(J-373) Cancer Therapeutic Efficacy of Immune Booster Peptides

Cancer immunotherapy has emerged as a promising approach in the fight against cancer, offering the potential for targeted and more effective treatment options. Successful outcomes are often linked to immunogenic cell death (ICD), achieved by applying damaging stress to release danger signals called damage-associated molecular patterns (DAMPs) which prompt the immune system to target the affected area^1,2. However, ICD-inducing chemotherapeutics can cause devastating side effects by killing incoming immune cells, even when directly administered to tumor tissue².

The Acar Lab developed a novel self-assembling peptide [II] that induces immunogenic cell death (ICD) by accumulating stress on cell membranes and triggering an immune response using biodegradable amino acids, which we call peptide-aggregation induced immunogenic rupture (PAIIR)³. PAIIR activity stresses cell membranes, therefore cancer cell membranes' higher fluidity could enhance peptide toxicity to cancerous cells³.  Importantly, the activity of these peptides is limited by their aggregation kinetics, which was controlled via the addition of albumin in different concentrations. Due to limited activity, they do not remain toxic to incoming cells⁴.

To test efficacy, we studied cancer cells as multicellular aggregates (spheroids) to represent accurate in vivo models. We identified IC50 levels of the [II] peptide in different aggregation kinetics. Incubating macrophages with peptide-treated cells' supernatant demonstrated ongoing toxicity and activation effects. The cytotoxicity of [II] and ICD-inducing chemotherapeutics was compared on both cancerous EMT6 and healthy C2C12 cell lines to examine possible differences. This peptide technology represents the first and only peptide-based immune system booster suitable for vaccine development and immunotherapies.

Cell Culture Treatment and Cytotoxicity:

Murine mammary carcinoma EMT6 and murine myoblast C2C12 cell lines were utilized in the study. Different concentrations of [II] peptides, as well as chemotherapeutics Mitoxantrone (Millipore Sigma M6545) and cisplatin (Millipore Sigma 232120), were employed.

To assess the efficacy of released signals from cells dying by [II] peptides and chemotherapeutics, we determined the 50% cell death ratio (IC50 values) within 24 hours for all experimental groups. Spheroids were formed using the ultra-low attachment technique in U-bottom 96-well plates.

Cell Death Determination:

Cell viability was assessed using AlamarBlue HS Cell Viability Reagent (Thermo Fisher Scientific A50100), which detects live cells through fluorescence intensity with a BioTek Neo2SM microplate reader. Membrane damage in dying cells was evaluated through 24-hour treatment using Propidium Iodide (PI) (Invitrogen P1304MP) and visualized through fluorescent microscope images.

Quantification of membrane damage was performed using the Cytoscan-LDH Cytotoxicity Assay Kit (G-BIOSCIENCES 786-210), and analyzed with BioTek Neo2SM microplate reader.

Released extracellular ATP and ATP consumption was measured in supernatant of the treated cells via CellTiter-Glo 2.0 (Promega G9248) luminescence.

Peptide and Chemotherapeutic Preparation and Treatment:

[II] peptides were prepared in different concentrations with varying albumin concentrations. The preparation technique of [II] peptides was established previously³. Individually charged counterparts were prepared in albumin solutions and mixed for 30 minutes before treatment. Mitoxantrone (Millipore Sigma M6545) and cisplatin (Millipore Sigma 232120) were used at indicated concentrations and prepared in respective cell culture media. One-way ANOVA was used for statistical significance analysis.

In this study, we investigated the impact of [II] peptides on immune cells and their potential as cancer therapeutics. We identified the IC50 of [II] in different concentrations, the concentrations of [II] peptides and chemotherapeutics required for 50% cell death in each time (Figure 1, Figure 2). Chemotherapeutics (mitoxantrone and cisplatin) required higher concentrations to achieve 50% of the cell death in spheroids (Figure 1), while the aggregation kinetics change at the same concentration of [II] as in monolayer was effective on spheroids as well (Figure 2).  The IC50 concentrations varied based on the in vitro model used, suggesting potential variations in drug sensitivity (Figure 1). The 50% cell death ratio within both monocultures and spheroids indicated the efficacy of released signals from cells dying by [II] peptides and chemotherapeutics.

Supernatant  from treated cultures was tested on IBMDM cells, demonstrating the activation of immune cells without cytotoxic effects (Figure 3). Statistical analyses revealed significant differences between experimental groups, and ongoing investigations explore the implications of [II] peptides in cancer treatment and immune response modulation.

The potential of [II] peptides as immunomodulatory agents is evident in their ability to activate immune cells while preserving cell viability. The cytotoxicity comparison of [II] peptides and mitoxantrone on cancer and healthy cells are ongoing to explore molecular mechanisms and assess efficacy in different cancer types and models.

In conclusion, our study highlights [II] peptides as promising immunomodulatory agents for cancer treatment. Their ability to induce immunogenic cell death, lack of drug resistance, and immune cell safety profile make them potential candidates for further research and clinical applications. 

[1] J. Zhou, G. Wang, Y. Chen, H. Wang, Y. Hua, Z. Cai, “Immunogenic cell death in cancer

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2019. Available: https://onlinelibrary.wiley.com/doi/full/10.1111/jcmm.14356

[2] D. V. Krysko, A. D. Garg, A. Kaczmarek, O. Krysko, P. Agostinis, P. Vandenabeele,

“Immunogenic cell death and DAMPs in cancer therapy,” Nat. Rev. Cancer, vol. 12, pp.

860–875, Nov 2012. Available: https://www.nature.com/articles/ncr3380

[3] G. Gunay, S. Hamsici, M. L. Lang, G. A. Lang, S. Kovats, H. Acar, “Peptide Aggregation

Induced Immunogenic Rupture (PAIIR).” Advanced Science, 2022. Available:

10.1002/advs.202105868.

[4] S. Hamsici, G. Gunay, and H. Acar, “Controllable membrane damage by tunable peptide

aggregation with albumin,” AIChE Journal, vol. 68, no. 12, p. e17893, 2022, doi: 10.1002/aic.17893.