centrifugation and washing to get purified PPMS and PLGAMS. Surface and cross-sectional morphologies of PPMS and PLGAMS were observed using scanning electron microscopy (SEM). The microspheres were treated with A549 lung adenocarcinoma cells, which were then removed by trypsinization, to evaluate the degradation pattern of the MPs following cell attachment. Samples were analyzed at predetermined intervals (1, 2, 4, 7, 14, 21 and 28 days) for the change in weight and surface morphology. A549 cells cultured on PPMS and PLGAMS were also evaluated for cell viability using live/ dead staining and cell seeding density was optimized by WST-1 assay. Optimized cell density was used to develop a co-culture (lung adenocarcinoma cells (A549) and human lung fibroblasts (HLF) cell lines) spheroid. Co-culture spheroids were finally evaluated by live dead staining, cell trace dyes (DiO for HLF and DiD for A549) and WST-1 assay for viability before drug testing.
Results: The AMS had the diameter of 10 ± 4 µm whereas PLGAMS and PPMS had diameters of 79 ± 22 and 103 ± 30 µm. Both microspheres had spherical morphologies, and the pores on PPMS can be seen in Figure 1A and 1B. Interconnected pores within the PPMS was visualized by cryo-SEM (Figure 1C and 1D). Following cell attachment, PPMS degrades faster than PLGAMS and the particle morphology was distorted with time. SEM images of PPMS with A549 and after removal of cells by trysinization at day 14 demonstrated that PPMS can serve as a potential/better scaffold system than PLGAMS for the growth of cancer cells. WST-1 assay confirms that the lung cancer cells could attach and proliferate on both PPMS (Figure 1G) and PLGAMS (Figure 1H) at all the cell seeding densities (5 x 104, 10 x 104 and 25 x 104 cells/well/mg of particles). Co-culture studies without particles (figure 1E) and with particles (Figure 1F) using cell trace dyes suggests that both the cell lines can attach and proliferate simultaneously on particles over time, which mimics in vivo environment for drug testing.
Conclusion: PPMS formulated in the present investigation had a spherical morphology and large pores. It degraded faster than non-porous particles and supported A549 cell attachment and growth. Although porous microparticles have been used in various research applications, we report a novel method of safe and controlled pore formation using AMS and EDTA. The PPMS thus formed was demonstrated as a robust and favorable architecture for cell attachment and development of in vitro co-culture lung cancer models for drug screening application. Future studies include studying A549-HLF co-cultures on the microspheres over time and using the PPMS to develop tumor models using human patient-derived cells for predictive screening of therapeutic agents.
Duong Le– Kingston, Rhode Island
Rachel Movsas– Kingston, Rhode Island
Andrea Gonsalves– Kingston, Rhode Island
Jyothi Menon– Assistant Professor, Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of Rhode Island, Kingston, Rhode Island