Perspectives on Clinical Adoption Barriers to Blood-Based Multi-Cancer Early Detection Tests across Stakeholders.

Current United States Preventive Services Task Force (USPSTF) recommendations include routine screening for breast, cervical, colorectal, and lung cancer; however, two out of every three cancer cases occur in other indications, leading to diagnoses in advanced stages of the disease and a higher likelihood of mortality. Blood-based multi-cancer early detection (MCED) tests can impact cancer screening and early detection by monitoring for multiple different cancer types at once, including indications where screening is not performed routinely today. We conducted a survey amongst healthcare providers (HCPs), payers, and patients within the U.S. health system to understand the current utilization of cancer screening tests and the anticipated barriers to widespread adoption of blood-based MCED tests. The results indicated that the community favors the adoption of blood-based MCED tests and that there is broad agreement on the value proposition. Despite this recognition, the survey highlighted that there is limited use today due to the perceived lack of clinical accuracy and utility data, high out-of-pocket patient costs, and lack of payer coverage. To overcome the hurdles for future widespread adoption of blood-based MCED tests, increased investment in data generation, education, and implementation of logistical support for HCPs will be critical.

Stakeholders Perceptions of Barriers to Precision Medicine Adoption in the United States.

Despite evidence that precision medicine (PM) results in improved patient care, the broad adoption and implementation has been challenging across the United States (US). To better understand the perceived barriers associated with PM adoption, a quantitative survey was conducted across five stakeholders including medical oncologists, surgeons, lab directors, payers, and patients. The results of the survey reveal that stakeholders are often not aligned on the perceived challenges with PM awareness, education and reimbursement, with there being stark contrast in viewpoints particularly between clinicians, payers, and patients. The output of this study aims to help raise the awareness that misalignment on the challenges to PM adoption is contributing to broader lack of implementation that ultimately impacts patients. With better understanding of stakeholder viewpoints, we can help alleviate the challenges by focusing on multi-disciplinary education and awareness to ultimately improve patient outcomes.

Investigation of candidate genes involved in the rhodoquinone biosynthetic pathway in Rhodospirillum rubrum.

The lipophilic electron-transport cofactor rhodoquinone (RQ) facilitates anaerobic metabolism in a variety of bacteria and selected eukaryotic organisms in hypoxic environments. We have shown that an intact rquA gene in Rhodospirillum rubrum is required for RQ production and efficient growth of the bacterium under anoxic conditions. While the explicit details of RQ biosynthesis have yet to be fully delineated, ubiquinone (Q) is a required precursor to RQ in R. rubrum, and the RquA gene product is homologous to a class I methyltransferase. In order to identify any additional requirements for RQ biosynthesis or factors influencing RQ production in R. rubrum, we performed transcriptome analysis to identify differentially expressed genes in anoxic, illuminated R. rubrum cultures, compared with those aerobically grown in the dark. To further select target genes, we employed a bioinformatics approach to assess the likelihood that a given differentially expressed gene under anoxic conditions may also have a direct role in RQ production or regulation of its levels in vivo. Having thus compiled a list of candidate genes, nine were chosen for further study by generation of knockout strains. RQ and Q levels were quantified using liquid chromatography-mass spectrometry, and rquA gene expression was measured using the real-time quantitative polymerase chain reaction. In one case, Q and RQ levels were decreased relative to wild type; in another case, the opposite effect was observed. These results comport with the crucial roles of rquA and Q in RQ biosynthesis, and reveal the existence of potential modulators of RQ levels in R. rubrum.

Employing proteomics to understand the effects of nutritional intervention in cancer treatment.

Lifestyle optimizations are implementable changes that can have an impact on health and disease. Nutrition is a lifestyle optimization that has been shown to be of great importance in cancer initiation, progression, and metastasis. Dozens of clinical trials are currently in progress that focus on the nutritional modifications that cancer patients can make prior to and during medical care that increase the efficacy of treatment. In this review, we discuss various nutritional inventions for cancer patients and the analytical approaches to characterize the downstream molecular effects. We first begin by briefly explaining the many different forms of nutritional intervention currently being used in cancer treatment as well as their motivating biology. The forms of nutrient modulation described in this review include calorie restriction, the different practices of fasting, and carbohydrate restriction. The review then shifts to explain how proteomics is used to determine biomarkers of cancer and how it can be utilized in the future to determine the metabolic phenotype of a tumor, and inform physicians if nutritional intervention should be recommended for a cancer patient. Nutrigenomics aims to understand the relationship of nutrients and gene expression and can be used to understand the downstream molecular effects of nutrition restriction, partially through proteomic analysis. Proteomics is just beginning to be used as cancer diagnostic and predictive tools. However, these approaches have not been used to their full potential to understand nutritional intervention in cancer. Graphical abstract ᅟ.

