Communication Cytomorphological Characterization of Individual Metastatic Tumor Cells from Gastrointestinal Cancer Patient Lymph Nodes with Imaging Flow Cytometry Marnie Winter 1 , Rachel Gibson 2, Andrew Ruszkiewicz 3 and Benjamin Thierry 1,* 1 Future Industries Institute and ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, University of South Australia, Adelaide 5095, Australia; [email protected] 2 Faculty of Health and Medical Sciences, University of Adelaide, Adelaide 5000, Australia; [email protected] 3 Department of Surgical Pathology, SA Pathology, Centre for Cancer Biology, University of South Australia, Adelaide 5000, Australia; [email protected] * Correspondence: [email protected]; Tel.: +61-8-8302-368 Received: 16 July 2019; Accepted: 19 September 2019; Published: 26 September 2019 Abstract: The presence or absence of tumor cells within patient lymph nodes is an important prognostic indicator in a number of cancer types and an essential element of the staging process. However, patients with the same pathological stage will not necessarily have the same outcome. Therefore, additional factors may aid in identifying patients at a greater risk of developing metastasis. In this proof of principle study, initially, spiked tumor cells in rat lymph nodes were used to mimic a node with a small cancer deposit. Next, human lymph nodes were obtained from cancer patients for morphological characterization. Nodes were dissociated with a manual tissue homogenizer and stained with fluorescent antibodies against CD45 and Pan-Cytokeratin and then imaging flow cytometry (AMNIS ImageStreamX Mark II) was performed. We show here that imaging flow cytometry can be used for the detection and characterization of small numbers of cancer cells in lymph nodes and we also demonstrate the phenotypical and morphological characterization of cancer cells in gastrointestinal cancer patient lymph nodes. When used in addition to conventional histological techniques, this high throughput detection of tumor cells in lymph nodes may offer additional information assisting in the staging process with therapeutic and prognostic applications. Keywords: imaging flow cytometry; lymph node; metastasis; cellular morphology; cancer 1. Introduction The presence of metastatic cancer cells within regional lymph nodes, including in sentinel lymph nodes (SLNs), is a significant prognostic indicator in a number of cancer types including gastrointestinal (GI) cancers [1]. Importantly, positive identification of metastatic tumor cells in lymph nodes may indicate the need for adjuvant therapy [2]. The size of cancer deposits in lymph nodes is also clinically relevant. These can be defined as either (1): Macrometastasis (>2 mm), (2): Micrometastasis (0.2–2 mm), or (3): Isolated tumor cells (ITCs; <0.2 mm) [1]. Prognostic significance for the smaller deposits (including micrometastasis and ITC) is highly debated and is likely to vary between cancer types [3–7]. Determining which patients with positive nodes require Axillary Lymph Node Dissection (ALND) and that would benefit from adjuvant radiotherapy and systemic treatment is a problem [8] as some lymph node metastasis may never develop into clinically detectable disease [9]. Therefore, additional approaches to identify individuals with a higher likelihood of developing metastatic disease are required to provide better prognostication and selection for adjuvant treatment [10]. Gastrointest. Disord. 2019, 1, 372–384; doi:10.3390/gidisord1040030 www.mdpi.com/journal/gastrointestdisord Gastrointest.Gastrointest. 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