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.

The ability of immune cells to sense changes associated with malignant transformation as early as possible is likely to be important for the successful outcome of cancer immunosurveillance. In this process, the immune system faces a trade-off between elimination of cells harboring premalignant or malignant changes, and autoimmune pathologies. We hypothesized that the immune system has therefore evolved a threshold for the stage of transformation from normal to fully malignant cells that first provides a threat (danger) signal requiring a response. We co-cultured human macrophages with a unique set of genetically related human cell lines that recapitulate successive stages in breast cancer development: MCF10A (immortalized, normal); MCFNeoT (benign hyperplasia); MCFT1 (atypical hyperplasia); MCFCA1 (invasive cancer). Using cytokines-based assays, we found that macrophages were inert towards MCF10A and MCFNeoT but were strongly activated by MCFT1 and MCFCA1 to produce inflammatory cytokines, placing the threshold for recognition between two premalignant stages, the earlier stage MCFNeoT and the more advanced MCFT1. The cytokine activation threshold paralleled the threshold for enhanced phagocytosis. Using proteomic and transcriptomic approaches, we identified surface molecules, some of which are well-known tumor-associated antigens, that were absent or expressed at low levels in MCF10A and MCFNeoT but turned on or over-expressed in MCFT1 and MCFCA1. Adding antibodies specific for two of these molecules, Annexin-A1 and CEACAM1, inhibited macrophage activation, supporting their role as cancer “danger signals” recognized by macrophages.

ObjectivesBreastfeeding is an energetically costly and intense form of human parental investment, providing sole‐source nutrition in early infancy and bioactive components, including immune factors. Given the energetic cost of lactation, milk factors may be subject to tradeoffs, and variation in concentrations have been explored utilizing the Trivers‐Willard hypothesis. As human milk immune factors are critical to developing immune system and protect infants against pathogens, we tested whether concentrations of milk immune factors (IgA, IgM, IgG, EGF, TGFβ2, and IL‐10) vary in response to infant sex and maternal condition (proxied by maternal diet diversity [DD] and body mass index [BMI]) as posited in the Trivers‐Willard hypothesis and consider the application of the hypothesis to milk composition.MethodsWe analyzed concentrations of immune factors in 358 milk samples collected from women residing in 10 international sites using linear mixed‐effects models to test for an interaction between maternal condition, including population as a random effect and infant age and maternal age as fixed effects.ResultsIgG concentrations were significantly lower in milk produced by women consuming diets with low diversity with male infants than those with female infants. No other significant associations were identified.ConclusionsIgG concentrations were related to infant sex and maternal diet diversity, providing minimal support for the hypothesis. Given the lack of associations across other select immune factors, results suggest that the Trivers‐Willard hypothesis may not be broadly applied to human milk immune factors as a measure of maternal investment, which are likely buffered against perturbations in maternal condition.

Therapeutic cancer vaccines have been tested for years but showed limited immunogenicity and clinical efficacy mainly because of the immunosuppressive environment generated by tumors. One approach for improving cancer vaccine efficacy would be to use them prior to cancer occurrence to strengthen immunosurveillance. This would ensure cancer elimination at the earliest stages of carcinogenesis and avoid cancer editing and escape. However, identifying antigens that can be incorporated into safe and effective prophylactic cancer vaccines remains a major challenge. Best candidates would be shared tumor-associated antigens (TAA) that are reproducibly and stably expressed on advanced tumors and premalignant lesions but not on normal tissues and that would induce antibodies and T cells causing tumor rejection without danger of autoimmunity. Antibodies and T cells specific for some well-known TAA have been found in individuals without cancer but with a history of acute infections and this pre-existing immunity has been associated with lowered lifetime risk for developing cancer while causing no obvious toxicity. We hypothesized that those immune responses were generated to self-antigens that are abnormally expressed on infected cells and again later on tumor cells, which we named disease-associated antigens (DAA)/tumor associated antigens (TAA), DAA/TAA. We tested this hypothesis here using two strains of lymphocytic choriomeningitis virus (LCMV): Armstrong (Arm) strain and CL-13 that cause acute and chronic infection in mice, respectively. Both strains elicited antibodies that recognized antigens on mouse lung (LLC) and lymphoid (EL4) tumors. Mice were later injected with tumor cells and Arm-infected mice controlled tumor challenge better than naïve controls, whereas the Cl-13 infection in mice had either no effect or promoted tumor growth. We characterized 5 DAA/TAA that were targets of this virus infection-elicited anti-tumor immunity using immunoprecipitation followed by 2D DIGE and mass spectrometry. Our results suggest that a vaccine based on DAA/TAA that are expressed on tumors and infected cells could confer protection against both cancer and a viral infection. Accordingly, we are currently immunizing mice with peptides derived from the 5 DAA/TAA. Mice responding to the vaccine will be given either tumors or LCMV and we will measure their response to both and the immune effector mechanisms mediating the response. Citation Format: Camille Jacqueline, Sarah Boothman, Jonathan S. Minden, Olivera J. Finn. Acute but not chronic LCMV infection generates immunity against abnormally expressed self-antigens on infected and tumor cells and protects against lung and lymphoid cancers in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1763.