Combined Short-Term Glucose Starvation and Chemotherapy in 3D Colorectal Cancer Cell Culture Decreases 14-3-3 Family Protein Expression and Phenotypic Response to Therapy.

Short-term glucose starvation prior to chemotherapy has the potential to preferentially weaken cancer cells, making them more likely to succumb to treatment, while protecting normal cells. In this study, we used 3D cell cultures of colorectal cancer and assessed the effects of short-term glucose starvation and chemotherapy compared to treatment of either individually. We evaluated both phenotypic changes and protein expression levels. Our findings indicate that the combined treatment results in more significant phenotypic responses, including decreased cell viability and clonogenicity. These phenotypic responses can be explained by the decreased expression of LDHA and 14-3-3 family proteins, which were found only in the combined treatment groups. This study indicates that short-term glucose starvation has the potential to increase the efficacy of current cancer treatment regimes. Graphical Abstract ᅟ.

Comparison of In-Solution, FASP, and S-Trap Based Digestion Methods for Bottom-Up Proteomic Studies.

Bottom-up proteomic strategies rely on efficient digestion of proteins into peptides for mass spectrometry analysis. In-solution and filter-based strategies are commonly used for proteomic analysis. In recent years, filter-aided sample preparation (FASP) has become the dominant filter-based method due to its ability to remove SDS prior to mass spectrometry analysis. However, the time-consuming nature of FASP protocols have led to the development of new filter-based strategies. Suspension traps (S-Traps) were recently reported as an alternative to FASP and in-solution strategies as they allow for high concentrations of SDS in a fraction of the time of a typical FASP protocol. In this study, we compare the yields from in-solution, FASP, and S-Trap based digestions of proteins extracted in SDS and urea-based lysis buffers. We performed label-free quantification to analyze the differences in the portions of the proteome identified using each method. Overall, our results show that each digestion method had a high degree of reproducibility within the method type. However, S-Traps outperformed FASP and in-solution digestions by providing the most efficient digestion with the greatest number of unique protein identifications. This is the first work to provide a direct quantitative comparison of two filter-based digestion methods and a traditional in-solution approach to provide information regarding the most efficient proteomic preparation.

Calcitriol Supplementation Causes Decreases in Tumorigenic Proteins and Different Proteomic and Metabolomic Signatures in Right versus Left-Sided Colon Cancer.

Vitamin D deficiency is a common problem worldwide. In particular, it is an issue in the Northern Hemisphere where UVB radiation does not penetrate the atmosphere as readily. There is a correlation between vitamin D deficiency and colorectal cancer incidence and mortality. Furthermore, there is strong evidence that cancer of the ascending (right side) colon is different from cancer of the descending (left side) colon in terms of prognosis, tumor differentiation, and polyp type, as well as at the molecular level. Right-side tumors have elevated Wnt signaling and are more likely to relapse, whereas left-side tumors have reduced expression of tumor suppressor genes. This study seeks to understand both the proteomic and metabolomic changes resulting from treatment of the active metabolite of vitamin D, calcitriol, in right-sided and left-sided colon cancer. Our results show that left-sided colon cancer treated with calcitriol has a substantially greater number of changes in both the proteome and the metabolome than right-sided colon cancer. We found that calcitriol treatment in both right-sided and left-sided colon cancer causes a downregulation of ribosomal protein L37 and protein S100A10. Both of these proteins are heavily involved in tumorigenesis, suggesting a possible mechanism for the correlation between low vitamin D levels and colon cancer.

Glucose Restriction Combined with Autophagy Inhibition and Chemotherapy in HCT 116 Spheroids Decreases Cell Clonogenicity and Viability Regulated by Tumor Suppressor Genes.

Drug resistance is a prevalent phenomenon that decreases the efficacy of cancer treatments and contributes to cancer progression and metastasis. Weakening drug-resistant cancer cells prior to chemotherapy is a potential strategy to combat chemoresistance. One approach to damage resistant cancer cells is modulation of nutritional intake. The combination of nutrient restriction with targeted compound treatment results in pronounced molecular changes. This study provides valuable information about augmenting existing chemotherapeutic regimes with simultaneous glucose restriction and autophagy inhibition in colorectal cancer cells. In this study, we explore the chemical pathways that drive the cellular response to nutrient restriction, autophagy inhibition, and the chemotherapy irinotecan using global quantitative proteomics and imaging mass spectrometry. We determined that significant pathways were altered including autophagy and metabolism via glycolysis, gluconeogenesis, and sucrose degradation. We also found that period circadian clock 2 (PER2), a tumor suppressor protein, was significantly up-regulated only when glucose was restricted with autophagy inhibition and chemotherapy. The upstream regulators of these differentially regulated pathways were determined to have implications in cancer, showing an increase in tumor suppressor proteins and a decrease in nuclear protein 1 (NUPR1) an important protein in chemoresistance. We also evaluated the phenotypic response of these cells and discovered autophagy inhibition and chemotherapy treatment increased apoptosis and decreased cell clonogenicity and viability. When glucose restriction was combined with autophagy inhibition and chemotherapy, all of the phenotypic results were intensified. In sum, our results indicate that glucose metabolism is of great importance in the ability of cancer cells to survive chemotherapy. By weakening cancer cells with glucose restriction and autophagy inhibition prior to chemotherapy, cancer cells become more sensitive to therapy.

