The development and characterization of a human mesothelioma in vitro 3D model to investigate immunotoxin therapy

Abstract

Background: Tumor microenvironments present significant barriers to penetration by antibodies and immunoconjugates. Tumor microenvironments, however, are difficult to study in vitro. Cells cultured as monolayers exhibit less resistance to therapy than those grown in vivo and an alternative research model more representative of the in vivo tumor is more desirable. SS1P is an immunotoxin composed of the Fv portion of a mesothelin-specific antibody fused to a bacterial toxin that is presently undergoing clinical trials in mesothelioma.

Methodology/principal findings: Here, we examined how the tumor microenvironment affects the penetration and killing activity of SS1P in a new three-dimensional (3D) spheroid model cultured in vitro using the human mesothelioma cell line (NCI-H226) and two primary cell lines isolated from the ascites of malignant mesothelioma patients. Mesothelioma cells grown as monolayers or as spheroids expressed comparable levels of mesothelin; however, spheroids were at least 100 times less affected by SS1P. To understand this disparity in cytotoxicity, we made fluorescence-labeled SS1P molecules and used confocal microscopy to examine the time course of SS1P penetration within spheroids. The penetration was limited after 4 hours. Interestingly, we found a significant increase in the number of tight junctions in the core area of spheroids by electron microscopy. Expression of E-Cadherin, a protein involved in the assembly and sealing of tight junctions and highly expressed in malignant mesothelioma, was found significantly increased in spheroids as compared to monolayers. Moreover, we found that siRNA silencing and antibody inhibition targeting E-Cadherin could enhance SS1P immunotoxin therapy in vitro.

Conclusion/significance: This work is one of the first to investigate immunotoxins in 3D tumor spheroids in vitro. This initial description of an in vitro tumor model may offer a simple and more representative model of in vivo tumors and will allow for further investigations of the microenvironmental effects on drug penetration and tumor cell killing. We believe that the methods developed here may apply to the studies of other tumor-targeting antibodies and immunoconjugates in vitro.

Figure 1. Establishment of human mesothelioma spheroids.

A. Monolayers and spheroids of human mesothelioma cell line, NCI-H226, and primary mesothelioma lines NCI-M-03 and NCI-M-13. Scale bars, 400 µm (except 200 µm for NCI-M-13 spheroids). B. Immunohistochemical staining of an NCI-H226 spheroid (see Methods ). (a) Mesothelin stains both the membrane and cytoplasm. (b) Calretinin stains both the nuclei and cytoplasm. (c) HBME-1 exhibits a focal membranous staining pattern (arrows). (d) Cytokeratin 5/6 exhibits a focal cytoplasmic staining pattern. (e) WT-1 stains the nuclei. (f) Thrombomodulin exhibits a focal membranous staining pattern close to the rim (arrows).

Figure 2. Anti-tumor immunotoxin activity on mesothelioma monolayers and spheroids.

Cells treated with SS1P and BL22. A. Cell growth inhibition (WST-8 assays) of NCI-H226. IC₅₀ of SS1P for spheroids was >1000 ng/mL. IC₅₀ for monolayers was ∼10 ng/mL. B. Cell viability (CellTiter-Glo Luminescent assays) of NCI-H226. IC₅₀ of SS1P for spheroids was >1000 ng/mL. IC₅₀ for monolayers was ∼5 ng/mL. C. Primary mesothelioma lines NCI-M-03 and NCI-M-13 treated with 100 ng/mL of SS1P. 2D, monolayers; 3D, spheroids. *p<0.01.

Figure 3. Mesothelin expression in mesothelioma monolayers and spheroids.

NCI-H226 cells incubated with an anti-mesothelin mAb (MN) and detected with goat anti-mouse IgG conjugated with Alexa488 by flow cytometry.

Figure 4. Penetration of Alexa488-labeled immunotoxin SS1P in tumor spheroids.

A. SS1P labeled with Alexa488 (green fluorescence). A cross section close to the middle of an NCI-H226 spheroid evaluated at hours 0, 8 and 16 using confocal microscopy. B. Overlay of bright field and fluorescence images of a spheroid. C. Mean fluorescence intensity of SS1P (see Methods ). Scale bars, 400 µm.

Figure 5. Electron microscopy of cell contacts in NCI-H226 mesothelioma spheroids.

A and B. SEM analysis showing microvilli (arrow). Scale bars, 200 µm (A) and 10 µm (B). C and D. Ultrathin sections (TEM). Presence of intracellular tight junctions (arrow). Scale bar, 0.5 µm. E. Number of cellular junctions in the core and rim areas.

Figure 6. Expression of cell junction and Bcl-2 signaling proteins in NCI-H226 monolayers and spheroids.

A. Protein expression of E-Cadherin, ZO-1, Connexin-32, Mcl-1, Bcl-xL, Bid, Bak, Bax, and β-actin examined by Western blot. B. Molecular mechanisms underlying drug resistance in tumor spheroids.

Figure 7. Sensitizing spheroids to immunotoxin therapy in vitro.

A. Silencing E-Cadherin expression by siRNA. siE-Cad, E-Cadherin siRNA. 2D: monolayers; 3D: spheroids. B. Silencing E-Cadherin enhanced the anti-tumor activity of SS1P. Cell growth was assessed by incubation with WST-8 with values normalized (%) relative to the growth of the non-silencing siRNA (* p<0.05). C and D. An anti-adhesive mAb targeting E-Cadherin (SHE78-7) enhanced the anti-tumor activity of SS1P when added before the formation of spheroids (Pre) (* p<0.05), but not after (Post). Arrow, lysed cell debris. Scale bars, 400 µm. NS, not significant.