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The concept of the stem cell of origin for cancers was first proposed more than 100 years ago. In this theoretical model, certain cells with self-renewal capacity would form tumors from “embryonic rests.” More evidence has accumulated since that time as a result of extensive research that strongly supports the cancer stem cell (CSC) hypothesis, suggesting the existence of self-renewing cells that generate heterogenous populations of cells within the tumor mass. The preponderance of evidence now suggests that the majority of cancers are hierarchically organized and sustained by a population of cells that display stem cell properties—CSCs. As is the case for normal tissue stem cells, CSCs are able to self-renew and differentiate, generating cells that comprise the tumor bulk. Furthermore, preclinical and clinical studies demonstrate that CSCs mediate tumor metastasis and contribute to resistance to chemotherapy and radiation therapy. The CSC hypothesis has fundamental biological and clinical implications that are discussed in detail in this chapter.

Infection negatively impacts mental health, as evidenced by the lethargy, malaise, and cognitive deficits experienced during illness. These changes in central nervous system processes, collectively termed sickness behavior, have been shown in animal models to be mediated primarily by the actions of cytokines in brain. Most studies of sickness behavior to date have used bolus injection of bacterial lipopolysaccharide (LPS) or selective administration of the proinflammatory cytokines interleukin-1β (IL-1β) or IL-6 as the immune challenge. Such models, although useful for determining mechanisms responsible for acute changes in physiology and behavior, do not adequately represent the more complex effects on central nervous system (CNS) processes of a true infection with replicating pathogens. In the present study, we used the cecal ligation and puncture (CLP) model to quantify sepsis-induced alterations in several facets of physiology and behavior of mice. We determined the impact of sepsis on cage activity, body temperature, food and water consumption and body weights of mice. Because cytokines are critical mediators of changes in behavior and temperature regulation during immune challenge, we also quantified sepsis-induced alterations in cytokine mRNA and protein in brain during the acute period of sepsis onset. We now report that cage activity and temperature regulation in mice that survive are altered for up to 23 days after sepsis induction. Food and water consumption are transiently reduced, and body weight is lost during sepsis. Furthermore, sepsis decreases social interactions for 24 – 48 hours. Finally, mRNA and protein for IL-1β, IL-6, and tumor necrosis factor-α (TNFα) are upregulated in the hypothalamus, hippocampus, and brain stem during sepsis onset, from 6–72 hour post sepsis induction. Collectively, these data indicate that sepsis not only acutely alters physiology, behavior and cytokine profiles in brain, but that some brain functions are impaired for long periods in animals that survive.

Standard protocols for sepsis treatment, relying on broad-spectrum antibiotics, fluid resuscitation, and ventilation, have remained essentially unchanged for the last three decades. Although decreased mortality has been observed with early goal-directed therapy and activated protein C (1), aggressive anti-inflammatory trials, either in the form of corticosteroid-based therapy (2–5) or more recent specific anti-cytokine approaches (6), not only failed to improve survival but some were even associated with increased mortality (7). Corticosteroids have a long history in the therapy of sepsis, beginning with the landmark study by Schumer (8), which showed a 30% improvement in survival after early dexamethasone (DEX) and prednisolone treatment in patients with septic shock. However, subsequent clinical trials contradicted the original findings by showing that high-dose glucocorticoids did not improve outcome in septic patients (3, 4, 9, 10). It should be noted that low-dose steroids were reintroduced as an adjunctive therapy in patients with adrenal insufficiency (11). There is a growing consensus that appropriate therapy for sepsis cannot rely on the “magic bullet” paradigm where specific, targeted therapy directed against a single mediator will be successful. Two major immune conditions proposed to coexist during the course of sepsis, the systemic inflammatory response syndrome and the compensatory anti-inflammatory response syndrome, emphasize that sepsis constantly fluctuates (12). Thus, successful treatment needs to be frequently adjusted based on the patient’s current immunoinflammatory status. Statistical analysis suggested that the failure of the anticytokine trials may have been related to heterogeneous patient populations, because cohorts with the highest mortality risk were more likely to benefit from anti-inflammatory interventions (13). Using inflammatory cytokines as biomarkers, we developed an approach that allows accurate mortality prediction in the cecal ligation and puncture (CLP) model of murine sepsis (14, 15). To verify this approach in a scenario of targeted treatment, we hypothesized that nonspecific corticosteroid (DEX) suppression of inflammation in subjects predicted to die (P-DIE) would improve survival, whereas the identical treatment applied to animals predicted to live (P-LIVE) will produce little benefit.

The use of proteomics for efficient, accurate, and complete analysis of clinical samples poses a variety of technical challenges. The presence of higher abundance proteins in the plasma, such as albumin, may mask the detection of lower abundance proteins such as the cytokines. Methods have been proposed to deplete the sample of these higher abundance proteins to facilitate detection of those with lower abundance. In this study, a commercially available albumin depletion kit was used to determine if removal of albumin would measurably reduce detection of lower abundance cytokine proteins in human plasma. The Montage Albumin Deplete Kit (Millipore) was used to deplete albumin from LPS-stimulated whole blood from 15 normal human donors. Albumin depletion was measured using the BCG reagent and SDS-PAGE, and cytokine recovery was determined by a microassay immunoassay that measures both pro- and anti-inflammatory cytokines. Average albumin depletion from the samples was 72%. However, several cytokines were also significantly reduced when the albumin was removed from the plasma. Additionally, there was a variable reduction in cytokine recovery from a known mixture of cytokines in a minimal amount of plasma that were loaded onto the columns. These data demonstrate that there may be a non-specific loss of cytokines following albumin depletion, which may confound subsequent proteomic analysis.

