New microfluidic systems for whole organism analysis and experimentation are catalyzing biological breakthroughs across many fields, from human health to fundamental biology principles. This perspective discusses recent microfluidic tools to study intact model organisms to demonstrate the tremendous potential for these integrated approaches now and into the future. We describe these microsystems' technical features and highlight the unique advantages for precise manipulation in areas including immobilization, automated alignment, sorting, sensory, mechanical and chemical stimulation, and genetic and thermal perturbation. Our aim is to familiarize technologically focused researchers with microfluidics applications in biology research, while providing biologists an entrée to advanced microengineering techniques for model organisms.

Tumor-associated antigens (TAAs) are self-molecules abnormally expressed on tumor cells, which elicit humoral and cellular immunity and are targets of immunosurveillance. Immunity to TAAs is found in some healthy individuals with no history of cancer and correlates positively with a history of acute inflammatory and infectious events and cancer risk reduction. This suggests a potential role in cancer immunosurveillance for the immune memory elicited against disease-associated antigens (DAAs) expressed on infected and inflammed tissues that are later recognized on tumors as TAAs. To understand probable sources for DAA generation, we investigated in vitro the role of inflammation that accompanies both infection and carcinogenesis. After exposure of normal primary breast epithelial cells to pro-inflammatory cytokines IL1β, IL6, and TNFα, or macrophages producing these cytokines, we saw transient overexpression of well-known TAAs, carcinoembryonic antigen (CEA) and Her-2/neu, and overexpression and hypoglycosylation of MUC1. We documented inflammation-induced changes in the global cellular proteome by 2D Difference Gel Electrophoresis (2D-DIGE) combined with mass spectrometry and identified seven new DAAs. Through gene profiling, we showed that the cytokine treatment activated NF-κB and transcription of the identified DAAs. We tested three in vitro-identified DAAs, Serpin B1, S100A9, and SOD2, and found them overexpressed in premalignant and malignant breast tissues as well as in inflammatory conditions of the colon, stomach, and liver. This new category of TAAs, which are also DAAs, represent a potentially large number of predictable, shared, immunogenic, and safe antigens to use in preventative cancer vaccines and as targets for cancer therapies.

Successful proteome analysis requires reliable sample preparation beginning with protein solubilization and ending with a sample free of contaminants, ready for downstream analysis. Most proteome sample preparation technologies utilize precipitation or filter-based separation, both of which have significant disadvantages. None of the current technologies are able to prepare both intact proteins or digested peptides. Here, we introduce a reversible protein tag, ProMTag, that enables whole proteome capture, cleanup, and release of intact proteins for top-down analysis. Alternatively, the addition of a novel Trypsin derivative to the workflow generates peptides for bottom-up analysis. We show that the ProMTag workflow yields >90% for intact proteins and >85% for proteome digests. For top-down analysis, ProMTag cleanup improves resolution on 2D gels; for bottom-up exploration, this methodology produced reproducible mass spectrometry results, demonstrating that the ProMTag method is a truly universal approach that produces high-quality proteome samples compatible with multiple downstream analytical techniques. Data are available via ProteomeXchange with identifier PXD027799.

<div>Abstract<p>Tumor-associated antigens (TAA) are self-molecules abnormally expressed on tumor cells, which elicit humoral and cellular immunity and are targets of immunosurveillance. Immunity to TAAs is found in some healthy individuals with no history of cancer and correlates positively with a history of acute inflammatory and infectious events and cancer risk reduction. This suggests a potential role in cancer immunosurveillance for the immune memory elicited against disease-associated antigens (DAA) expressed on infected and inflamed tissues that are later recognized on tumors as TAAs. To understand probable sources for DAA generation, we investigated <i>in vitro</i> the role of inflammation that accompanies both infection and carcinogenesis. After exposure of normal primary breast epithelial cells to proinflammatory cytokines IL1β, IL6, and TNFα, or macrophages producing these cytokines, we saw transient overexpression of well-known TAAs, carcinoembryonic antigen and Her-2/neu, and overexpression and hypoglycosylation of MUC1. We documented inflammation-induced changes in the global cellular proteome by 2D difference gel electrophoresis combined with mass spectrometry and identified seven new DAAs. Through gene profiling, we showed that the cytokine treatment activated NF-κB and transcription of the identified DAAs. We tested three <i>in vitro</i>–identified DAAs, Serpin B1, S100A9, and SOD2, and found them overexpressed in premalignant and malignant breast tissues as well as in inflammatory conditions of the colon, stomach, and liver. This new category of TAAs, which are also DAAs, represent a potentially large number of predictable, shared, immunogenic, and safe antigens to use in preventative cancer vaccines and as targets for cancer therapies.</p></div>

Tumor-associated antigens (TAAs) are self-molecules abnormally expressed on tumor cells that elicit humoral and cellular immunity and are targets of effective cancer immunosurveillance. Surprisingly, immunity to TAAs is found in many healthy individuals with no history of cancer. We previously showed that other events in addition to cancer, such as viral and bacterial infections, can cause the expression of some TAAs and correlate with effective tumor immunosurveillance. We are now testing in mice the ability of two LCMV strains, one that causes acute (Armstrong) and one that causes chronic (Cl-13) infection, to elicit expression of and immune memory to TAAs and protect from a later tumor challenge. We observed that infection with the acute strain elicits immunity to specific TAAs expressed by two tumor cell lines that were then used in tumor challenge experiments. So far, we were able to show a protective effect of a previous Armstrong infection against our model of lymphoid tumor (EL4). We also developed a highly reproducible in vitro model of acute inflammation of primary epithelial cells and confirmed that short-term exposure to pro-inflammatory cytokines IL-1β, IL-6 and TNF-α leads to abnormal expression of three well known TAAs, MUC1, CEA and HER2/neu. These cells were found to activate macrophages, which should promote antigen uptake and presentation of these and other TAAs. We have used this system and 2D Difference Gel Electrophoresis (2D-DIGE) combined with mass spectrometry to identified so far seven new TAAs. Because these TAAs are generated first as disease-associated antigens (DAAs), they represent highly predictable shared antigens for safe and effective preventative cancer vaccines.