Selective Cytotoxicity of a Novel Trp‐Rich Peptide against Lung Tumor Spheroids Encapsulated inside a 3D Microfluidic Device

Abstract

There is a globally rising healthcare need to develop new anticancer therapies as well as to test them on biologically relevant in vitro cancer models instead of overly simplistic 2D models. To address both these needs, a 3D lung cancer spheroid model is developed using human A549 cells trapped inside a collagen gel in a compartmentalized microfluidic device and homogenously sized (35–45 µm) multicellular tumor spheroids are obtained in 5 days. The novel tryptophan‐rich peptide P1, identified earlier as a potential anticancer peptide (ACP), shows enhanced cytotoxic efficacy against A549 tumor spheroids (>75%) in clinically relevant low concentrations, while it does not affect human amniotic membrane mesenchymal stem cells at the same concentrations (<15%). The peptide also inhibits the formation of tumor spheroids by reducing cell viability as well as lowering the proliferative capacity, which is confirmed by the expression of cell proliferation marker Ki‐67. The ACP offers a novel therapeutic strategy against lung cancer cells without affecting healthy cells. The microfluidic device used is likely to be useful in helping develop models for several other cancer types to test new anticancer agents.

Indirect co-culture of lung carcinoma cells with hyperthermia-treated mesenchymal stem cells influences tumor spheroid growth in a collagen-based 3-dimensional microfluidic model

“…On day 5, the average diameter for the co-culture group was 44 mm, whereas it was only 38 mm for the monoculture group, indicating a significant increase of 15.7% (P< 0.005). The authors have shown in an earlier study that cancer cells do not migrate to form tumor spheroids; instead, they undergo proliferation to form cellular aggregates, as confirmed by non-significant variations in the total number of cellular aggregates [22]. Therefore, it can be concluded that most of the cell aggregates in the monoculture group had a slower growth rate compared with the co-culture groups and reached a diameter of >35 mm only after 5 days of culture.…”

“…The SU-8 100 (MicroChem, Texas, USA) photoresist-based UV photolithography was used to generate a silicon-SU-8 (Si-SU-8) master that was then replica-molded to create polydimethylsiloxane (PDMS) (Sylgard 184; Dow Chemical Pty Ltd, Victoria, Australia) microfluidic chips, as described in the authors' previous work [22]. Briefly, after overnight silanization of the Si-SU-8 master using trichloro (1h,1h,2h,2h-perfluorooctyl)silane (Sigma-Aldrich, New South Wales, Australia), PDMS at a ratio of 1:10 was mixed, degassed and poured over the SI-SU-8 master and cured at 70°C for 6 h. The cured PDMS was then peeled off carefully, punched at the inlets/outlets, cleaned and dried.…”

“…Next, 3 mg/mL À1 collagen gel solution (Life Technologies Australia Pty Ltd, Victoria, Australia) was diluted to 2 mg/mL À1 using sterile distilled water and 10X PBS and neutralized to pH 7.4 using 1N sodium hydroxide. The cell suspension was mixed with the collagen gel at a ratio of 1:10 to generate a final cell concentration of 0.5 £ 10 6 cells/mL À1 , as described previously [22]. Next, 10 mL of the cell-hydrogel mixture was injected into the gel channel of the microfluidic devices, introducing cells at a concentration of 5 £ 10 3 cells/mL À1 .…”

“…The ACP used in this study was a cationic tryptophan-rich antimicrobial peptide called P1 (RKRWWRWWKWWKR-NH 2 , where R is arginine, K is lysine and W is tryptophan). The synthesis and design of this particular peptide were described in detail in the authors' previous work, in which a charge-based mechanism attacked the cancer cells [22,30]. ACP was added to the complete culture medium (without penicillin/streptomycin) to prepare the final ACP media at 2 different test concentrations: 30 mM and 58 mM.…”

“…Live cells were distinguished from dead cells by simultaneously staining the 3D culture using Calcein AM dye (green fluorescence), which acted on the intracellular esterase activity, and Ethidium Homodimer I dye (red fluorescence), which entered the cells that had lost the integrity of their plasma membrane. Previous studies, including the authors' own [22], have shown that both Calcein AM and Ethidium Homodimer I work very well with hydrogels, including collagen type 1, in both macroscale [31] and microscale cell cultures [28,29,32]. The channels were washed twice with prewarmed PBS to remove background fluorescence and then imaged using a confocal laser scanning microscope.…”