Although sepsis causes significant morbidity and mortality, its basic pathology is still not well understood. We investigated the inflammatory and physiologic alterations of non-lethal sepsis using cecal ligation and puncture (CLP), a model that induces peritonitis due to mixed intestinal flora, reproducing the complex immunology of sepsis. Groups of mice were subjected to CLP (25G needle) or sham surgery, had minimitters implanted to continuously monitor temperature and activity, and were sacrificed daily for 6 days. There was significant hypothermia (6-13 hrs post-surgery), and decreases in activity (to day 4) and weight (to day 3) but no mortality in the CLP group. Blood analyses of the CLP-treated mice showed reduced hemoglobin, platelets, lymphocytes, monocytes, and neutrophils, compared to sham animals. Both groups had nearly equivalent neutrophil influx into the peritoneum. Plasma and peritoneal G-CSF, IL-6, as well as the murine chemokines KC and MIP2-alpha were significantly higher in the CLP-treated mice at day 1. Plasma and peritoneal TNF were low (

Most tumors are hierarchically organized and driven by a population of cells that display stem cell properties. These cancer stem cells (CSCs) have been characterized by in vitro assays such as sphere formation and in vivo by tumor initiation. In addition cell surface markers have been utilized to enrich for CSC populations. More recently a subtype of CSCs have been reported to display autofluorescence (AF), a property mediated by cellular uptake and concentration of riboflavin. In order to characterize the molecular heterogeneity of CSCs, we studied AF positive sorted single cells in different subtypes of breast cancer cell lines and also patient derived xenograft (PDX) breast tumor cells. Exposure of cell lines and PDXs to riboflavin significantly increased the percentage of AF+ cells 3-5 fold. These sorted AF+ cells were enriched for tumorsphere forming capacity in vitro. Flow cytometry analysis revealed partial overlap of AF+ cells with the previously characterized EMT-CSC phenotype CD44+/CD24- cells. We also utilized the microfluidic C1 and BioMark HD instruments to determine the expression patterns of 96 target genes using TaqMan assays in a multiplex RT-qPCR setting at single cell resolution. Single cell gene expression data showed a distinct signature in AF+ cells with high expression levels of MKI67, PCNA, ABCG2, BRCA1, Jag2, EZH2, and MMP9 genes. Since AF is mediated by specific cellular uptake and concentration of riboflavin, and the report that riboflavin is capable of generating cytotoxic free radicals upon light exposure, we determined whether this property could be exploited to selectively target CSCs. The AF+ cells were cultured in the presence and absence of riboflavin, exposed to visible light and analyzed by MTT cytotoxicity assay. Data revealed significant cytotoxicity of riboflavin on AF+ cells that were exposed to visible light. In conclusion, AF+ cells show distinct cancer stem cell features and are sensitive to light-activated riboflavin. These findings will help better understanding of tumor biology to identify new target therapies for treatment of cancer patients. Citation Format: Shamileh Fouladdel, Tahra Luther, Kaitlin Harvey, Rachel Martin-Trevino, Jill Granger, Ebrahim Azizi, Max S. Wicha. Riboflavin targets autofluorescent-positive breast cancer stem cells upon light exposure. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3310.

Granulocyte colony-stimulating factor (G-CSF) stimulates the proliferation and differentiation of hematopoietic progenitor cells of the neutrophil lineage. Measurement of murine G-CSF levels will allow examination of its role in host defense using murine models. Therefore, we developed a sensitive sandwich enzyme-linked immunoabsorbent assay (ELISA) for murine G-CSF. A polyclonal antibody to recombinant murine G-CSF was produced in rabbits and isolated using a protein A column. This purified native IgG served as the capture antibody and a portion of the IgG was biotinylated to serve as the developing antibody. Specificity was verified by lack of reactivity to GM-CSF, IL-6, IL-3, prolactin, and growth hormone. The lower limit of sensitivity routinely extended to 16 pg/ml in multiple ELISAs. Intra-assay coefficient of variation (CV) ranged from 3.4 to 21.5% across the detection limits of the assay, with the greatest variance occurring near the standard curve maximum. Interassay CV ranged from 11.5 to 23.3%. The ability of the ELISA to detect G-CSF in different sample preparations was examined in RPMI 1640 with 10% FCS, Hanks balanced salt solution, PBS/Tween-20/2% FCS, and the dilution media for ELISA (10% BLOTTO/PBS/0.05% Tween-20). Average recovery in these media ranged from 98 to 107%. Heparin anti-coagulated normal mouse plasma had a suppressive effect on the ELISA that varied between individual mice. Recovery was also determined from liver, spleen, and lung homogenate suspensions at dilutions of 1:5, 1:10, and 1:20 in dilution buffer. Recovery from liver was optimal at the 1:10 and 1:20 dilutions at 105%, with that of the 1:5 dilution at 135%. Recovery from spleen ranged from 94 to 96%. Lung homogenate displayed enhanced recovery (139% or greater) across all dilutions. The ability of the assay to detect G-CSF was explored by measurement of G-CSF levels in peritoneal lavage following polymicrobial intra-abdominal infection. Peak levels of G-CSF production occurred at 16 h after cecal ligation and puncture surgery with 18- and 21-guage needles (75.7 ng/ml and 111.4 ng/ml, respectively) as compared to the sham animals (0.61 ng/ml). The assay was found to be specific, sensitive, and accurate for measurement of murine G-CSF in a variety of sample types.