(E-198) Enhancing Immunotherapy via Chemokine-Driven Natural Killer Cells in 3D Melanoma Organ-on-a-Chip
Platform in Presence of Fibroblasts

Introduction:: Critical mutations in invasive and metastatic tumors enable evasion of the immune system. Traditionally used therapeutic antic-cancer modalities often exhibit poor efficacy. Of cellular immunotherapies, T cells have been most widely explored, but are not always effective. Moreover, tumors can suffer from immune suppression, forming ‘immune deserts’, and the deficiency of tumor antigens or high variability in those present, make T cell-based techniques like Chimeric Antigen Receptor T cells (CAR-T) and Immune Checkpoint Blockade (ICB), ineffective. Using sentinel lymphocytes like natural killer (NK) cells in cell-mediated immune therapy can be an effective alternative as they target cells based not on tumor antigen recognition (like T cells), but rather by their MHC class I expression; a feature that most cancer cells lose during their tumorigenesis. However, the tumor microenvironment, which consists of various stromal components, can impact treatment effectiveness. Hence, it is crucial to consider the role of such components – such as often abundantly found fibroblasts – while designing therapies to boost overall
therapeutic efficiency.
This study uses a 3D microfluidic device – a Natural Killer cell-containing Tumor-on-a-Chip (NK-TOC) – to effectively study homing and migration patterns of NK cells towards tumors organoids to explore methods by which to enhance NK cell immunotherapy while also evaluating contribution of stromal components like fibroblasts to overall therapeutic efficacy.

Materials and Methods:: Organoid preparation- Fifty-two μL of methacrylated collagen type I is added to 18.75 μL of the thiolated HA and 4.41 μL of pH neutralizing solution in a small conical tube with A-375 (highly metastatic melanoma) tumor cells. From this resulting solution, cells are injected into the chambers of the microfluidic device using a small gauge needle. Organoids are photopolymerized in place within the device using a brief UV light pulse.
This kind of a set-up challenges NK cells to leave their natural path of circulation and move towards the organoids to carry out their effector function.
To assess cell immunotherapy efficacy, fibroblasts are added to A-375 organoids constructed using the same hydrogel, and NK-TOC systems are treated with small molecule kinase inhibitor drugs alisertib (ALS) and abemaciclib (ABE).
NK staining and flow- Fluorescently labeled NK-92 cells are infused through the chambers via a peristaltic pump setup. The organoids are treated with ALS and ABE, which render the tumor cells senescent, but induce production and secretion of potent chemokines (e.g., CCL5, CXCL10 etc.) that NK cells utilize to guide migration. NK cells are added to the device and organoids on day 5 and imaging and viability assays are performed on day 6 to assess NK cell homing and effector function.
Phenotypic and functional assays- NK cell-mediated killing of melanoma cells is confirmed using perforin and granzyme B ELISA assays, performed on media collected from the samples. Fluorescent microscopy imaging enables tracking extent and migration patterns of NK cells to tumor organoids.

Results, Conclusions, and Discussions:: Our initial results have shown that migration occurs within 24 hours of NK cell addition and more importantly (a-b), greater NK cell migration towards drug treated organoids than those without any drug (c). We also observed greater secretion of CCL5 and CXCL10 chemokines in the drug-treated samples than those without drug (d,e).
In contrast, for the A-375-HNLF organoids, viability was greater in samples without any drug. Also, IL-6 (fibroblast interaction in presence of immune cells) and SDF1-α (confirmatory action of fibroblasts) were present in greater quantities for drug treated samples (f-k). We compared this to our studies with colorectal cancer organoids- mildly metastatic Caco-2, endothelial/intermediary SW480, and highly aggressive HCT-116 cells treated with cytotoxic Cisplatin, 5-Flurouracil and Regorafenib-and found greater viability in samples treated with these different drugs (l).
Thus, chemokine-fueled NK cell migration can boost effector function and augment existing chemotherapies. In some cases, despite the type of drug treatment, fibroblast behavior can negatively affect therapeutic efficacy notwithstanding the extent of tumorigenesis (as seen with our colorectal cancer organoids). However, with respect to the cell cycle inhibitors that we have looked at, the tumor-associated fibroblasts seem to be assisting chemotherapy while also giving a boost to immunotherapy. This merits further studies as it is usually one or the other form of therapy which is boosted.

Acknowledgements (Optional): : The authors acknowledge funding from NIH grantR21CA229027, the Ohio State University Comprehensive Cancer Centre (OSUCCC) and the OSUCCC Translational Therapeutics Program Seed Award.

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