Multicellular Tumor Spheroids Combined with Mass Spectrometric Histone Analysis To Evaluate Epigenetic Drugs.

Multicellular tumor spheroids (MCTS) are valuable in vitro tumor models frequently used to evaluate the penetration and efficacy of therapeutics. In this study, we evaluated potential differences in epigenetic markers, i.e., histone post-translational modifications (PTMs), in the layers of the HCT116 colon carcinoma MCTS. Cells were grown in agarose-coated 96 well plates, forming reproducible 1-mm-diameter MCTS. The MCTS were fractionated into three radially concentric portions, generating samples containing cells from the core, the mid and the external layers. Using mass spectrometry (MS)-based proteomics and EpiProfile, we quantified hundreds of histone peptides in different modified forms; by combining the results of all experiments, we quantified the abundance of 258 differently modified peptides, finding significant differences in their relative abundance across layers. Among these differences, we detected higher amounts of the repressive mark H3K27me3 in the external layers, compared to the core. We then evaluated the epigenetic response of MCTS following UNC1999 treatment, a drug targeting the enzymes that catalyze H3K27me3, namely, the polycomb repressive complex 2 (PRC2) subunits enhancer of zeste 1 (EZH1) and enhancer of zeste 2 (EZH2). UNC1999 treatment resulted in significant differences in MCTS diameter under drug treatment of varying duration. Using matrix-assisted laser desorption/ionization (MALDI) imaging, we determined that the drug penetrates the entire MCTS. Proteomic analysis revealed a decrease in abundance of H3K27me3, compared to the untreated sample, as expected. Interestingly, we observed a comparable growth curve for MCTS under constant drug treatment over 13 days with those treated for only 4 days at the beginning of their growth. We thus demonstrate that MS-based proteomics can define significant differences in histone PTM patterns in submillimetric layers of three-dimensional (3D) cultures. Moreover, we show that our model is suitable for monitoring drug localization and regulation of histone PTMs after drug treatment.

Nutrient restriction of glucose or serum results in similar proteomic expression changes in 3D colon cancer cell cultures.

Nutrient restriction, also known as caloric restriction, has been extensively examined for its positive impact on lifespan, immune system boost, and aging. In addition, nutrient restriction is implicated in decreasing cancer initiation and progression. Given the phenotypic changes associated with nutrient restriction, we hypothesized significant protein expression alterations must be associated with caloric restriction. To compare the molecular and phenotypic changes caused by glucose restriction and fetal bovine serum restriction there is need for an efficient model system. We establish 3-dimensional cell culture models, known as spheroids, in the HCT 116 colorectal cancer cell line as a high throughput model for studying the proteomic changes associated with nutrient restriction. Flow cytometry was used to assess apoptosis and autophagy levels in the spheroids under nutrient restriction. Isobaric tags for relative and absolute quantification and liquid chromatography tandem mass spectrometry were used to determine differential protein abundances between the nutrient restriction conditions. We identified specific proteins that have implications in cancer progression and metastasis that are differentially regulated by restriction of either glucose or serum. These proteins include the up-regulation of sirtuin 1 and protein inhibitor of activated STAT 1 and down-regulation of multi-drug resistance protein and Zinc finger and BTB domain-containing protein 7A. The results indicate nutrient restriction causes lower apoptotic and higher autophagy rates in HCT 116 spheroids. In addition, proteins shown to be differentially regulated by both glucose and serum restriction were similarly regulated.

Surface histidine mutations for the metal affinity purification of a ß-carbonic anhydrase.

Metal affinity chromatography using polyhistidine tags is a standard laboratory technique for the general purification of proteins from cellular systems, but there have been no attempts to explore whether the surface character of a protein may be engineered to similar affinity. We present the Arg160His mutation of Haemophilus influenzae carbonic anhydrase (HICA), which mimics the endogenous metal affinity of Escherichia coli carbonic anhydrase (ECCA). The purity and activity of the mutant are reported, and the purification is discussed. This is the first step toward developing a general method to engineer surface metal affinity for use in purification and metal labeling techniques.