Introduction: This project focuses on head and neck squamous cell carcinomas (HNSCCs), which are classified as a group of curative cancers that cause >300k deaths worldwide each year. Patients typically find this cancer as a solid tumor where, in most cases, complete surgical resection is the primary treatment and, assuming no spread of the disease outside of the primary tumor, is potentially curative. However, identifying whether the cancer is curative is a tedious process and involves a highly invasive neck dissection to screen all draining lymph nodes for the presence of satellite growths. This time-consuming process requires histopathology, including formalin fixation, paraffin embedding, microtomy, and staining with dyes such as hematoxylin and eosin (H&E). This process can take up to a week to complete and allows for < 1% of the total lymph node volume to be sampled and screened. This is where our project comes into play; in aiding in the lymph node dissection, we have developed a metastatic lymph node model and a paired-agent widefield fluorescence tomography imaging protocol where we can rapidly identify the presence of Epidermal Growth Factor Receptor (EGFR) overexpressing human cancer cell aggregates as small as 300-micrometers in diameter. The project presented here aims to optimize the lymph node model, concentrations, timing of imaging agent infusion, and imaging and tomographic reconstruction protocols.
Materials and
Methods: The model being used within this experiment is referred to as a metastatic lymph node model, and to start, porcine tissue is acquired from a local slaughter facility from the head and neck region, followed by dissection and isolation of lymph nodes. The human squamous cell carcinoma cell line (FaDu) is cultured from here. Then, those cells will be aggregated into 250-500 micrometer diameter spheroids using an established methyl-cellulose supplemented cell culture medium-based spheroid culture protocol. These spheroids that have been made are injected with a needle into the lymph nodes' core, representing the model “metastatic” node. Once the node has been created, imaging agents are infused with initial infusion parameters from previous work in the Molecular Imaging Laboratory at IIT. These imaging agents consist of an EGFR-targeted agent, ABY-029, that has a fluorescence emission at 800 nm, as well as an untargeted agent, IRDye 700Dx-conjugated affibody isotype, which has a fluorescence emission at 700 nm. The infused lymph nodes are then embedded in an agarose cylinder, which is mounted to an optical post and submerged in a water bath. The agarose cylinder with lymph node encased is then fluorescence images taken at 700 and 800 nm at 12 different projections, followed by tomographic reconstruction of each channel and calculation of a “binding potential (BP) map,” where BP is a quantity that is proportional to the concentration of the targeted agent in that voxel, and BP= Intensitytargeted agent (800 nm)Intensityuntargeted agent (700 nm)-1.
Results, Conclusions, and Discussions: Full tomographic reconstructions and resulting BP maps are assessed for BP voxels > 0 presence. The resulting location in the space of these voxels is isolated in the lymph node, and the lymph node is sliced into 200-micron-thick sections. The slice that contains the suspicious lesion is then sectioned at a 10-micron diameter, followed by H&E staining and color imaging via confocal microscopy to delineate the boundaries of the spheroid relative to healthy tissue. This is then compared with the reconstruction. Optimization of imaging agent concentration and infusion times is carried out to yield peak contrast and spheroid localization relative to the position identified via traditional histopathology. While results presented here entail using a moderate EGFR-expressing cell line, FaDu, and while not all cell lines overexpress EGFR, it is thought that >90% of HNSCCs overexpress this specific biomarker. However, it is possible to utilize tracers with other biomolecular targets, such as those that target CD-34, as a replacement for, or possibly in conjunction with, an EGFR-targeted tracer. A small molecular-weight tracer is utilized here, where ABY-029 is a seven kDa affibody. These results may not necessarily be relevant to the use of much larger molecular weight monoclonal antibodies (mABs), such as the EGFR-targeted FDA-approved mABs Erbitux (cetuximab) or Herceptin (trastuzumab). These high molecular weight tracers have high specificity yet low penetration, and further testing needs to be done to ensure their use in this model. In conclusion, this model presents a screening technique that is faster and more convenient to patients and offers a higher molecular sensitivity and specificity for identifying metastatic lymph nodes. Once thoroughly tested, this technology could bring significant clinical benefits when used intraoperatively during primary tumor resections. It could help determine the presence of metastasis while still in the operating room, guide pathologists toward suspicious nodes, and expedite sectioning and histopathology, potentially saving lives and improving patient outcomes.
Acknowledgements (Optional): Dr. Kenneth Tichauer, PhD, and PhD student Cody Rouds advised and afflicted this research project.
