(G-255) Spheroid Tumor Models for Assessing Peptide-Induced Immunogenic Cell Death

Immunogenic Cell Death (ICD) is a systematic biological pathway that if controlled, could address a multitude of challenges in cancer therapy. The release of specific damage-associated molecular patterns (DAMPs), such as ATP, nucleic acids, and high mobility group box 1 (HMGB-1), into the extracellular environment and the display of stress-related proteins such as calreticulin (CRT) on the cell membrane act as signals, triggering an otherwise dysfunctional anti-tumor immune system and prompting phagocytes to mobilize and initiate an immune response. While immunotherapeutic cytotoxicity and DAMP release profiles are commonly studied using conventional monolayer culture, spheroids are 3D cell constructs that serve as suitable high-throughput in vitro test subjects due to their ability to better mimic the complex 3D in vivo tumor microenvironment via their cell-to-cell interactions, cell-to-extracellular matrix interactions, drug resistance, and chemical gradient-induced necrotic zone formation.¹ Developed from the Acar lab’s self-assembling peptide strategy, “co-assembly of oppositely charged peptides (CoOP)”², [II] is a peptide duo capable of inducing stress-related cell membrane rupture and DAMP release on monolayer culture in a process we termed “PAIIR” (Peptide-Aggregation Induced Immunogenic Rupture).³ PAIIR is a modulatory immunotherapeutic platform suitable for drug development due to its controllable aggregation kinetics through bovine serum albumin (BSA) concentration manipulation.⁴  In this study, we compared [II] to commonly used chemotherapeutics mitoxantrone and cisplatin, clinically known ICD-inducer and non-inducer respectively, on spheroid tumor models to assess PAIIR-induced ICD and DAMP release in a 3D system that closely mimics in vivo physiology.

Materials and Methods::

Spheroid tumor model formation, culture, and characterization:

EMT6 murine mammary carcinoma cell spheroids were formed using the ultra-low attachment technique in U-bottom 96-well plates.¹ Media was changed daily, excluding the first day, and spheroids were imaged every 24 hours and later analyzed for area, diameter, and circularity using ImageJ.

Peptide and chemotherapeutics preparation and treatment:

As previously established, PAIIR peptides were prepared in albumin solutions and mixed for 30 minutes before treatment to create [II].³ Peptides, mitoxantrone (Millipore Sigma M6545), and cisplatin (Millipore Sigma 232120) were applied in various concentrations to determine the half-maximal inhibitory concentrations (IC­₅₀) in spheroids at 24 hours. Spheroids were then treated for 24 hours after 7 days of growth.

Viability assessment:

Spheroid relative viability was assessed using CellTiter-Glo® 3D Cell Viability Assay. Spheroids were subjected to a Calcein AM and Ethidium homodimer-1 staining solution, incubated, and imaged through fluorescent microscopy. Lactate dehydrogenase (LDH), a cytoplasmic enzyme released upon cell membrane damage, was quantified using Cytoscan-LDH cytotoxicity assay kit on culture supernatant.

DAMP release:

Post-treatment culture supernatant was collected and analyzed for extracellular ATP release using CellTiter-Glo® 2.0 Cell Viability Assay. Extracellular double-stranded DNA (dsDNA) release from spheroids was measured using AccuGreen™ High Sensitivity dsDNA Quantitation Kit. HMGB-1 was detected through immunoblotting. Statistical analysis was done with one-way ANOVA test.

Immunostaining:

Calreticulin (CRT) expression on the spheroid exterior was shown through fixing and staining with a primary anti-calreticulin antibody conjugated with a fluorescent secondary antibody. Samples were counter-stained with DAPI.

Results, Conclusions, and Discussions::

Spheroid Formation and Characterization:

Prior to their treatment on day 7, where their sizes were around 800 µm in diameter (Figure 1A), spheroid surface area increased over time (Figure 1B) and displayed high circularity (Figure 1C).

Peptide-induced DAMP release is superior to chemotherapeutics:

Spheroid LDH release was significantly (****p< 0.0001) higher in [II] and mitoxantrone than cisplatin (Figure 2A), indicating higher membrane damage with peptides. Significantly higher extracellular ATP (Figure 2B) and dsDNA (Figure 2C) release was observed in peptide treatment groups compared to chemotherapeutics, as well as higher levels of HMGB-1 detection (Figure 2D).

Staining:

To show PAIIR-induced cell death, calcein AM and ethidium homodimer-1 viability staining (Figure 3) was performed and a positive correlation with increasing [II] peptide concentration was identified. Calreticulin, a hallmark ICD biomarker, was shown to be expressed on the spheroid exterior (Figure 4) as a result of [II] treatment, further indicating peptide-induced cell stress and immunogenicity.

Conclusions:

Conventional monolayer culture offers limited insights into drug efficacy for in vivo studies and clinical trials. In contrast, spheroids provide a physiologically relevant model that mimics the complex tumor microenvironment, serving as a high-throughput tool for further studies. This study shows that PAIIR-induced ICD results in superior DAMP release compared to chemotherapeutics in spheroid models. Additionally, the establishment of the spheroid calreticulin immunostaining method encourages the exploration of alternative immunogenicity assessment approaches for spheroids. Therefore, these findings highlight PAIIR’s potential as a promising immunotherapeutic for further development.

Discussions:

While our data indicates that PAIIR is an effective ICD-inducer in spheroid tumor models, a direct 2D-to-3D comparative study has yet to be done. Unexpectedly, ICD-inducing mitoxantrone yielded no release of ATP. This may be due to the concentration used, as the chemotherapeutic concentration has been shown to influence ICD and DAMP release,⁵ alluding to [II]’s ability to achieve ATP release at lower dosages. Despite being non-ICD-inducing, cisplatin treatment also resulted in dsDNA release, possibly due to its role in inducing DNA double-strand breaks.⁶ Additional studies are required to thoroughly understand the impact of PAIIR-induced DAMPs on immune cells and validate the findings of this study via animal trials.

Acknowledgements (Optional): :

References (Optional): :

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(2)        Hamsici, S.; White, A. D.; Acar, H. Peptide Framework for Screening the Effects of Amino Acids on Assembly. Sci. Adv. 8 (3), eabj0305. https://doi.org/10.1126/sciadv.abj0305.

 

(3)        Gunay G, Hamsici S, Lang GA, Lang ML, Kovats S, Acar H. Peptideaggregation induced immunogenic rupture (PAIIR). Adv Sci. 2022; 9:2105868. doi:10.1002/advs.202105868

 

(4)        Hamsici, S., Gunay, G. & Acar, H. Controllable membrane damage by tunable peptide aggregation with albumin. https://biorxiv.org/lookup/doi/10.1101/2022.06.02.494564 (2022)

 

(5)        Sriram, G. et al. The Injury Response to DNA Damage Promotes Anti-Tumor Immunity. https://biorxiv.org/lookup/doi/10.1101/2020.04.26.062216 (2020) doi:10.1101/2020.04.26.062216

 

(6)        Chen, S.-H. et al. O6-methylguanine-DNA methyltransferase modulates cisplatin-induced DNA double-strand breaks by targeting the homologous recombination pathway in nasopharyngeal carcinoma. J Biomed Sci 28, 2 (2021).