Merkel cell carcinoma (MCC) is a rare and aggressive skin cancer with a mortality rate of ~30%. In ~80% of cases, MCC development is attributed to the integration of Merkel cell polyomavirus (MCPyV) DNA into the host’s genome, leading to the expression of viral oncoproteins and tumorigenesis. Developing treatments that sustain immunity against MCC is imperative to address recurrent and/or progressive disease. In many cancers, tumor-infiltrating B cells have been associated with better prognosis and response to immunotherapies. However, the mechanisms by which B cells contribute to tumor immunity in humans have been difficult to resolve in part due to the inter-patient heterogeneity of tumor-specific antigens. The shared nature of MCPyV tumor antigens in MCC allows for MCC-specific B cell responses to be studied across patients. Using DNA-barcoded and fluorescently labeled viral oncoprotein tetramers, we analyzed the transcriptome, proteome, and receptor repertoire of MCC tumor-infiltrating B cells in 12 patient samples at single-cell resolution. From paired heavy and light chain sequences, we cloned 8 antibodies from B cells specific for the MCPyV oncoproteins to confirm binding to MCC-specific antigens. Transcriptomic and proteomic analyses of MCPyV-specific B cells revealed heterogeneity of intra-tumoral B cell responses. Interestingly, we found that the absence of MCC-specific germinal center (GC) B cells in MCC tumors associates with disease progression: ~80% of patients with no detectable GC B cells had MCC progression within a year post-surgery, whereas patients with detectable GC B cells remained progression-free a year after surgery (n=12, p=0.0043). These results suggest strong synergy between B cells and T cells may regulate tumor growth, as B cells rely on signals presented by T cells to differentiate into GC cells. Our long-term objective is to identify B cell phenotypes associated with anti-MCC responses to develop therapeutics that boost cancer-specific immunity.

mr-DWL is an epoxy-based, negative photoresist that can be used in both the DWL66+ direct write laser system and the Nanoscribe two photon polymerization system. mr-DWL shares several advantageous properties with SU-8 resist, such as high aspect ratio imaging, robust thermal and chemical resistance, and excellent biocompatibility, but has the added advantage of being more sensitive at higher wavelengths. The use of SU-8 at the Washington Nanofabrication Facility (WNF) is problematic, as prototyping with it is expensive and inefficient. Here, we explore mr-DWL as a possible alternative. The properties of SU-8 make it suitable for a variety of technologies including MEMS devices, microfluidics molds, cantilevers, waveguides, and inkjet nozzles. However, it is incompatible with the DWL66+ direct laser write system at the WNF. As such, researchers must prepare a new photolithography mask and perform contact alignment for each design iteration. Repeated mask preparation can render process development slow and expensive if a device’s design changes often. mr-DWL is compatible with maskless direct write lithography, so would substantially lower the time and cost for researchers developing microfabrication-based technologies. In this study, we explore the viability of mr-DWL as an alternative to SU-8 photoresist by spin coating wafers with mr-DWL 5 and 40 to various thicknesses and conducting an exposure series using the WNF’s Heidelberg direct write tool. These wafers can then be measured and characterized by WNF's metrology suite to narrow down the exposure parameters required for film uniformity and feature accuracy.

Facile deposition of thick, even photoresist layers remains a challenge in microfabrication facilities. This difficulty stems from the spin-coating process, which often fails to yield uniform films at thicknesses on the order of hundreds of microns. The shortcomings of this deposition method pose a particular challenge to the development of microfluidic devices, which often rely on thick resist layers in their designs. Engineers at the Washington Nanofabrication Facility (WNF) are considering SUEX and ADEX dry film photoresists as an alternative to SU-8, a common epoxy photoresist that is frequently employed in the fabrication of microfluidics. Like most photoresists, SU-8 is supplied in the form of a liquid solution, which must be deposited onto substrates by spin-coating. By contrast, SUEX and ADEX are supplied as films that may be applied to substrates by hot-roll lamination, yielding resist layers with exceptional uniformity at thicknesses up to a millimeter. The lamination process offers several key advantages to spin-coating: less expensive equipment, reduced exposure to solvents, minimal mess and waste, and a shallow learning curve. As an undergraduate research assistant (URA) at WNF, I am incorporating SUEX and ADEX films into double-layered microfluidic device fabrication workflows.  I will determine whether these laminate photoresist films improve the overall process in terms of simplicity, speed, and cost compared to existing methods reliant on SU-8.  This work consists primarily of lamination, photolithography, and various metrology methods such as microscopy and stylus profilometry. If I am successful, the laminate-based process could be utilized by WNF users and staff to ramp up the production and testing of a wide range of technologies.