Poster S11 - A Microphysiological System to Quantify the Interactions of Human Neutrophil Subpopulations With Pancreatic Tumor Spheroids

Introduction: Cancer cells recruit neutrophils from the bloodstream into tumor tissues, where these immune cells can promote the progression of many solid tumors such as pancreatic cancer [1] (Fig. 1A). Studies in mice show that polarization of neutrophils from a pro-tumor “N2” phenotype to an anti-tumor “N1” phenotype could be a potential cancer immunotherapy [2]. However, most studies on polarization of neutrophils to date rely on murine models [2], and thus the mechanisms by which N1 and N2 human neutrophils are recruited from the blood vessel to tumor tissues and influence tumor progression remain elusive. It is also unknown whether N1 polarization of neutrophils could serve as a cancer therapy in humans. These knowledge gaps exist partially due to the lack of suitable tools to measure specific immune cell-cancer interactions in the complex tumor microenvironment. In this study, we engineered a human cell-based microphysiological system “NTI-chip” (neutrophil-tumor interactions on-a-chip) to monitor in real-time and quantify the interactions of N1-like and N2-like human neutrophil subtypes with pancreatic tumor spheroids in the presence of a vascular endothelium (Fig. 1B). We validated and characterized the phenotypes of N1-like and N2-like neutrophils (surface marker expressions, ROS production, NET release, and cytotoxicity), the endothelium in the NTI-chip (VE-cadherin and ICAM-1 expressions), and tumor spheroids in the NTI-chip (IL-8 secretion, Ki-67, and E-cadherin expressions). We will use the NTI-chip to quantify the behavioral differences between N1-like and N2-like neutrophils including extravasation, migration, tumor contact, tumor infiltration, and tumor cytotoxicity and reciprocal responses from tumor spheroids (Fig. 1C).

Materials and

Methods: HL-60 human promyelocytic leukemia cells were differentiated with 1.5% dimethyl sulfoxide into a neutrophil-like state (“dHL-60 neutrophils”) and then polarized with 100 ng/mL lipopolysaccharide (LPS), 50 ng/mL interferon-γ (IFN-γ), and 50 ng/mL interferon-β (IFN-β) toward an N1-like (anti-tumor) state or with 100 ng/mL transforming growth factor-β (TGF-β) toward an N2-like (pro-tumor) state [3] (Fig. 2A). The unpolarized control is denoted as N0 neutrophils. Surface marker expression and ROS production by N1-like and N2-like neutrophils were assessed by flow cytometry, IL-8 secretion by ELISA, and release of neutrophil extracellular traps (NETs) by imaging. PANC-1 human pancreatic cancer cells were used to generate 3D tumor spheroids on ultra-low attachment six-well plates. Polydimethylsiloxane (PDMS)-based NTI-chips were designed and fabricated using standard photolithography and soft lithography techniques. Tumor spheroids (~10-15) or single cancer cells (2 x 10^6 cells/mL) embedded in type I collagen hydrogel (2.5 mg/mL) were loaded into the tumor gel channel of the NTI-chip to mimic the tumor tissue (Fig. 1B). Cell-free hydrogel was loaded into the empty gel channel as negative control. Human umbilical vein endothelial cells (HUVECs) were loaded into the central channel at 5 x 10^6 cells/mL to form a confluent endothelium for 24 h. dHL-60 neutrophils (2.5 x 10^6 cells/mL) were loaded into the central channel after formation of the endothelium (Fig. 1B). Time-lapse imaging of the NTI-chip was performed to capture interactions between neutrophils and cancer cells over 24 h. Image analysis was performed using ImageJ and IMARIS and statistical analysis using Prism 9.

Results, Conclusions, and Discussions: HUVECs formed a confluent endothelial lumen in the central channel of the NTI-chip, as evidenced by the expression of VE-cadherin, an endothelial cell-cell adhesion molecule, at the junctions between cells (Fig. 1E). We found an upregulation of ICAM-1, a surface molecule crucial for enabling neutrophil extravasation, by HUVECs in the NTI-chip after being primed with TNF-α overnight (Fig. 1F).

We confirmed expressions of neutrophil marker CD11b, typical N1 markers CD54_high and CD62L_low, and N2 marker CXCR2_high by our polarized N1-like and N2-like neutrophils (Fig. 2Bi-iv). N1-like neutrophils showed higher levels of ROS production (Fig. 2C), IL-8 secretion (Fig. 2D), and NET release (Fig. 2E) than N2-like neutrophils, thus showing a more activated state. N1-like neutrophils induced apoptosis of cancer cells (p < 0.05), while N2-like neutrophils showed the trend of attenuating chemotherapy-induced cancer cell apoptosis (p=0.29) (Fig. 2F).

PANC-1 tumor spheroids were found to secrete neutrophil chemoattractant IL-8 (Fig. 3A). N0 neutrophils promoted the proliferation (Ki-67 expression) of tumor spheroids in the NTI-chip (Fig. 3B). Tumor spheroids in the NTI-chip downregulated epithelial cell marker E-cadherin after treatment with TGF-β, a cytokine known to induce epithelial-mesenchymal transition (EMT) of pancreatic cancer cells (Fig. 3C).

PANC-1 tumor spheroids induced directional migration of N0 neutrophils in the central channel without an endothelium, as evidenced by an increased y-forward migration index (y-FMI), mean velocity, and displacement (Fig. 4). We demonstrated the feasibility of quantifying neutrophil-tumor contact by showing a reduction in both frequency and duration of contact between N0 neutrophils and tumor spheroids by inhibition of neutrophil chemokine receptor CXCR2 (Fig. 5).

We validated the strategic loading of gel-embedded cancer cells, HUVECs, and N0 neutrophils into the NTI-chip (Fig. 1D). We will utilize the NTI-chip to quantify the behaviors of N0, N1-like, and N2-like neutrophils in terms of extravasation, migration, tumor contact, and tumor infiltration and the reciprocal responses of tumor spheroids including apoptosis, proliferation, and EMT. The device will shed new light on the “behaviorome” of human neutrophils in cancer, serve as an alternative tool to animal models for preclinical immunotherapy testing, and hold potential for personalized medicine by measuring patient-derived immune cell-cancer interactions.

Acknowledgements (Optional): Financial support was provided by Dr. Caroline Jones’ startup funds and NSF CBET CAREER Award Number:2240094. Shuai Shao was supported by the Jonsson Family Bioengineering Fellowship and the Bioengineering Convergence Grant from UT Dallas.

References:
1. Jaillon, S. et al. Neutrophil diversity and plasticity in tumour progression and therapy. Nature Reviews Cancer vol. 20 485–503 Preprint at https://doi.org/10.1038/s41568-020-0281-y (2020).
2. Antuamwine, B. B. et al. N1 versus N2 and PMN-MDSC: A critical appraisal of current concepts on tumor-associated neutrophils and new directions for human oncology. Immunol Rev 314, 250–279 (2023).
3. Ohms, M., Möller, S. & Laskay, T. An Attempt to Polarize Human Neutrophils Toward N1 and N2 Phenotypes in vitro. Front Immunol 11, (2020).